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A Platform for Generation of Chamber-Specific Cardiac Tissues and Disease Modeling
Abstract
Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.
... This has been achieved using modulated retinoic acid signalling during cardiomyocyte differentiation [78]. Other biomimetic approaches and tissue engineering techniques have been shown to enable the creation of electrophysiologically distinct atrial and ventricular 3D microtissues, with chamber-specific gene expression and drug responses [79]. ...
... Models are being developed that are able to provide biophysical stimulation of 3D tissues to model polygenic diseases over many months [79]. The complexity of such systems cannot be understated, combining, for example, microwells, conducting polymeric electrodes and myocardial tissues, created by combining CMs (ventricular, atrial, or both) and cardiac fibroblasts with hydrogel [79]. ...
... Models are being developed that are able to provide biophysical stimulation of 3D tissues to model polygenic diseases over many months [79]. The complexity of such systems cannot be understated, combining, for example, microwells, conducting polymeric electrodes and myocardial tissues, created by combining CMs (ventricular, atrial, or both) and cardiac fibroblasts with hydrogel [79]. Such systems rely upon combined directed cell differentiation, along with electrical field conditioning, in order to engineer electrophysiologically distinct atrial and ventricular tissues [79]. ...
Cardiovascular disease remains the leading cause of death worldwide, yet despite massive investment in drug discovery, the progress of cardiovascular drugs from lab to clinic remains slow. It is a complex, costly pathway from drug discovery to the clinic and failure becomes more expensive as a drug progresses along this pathway. The focus has begun to shift to optimisation of in vitro culture methodologies, not only because these must be undertaken are earlier on in the drug discovery pathway, but also because the principles of the 3Rs have become embedded in national and international legislation and regulation. Numerous studies have shown myocyte cell behaviour to be much more physiologically relevant in 3D culture compared to 2D culture, highlighting the advantages of using 3D-based models, whether microfluidic or otherwise, for preclinical drug screening. This review aims to provide an overview of the challenges in cardiovascular drug discovery, the limitations of traditional routes, and the successes in the field of preclinical models for cardiovascular drug discovery. It focuses on the particular role biomimicry can play, but also the challenges around implementation within commercial drug discovery.
... Various EHT platforms have been developed and used across different research groups [9][10][11]. Many of them are based on the model of cardiac tissue suspended between two or more elastic pillars. ...
... These models found application in fundamental research, drug development, disease modeling, and pharmaceutical safety. [7,9,16,41,42]. Many platforms support EHT formation and culture around rectangular or circular cross-section pillars, and mainly use image analysis to determine the contractile performance of tissues. ...
... Some of the main limitations of existing software for contractile analysis are that they are platform and pillar-shape specific [9,10,[43][44][45][46], which constrained their use within the scientific community. Specifically, Hansen et al. [10] uses software developed by a private company that tracks the top and bottom of the cylindrical pillars. ...
Engineered heart tissues (EHTs) have shown great potential in recapitulating tissue organization, functions, and cell-cell interactions of the human heart in vitro. Currently, multiple EHT platforms are used by both industry and academia for different applications, such as drug discovery, disease modelling, and fundamental research. The tissues’ contractile force, one of the main hallmarks of tissue function and maturation level of cardiomyocytes, can be read out from EHT platforms by optically tracking the movement of elastic pillars induced by the contractile tissues. However, existing optical tracking algorithms which focus on calculating the contractile force are customized and platform-specific, often not available to the broad research community, and thus hamper head-to-head comparison of the model output. Therefore, there is the need for robust, standardized and platform-independent software for tissues’ force assessment. To meet this need, we developed ForceTracker: a standalone and computationally efficient software for analyzing contractile properties of tissues in different EHT platforms. The software uses a shape-detection algorithm to single out and track the movement of pillars’ tips for the most common shapes of EHT platforms. In this way, we can obtain information about tissues’ contractile performance. ForceTracker is coded in Python and uses a multi-threading approach for time-efficient analysis of large data sets in multiple formats. The software efficiency to analyze circular and rectangular pillar shapes is successfully tested by analyzing different format videos from two EHT platforms, developed by different research groups. We demonstrate robust and reproducible performance of the software in the analysis of tissues over time and in various conditions. ForceTracker’s detection and tracking shows low sensitivity to common incidental defects, such as alteration of tissue shape or air bubbles. Detection accuracy is determined via comparison with manual measurements using the software ImageJ. We developed ForceTracker as a tool for standardized analysis of contractile performance in EHT platforms to facilitate research on disease modeling and drug discovery in academia and industry.
... Metabolic maturation can also provide maturation of key features including metabolism, cell cycle and sarcomeric isoforms 4 . In addition, pacing is one of the most potent inducers of functional maturation in terms of excitation-contraction coupling and drug responses [15][16][17] . Despite these advances, mechanistic understanding of the maturation stimuli are limited, and the induction of further maturation is challenging. ...
... We recently developed a serum-free protocol for hCO maturation (SF-hCO), featuring switching of metabolic substrates and vascularisation promoting growth factors 5 . We compared our SF-hCOs to various other hPSC-CM 3D cultures using mRNA expression ratios of sarcomeric proteins that are indicative of maturation 4,10,11,15,17 . MYH7 as a fraction of MYH7 and MYH6, did not correlate with maturation in any system, and is perhaps rate dependent (Extended Data 1a). ...
... MYH7 as a fraction of MYH7 and MYH6, did not correlate with maturation in any system, and is perhaps rate dependent (Extended Data 1a). MYL2 as a fraction of MYL2 and MYL7 (Extended Data 1b) and TNNI3 as a fraction of TNNI3 and TNNI1 (Extended Data 1c) were stronger indicators, with TNNI3 in particular correlating strongly with maturation and being elevated by pacing protocols that are proven methods of maturation 15,17 . It is noted that our SF-hCOs displayed advanced levels of maturation as a strong baseline to screen for further maturation conditions (Extended Data 1c). ...
Cardiac maturation is an important developmental phase culminating in profound biological and functional changes to adapt to the high demand environment after birth 1,2 . Maturation of human pluripotent stem cell-derived human cardiac organoids (hCO) to more closely resemble human heart tissue is critical for understanding disease pathology. Herein, we profile human heart maturation in vivo ³ to identify key signalling pathways that drive maturation in hCOs 4,5 . Transient activation of both the 5’ AMP-activated kinase (AMPK) and estrogen-related receptor (ERR) promoted hCO maturation by mimicking the increased functional demands of post-natal development. hCOs cultured under these directed maturation (DM) conditions (DM-hCOs) display robust transcriptional maturation including increased expression of mature sarcomeric and oxidative phosphorylation genes resulting in enhanced metabolic capacity. DM-hCOs have functionally mature properties such as sarcoplasmic reticulum-dependent calcium handling, accurate responses to drug treatments perturbing the excitation-coupling process and ability to detect ectopy CASQ2 and RYR2 mutants. Importantly, DM- hCOs permit modelling of complex human disease processes such as desmoplakin ( DSP ) cardiomyopathy, which is driven by multiple cell types. Subsequently, we deploy DM-hCOs to demonstrate that bromodomain extra-terminal inhibitor INCB054329 rescues the DSP phenotype. Together, this study demonstrates that recapitulating in vivo development promotes advanced maturation enabling disease modelling and the identification of a therapeutic strategy for DSP- cardiomyopathy.
... Importantly, the platform includes an array of microwells on polystyrene sheets secured at both ends with flexible wires made from poly (octamethylene maleate (anhydride) citrate) (POMaC) polymer using adhesive glue. Within these microwells, myocardial tissues are crafted by combining cardiomyocytes, either ventricular, atrial, or both, with cardiac fibroblasts, all embedded in the hydrogel [53]. ...
... The study of human cardiac tissue remodeling under pathophysiologically relevant mechanical loads and ECM compositions is further facilitated by decellularized heart matrices [48,50]. Microfluidic devices facilitate the modeling of ischemic gradients, mechanical stress, and inflammatory environments typically associated with heart failure [53]. Moreover, the immunological response to implanted materials, especially macrophage phenotype modulation, plays a central role in determining whether engineered cardiac tissues promote healing or exacerbate fibrotic outcomes [45]. ...
Cardiovascular diseases (CVD) are the primary cause of death and disability around the world. Over the past decades, several conventional model systems based on two-dimensional (3D) monolayer cultures or experimental animals have been adopted to dissect and understand heart diseases in order to develop treatment modalities. However, traditional models exhibit several limitations in recapitulating human-specific key physiological and pathological characteristics, which highlights the necessity of developing physiologically relevant models. In recent years, tissue engineering approaches have been extensively employed to generate revolutionary three-dimensional (3D) cardiac models. In particular, the combined use of various bioengineering strategies and cellular reprogramming approaches has facilitated the development of various models. This review presents an overview of different approaches (bioprinting, scaffolding, and electrospinning) for creating bioengineered cardiac tissue models. Next, a broad survey of recent research related to the modeling of various cardiac diseases is presented. Finally, current challenges and future directions are proposed to foster further developments in the field of cardiac tissue engineering.
... Patient-derived atrial and ventricular iPSC-CMs or human iPSC line BJ1D were co-embedded with CFs in hydrogel within the Biowire II platform and subjected to slow electrical conditioning. The electric stimulus effectively supported CM differentiation and promoted sarcomeric organization, the expression of chamber-specific proteins, calcium handling, and the reliance of differentiated CMs on glycolysis [47][48][49]. Such an approach, allowed for up to 8 months, enabled modelling of polygenic left ventricular hypertrophy starting from iPSC patient cells [49]. ...
... The electric stimulus effectively supported CM differentiation and promoted sarcomeric organization, the expression of chamber-specific proteins, calcium handling, and the reliance of differentiated CMs on glycolysis [47][48][49]. Such an approach, allowed for up to 8 months, enabled modelling of polygenic left ventricular hypertrophy starting from iPSC patient cells [49]. Additionally, a filtration layer-by-layer technique of 3D-bioprinting was applied to develop vascularized human iPSC-CM tissue combined with fabricated ECM-fibronectin-gelatin nanofilms [50]. ...
... Muscle on a Chip systems often comprise an elongated 3D miniature engineered muscle bundle, as these constructs have demonstrated more stability in long-term culture, higher myoblast fusion efficiency, [9,10] and enhanced transition from fetal to adult protein expression profiles [11,12] compared to planar 2D tissues. To fabricate 3D muscle bundles, muscle precursor cells are typically embedded in an extracellular matrix hydrogel and anchored to flexible pillars, [13][14][15][16] rods, [17] strings, [18] or frames. [19,20] In most examples, muscle bundle contraction causes deflection of the anchor points and thus the force of contraction can be estimated by capturing microscopy images of anchor deflection [13,16,17,19] or manually connecting the anchors to a force transducer. ...
... To fabricate 3D muscle bundles, muscle precursor cells are typically embedded in an extracellular matrix hydrogel and anchored to flexible pillars, [13][14][15][16] rods, [17] strings, [18] or frames. [19,20] In most examples, muscle bundle contraction causes deflection of the anchor points and thus the force of contraction can be estimated by capturing microscopy images of anchor deflection [13,16,17,19] or manually connecting the anchors to a force transducer. [21] However, the disadvantages of such approaches include low scalability and throughput and the absence of an immediate, in situ, readout of tissue performance. ...
Muscle on a Chip devices are valuable research tools for interrogating the structure and physiology of engineered heart, skeletal, or smooth muscle tissue constructs from the molecular to the multi‐cellular level. However, many existing devices rely on functional assays with limited throughput, such as optical microscopy, to measure contractility. Although electrical components have been integrated to automate recordings in advanced devices, their fabrication typically requires specialized equipment found in cleanroom facilities. In this work, miniature strain gauges are engineered to record the contractions of engineered skeletal muscle bundles using only benchtop fabrication equipment. A commercial CO2 laser is employed to generate patterns of laser‐induced graphene (LIG) on polyimide (PI) films. LIG is then transferred from PI to thin polydimethylsiloxane (PDMS) films to make conductive and intrinsically flexible and stretchable layers that demonstrate long‐term stability under repeated cycles of stretch. Engineered skeletal muscle bundles are anchored to LIG‐PDMS strain gauges and their contraction is sensed in response to electrical stimulation, which is delivered by LIG‐PI stimulation electrodes also integrated into the device. Collectively, these results demonstrate that LIG is an attractive material for rapidly and inexpensively integrating electrical components for in situ strain sensing and electrical stimulation in Muscle on a Chip devices.
... Isoproterenol is a positive chronotropic and inotropic compound used to treat bradycardia conditions 47 . After the addition of 10 µM isoproterenol (Fig. 4e), we observed an approximately 81% increase in field potential amplitude (Fig. 4f), an 84% increase in beat rate (Fig. 4g), and a 20% decrease in FPD ( Fig. 4h), findings that are consistent with previously reported data 41,42 . E-4031 is a hERG channelspecific blocker known to prolong atrial and ventricular refractoriness. ...
... Discussion (4-500 words) In this study, we present a new 3D activation mapping system for cardiac organoids using These advancements lay the groundwork for developing more physiologically relevant cardiac organoid models, and show promising applications for studying complex electrophysiological mechanisms, especially in disease models. Recent studies have highlighted the capacity of cardiac organoids to replicate more sophisticated structures of the heart 49 , including distinct atrial and ventricular regions by electrical conditioning 41,50 ; multiple chambers by co-developing progenitor subsets 51 ; and hypoxic gradients to model myocardial infarction 52 . Such cardiac organoids offer unparalleled possibilities to study tissue-scale and acquired arrhythmias that are beyond the reach of conventional 2D monolayer cultures (Fig. S16). ...
Cardiac organoids have emerged as transformative models for investigating cardiogenesis and cardiac diseases. While traditional 2D microelectrode arrays (MEAs) have been used to assess the functionality of cardiac organoids, they are limited to electrophysiological measurements from a single plane and do not capture the 3D propagation of electrical signals. Here, we present a programmable, shape-adaptive shell MEA designed to map the electrical activity across the entire surface of cardiac organoids. These shell MEAs are fabricated on-chip, with tunable dimensions and electrode layout, enabling precise encapsulation of spherical organoids. Using shell MEAs, we generated 3D isochrone maps with conduction velocity vectors, revealing the speed and trajectory of electrical signal propagation in spontaneously beating cardiac organoids. The optical transparency of the shell MEAs allowed for simultaneous calcium imaging, validating the electrophysiological propagation pattern. To demonstrate their utility in cardiotoxicity screening, we monitored the electrophysiological changes of organoids treated with isoproterenol and E-4031 over nine days. We anticipate that shell MEAs, combined with spatiotemporal mapping, can significantly advance the development of spatially organized cardiac organoids, structural disease models, and high-throughput drug screening platforms.
... For instance, iterations of mold design can be conceived, modeled, and 3D printed within a few hours, avoiding logistical constraints (e.g., time, material) of multi-step casting. We demonstrated this by creating hydrogel molds in different shapes (e.g., rectangular pillars, circular pillars, rectangular slabs) and with various modes of tissue anchoring, inspired by previous work (Extended Data Fig. 2) 12,[41][42][43] . To further illustrate the design flexibility, molds either without micropillars or with varying distance between micropillars (e.g., aspect ratios of 2:1, 4:1) were fabricated to control the microtissue aspect ratio, as visualized through bright field and F-actin staining of cells within the resulting EHTs (Fig. 2a). ...
... Therefore, a larger build plate would decrease the time necessary to print a single mold and thus increase the throughput. To place the current study in the context of previously reported EHTs [11][12][13][14]29,30,42 , we compared the throughput of fabrication to the size of the EHTs formed (Fig. 2e). Smaller EHTs reduce the number of iPSC-CMs required per EHT (e.g., as low as 500 cells) as compared to larger tissues that can require up to 1 to 2 million cells. ...
3D in vitro engineered heart tissue (EHT) models recapitulate aspects of native cardiac physiology but are often limited by scalability, cost, and reproducibility. Here, we report a simple, one-step method for rapid fabrication of molds using digital light processing (DLP)-based 3D printing that support the formation of EHTs by human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with high reproducibility (>95% efficiency) and varied designs (e.g., length, aspect ratio). Compared to 2D iPSC-CMs, 3D EHTs display enhanced maturity, including increased expression of B-oxidation genes, higher concentrations of sarcomeric myosins, improved sarcomere density and alignment, and enrichment of cardiac pathways (e.g., upregulation of sodium channels, action potentials, contraction). The technology is applied to model pathological cardiac hypertrophy in vitro, using either (i) acute adrenergic agonism or (ii) chronic culture within stiff hydrogel molds. Treated EHTs exhibit increased levels of pathology-associated gene expression and activation of signaling cascades involved in pathological remodeling compared to untreated controls or treated 2D iPSC-CMs. Thus, our method results in robust yet simpler, cheaper, and faster EHTs to study cardiac disease.
... Meanwhile, various types of EHTs have been developed, differing in their geometrical configurations. These include strip-shaped models [85][86][87][88][89], ring-shaped constructs [90,91], patches [92], cardiac biowires [93,94], spherical chambers [95], and tubular structures [96]. ...
Human induced pluripotent stem cell (hiPSC)-based disease modelling has significantly advanced the field of cardiogenetics, providing a precise, patient-specific platform for studying genetic causes of heart diseases. Coupled with genome editing technologies such as CRISPR/Cas, hiPSC-based models not only allow the creation of isogenic lines to study mutation-specific cardiac phenotypes, but also enable the targeted modulation of gene expression to explore the effects of genetic and epigenetic deficits at the cellular and molecular level.
hiPSC-based models of heart disease range from two-dimensional cultures of hiPSC-derived cardiovascular cell types, such as various cardiomyocyte subtypes, endothelial cells, pericytes, vascular smooth muscle cells, cardiac fibroblasts, immune cells, etc., to cardiac tissue cultures including organoids, microtissues, engineered heart tissues, and microphysiological systems. These models are further enhanced by multi-omics approaches, integrating genomic, transcriptomic, epigenomic, proteomic, and metabolomic data to provide a comprehensive view of disease mechanisms.
In particular, advances in cardiovascular tissue engineering enable the development of more physiologically relevant systems that recapitulate native heart architecture and function, allowing for more accurate modelling of cardiac disease, drug screening, and toxicity testing, with the overall goal of personalised medical approaches, where therapies can be tailored to individual genetic profiles.
Despite significant progress, challenges remain in the maturation of hiPSC-derived cardiomyocytes and the complexity of reproducing adult heart conditions. Here, we provide a concise update on the most advanced methods of hiPSC-based disease modelling in cardiogenetics, with a focus on genome editing and cardiac tissue engineering.
... However, it is worth noting that 3D-tissue generation is challenging and the experimental throughput is lower compared to 2D cultures 19 . Other approaches to enhance iPSC-CM maturation include supplementation of the culture medium with fatty acids (FA) 13,[20][21][22] , hormones or small molecules 10,23 , micro-or nanopatterning (NP) of culture surfaces 24,25 , and electrostimulation (ES) [26][27][28][29][30] . As these stimuli have been investigated independently, the most effective factor for enhancing iPSC-CM maturation remains unclear. ...
The immaturity of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is a major limitation for their use in drug screening to identify pro-arrhythmogenic or cardiotoxic molecules. Here, we demonstrate an approach that combines lipid-enriched maturation medium with a high concentration of calcium, nanopatterning of culture surfaces and electrostimulation to generate iPSC-CMs with advanced electrophysiological, structural and metabolic phenotypes. Systematic testing reveals that electrostimulation is the key driver of enhanced mitochondrial development and metabolic maturation and improved electrophysiological properties of iPSC-CMs. Increased calcium concentration strongly promotes electrophysiological maturation, while nanopatterning primarily facilitates sarcomere organisation with minor effect on electrophysiological properties. Transcriptome analysis reveals that activation of HMCES and TFAM targets contributes to mitochondrial development, whereas downregulation of MAPK/PI3K and SRF targets is associated with iPSC-CM polyploidy. These findings provide mechanistic insights into iPSC-CM maturation, paving the way for pharmacological responses that more closely resemble those of adult CMs.
... Therefore, electrical conditioning is widely used for in vitro modeling and induction of stress conditions. For example, Zhao et al. performed chronic electrical conditioning on hiPSC-CMs obtained from patients with left ventricle hypertrophy using an engineered biowire platform [75]. The stimulation frequency was gradually increased from 2 to 6 Hz to mimic chronic increased workloads arising from hypertension and then maintained at 3 Hz for up to 8 months. ...
Purpose of Review
This review aims to explore recent advancements in bioengineering approaches used in developing and testing in vitro cardiac disease models. It seeks to find out how these tools can address the limitations of traditional in vitro models and be applied to improve our understanding of cardiac disease mechanisms, facilitate preclinical drug screening, and equip the development of personalized therapeutics.
Recent Findings
Human induced pluripotent stem cells have enabled the generation of diverse cardiac cell types and patient-specific models. Techniques like 3D tissue engineering, heart-on-a-chip platforms, biomechanical conditioning, and CRISPR-based gene editing have enabled faithful recreation of complex cardiac microenvironments and disease conditions. These models have advanced the study of both genetic and acquired cardiac disorders.
Summary
Bioengineered in vitro models are transforming the basic science and clinical research in cardiovascular disease by improving the biomimicry and complexity of tissue analogues, increasing throughput and reproducibility of screening platforms, as well as offering patient and disease specificity. Despite challenges in scalability and functional maturity, integrating multiple bioengineering techniques with advanced analytical tools in in vitro modeling platforms holds promise for future precision and personalized medicine and therapeutic innovations.
... Natural biomaterials, such as ECM [28], fibrin [29], silk [30], collagen and gelatin, alginate, hyaluronic acid (HA), etc [31,32], have high biocompatibility and adhesion sites, but weak mechanical properties, leading to easy material denaturation [33]. With good biocompatibility and bioactivity, natural biomaterials play an important role in biology and SMTE, and are of great significance for the study of cell behavior, tissue repair and regenerative medicine [34]. ...
The growth and formation of tissues, such as skeletal muscle, involve a complex interplay of spatiotemporal events, including cell migration, orientation, proliferation, and differentiation. With the continuous advancement of in vitro construction techniques, many studies have contributed to skeletal muscle tissue engineering (STME). This review summarizes recent advances in the ordered construction of skeletal muscle tissues, and evaluates the impact of engineering strategies on cell behavior and maturation, including biomaterials, manufacturing methods and training means. Biomaterials are used as scaffolds to provide a good microenvironment for myoblasts, manufacturing methods to guide the alignment of myoblasts through construction techniques, and external stimulation to further promote the myoblast orientation and maturation after construction, resulting in oriented and functional skeletal muscle tissues. Subsequently, we critically examine recent advancements in engineered composite skeletal muscle constructs, with particular emphasis on essential functionalization strategies including skeletal muscle vascularization, innervation and others. Concurrently, we evaluate emerging applications of STME in diverse translational areas such as volumetric muscle loss treatment, muscle-related disease models, drug screening, biohybrid robots, and cultured meat. Finally, future perspectives are proposed to provide guidance for rational design based on engineering strategies in STME.
... The first landmark study in this field was the lung-on-a-chip, which successfully replicated an alveolar-capillary barrier that mimics breathing motion. [13,[44][45][46] To date, single-organ systems replicating the specific functions and structures of organs, such as the liver, [47] kidney, [48] gut, [49] heart, [50,51] bone, [52] and vasculature [53] have been successfully developed. In addition, multi-organ systems that integrate multiple tissues on a single chip facilitate the exchange of metabolites, secreted factors, extracellular vesicles, and circulating cells. ...
The high failure rates in clinical drug development based on animal models highlight the urgent need for more representative human models in biomedical research. In response to this demand, organoids and organ chips were integrated for greater physiological relevance and dynamic, controlled experimental conditions. This innovative platform—the organoids-on-a-chip technology—shows great promise in disease modeling, drug discovery, and personalized medicine, attracting interest from researchers, clinicians, regulatory authorities, and industry stakeholders. This review traces the evolution from organoids to organoids-on-a-chip, driven by the necessity for advanced biological models. We summarize the applications of organoids-on-a-chip in simulating physiological and pathological phenotypes and therapeutic evaluation of this technology. This section highlights how integrating technologies from organ chips, such as microfluidic systems, mechanical stimulation, and sensor integration, optimizes organoid cell types, spatial structure, and physiological functions, thereby expanding their biomedical applications. We conclude by addressing the current challenges in the development of organoids-on-a-chip and offering insights into the prospects. The advancement of organoids-on-a-chip is poised to enhance fidelity, standardization, and scalability. Furthermore, the integration of cutting-edge technologies and interdisciplinary collaborations will be crucial for the progression of organoids-on-a-chip technology.
... Extending culturing time from one to three weeks [42], or utilizing the fatty-acid-enriched media [43], can effectively boost CM connectivity and mitochondrial function, achieving peak contractility of 30 mN/mm 2 , which is comparable to the 25 -44 mN/mm 2 range observed in adult human myocardium [42]. Moreover, electrical stimulation [44,45] or mechanical stretching [46,47] can directly "train" the CM in hEHC to further mature. Noteworthy is the progressive increase of the electrical stimulation frequency from 2 Hz to 6 Hz, which enables CM to exhibit physiologic sarcomere length (2.2 μm) and large, well-organized T-tubules [44]. ...
Review Modeling Arrhythmia in a Dish: An Open View from Human-Engineered Heart Constructs Shiya Wang 1,†, Pengcheng Yang 1,2,†, Jonathan Nimal Selvaraj 1,* and Donghui Zhang 1,3,* 1 State Key Laboratory of Biocatalysts and Enzyme Engineering, Stem Cells and Tissue Engineering Manufacture Center, School of Life Sciences, Hubei University, Wuhan 430062, China 2 Biomanufacturing Center, Department of Mechanical Engineering, Tsinghua University, Beijing100084, China 3 Cardiovascular Research Institute, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China * Correspondence: corresponding author:jonathannimals@hubu.edu.cn (J.N.S.); dongh.zhang@hubu.edu.cn (D.Z.) † These authors contributed equally to this work. Received: 26 July 2024; Revised: 29 September 2024; Accepted: 30 September 2024; Published: 6 February 2025 Abstract: Human-engineered heart constructs (hEHC), comprising cardiac organoids and engineered heart tissues, have become essential for replicating pathological and physiological mechanisms associated with cardiac development and diseases. The ongoing advancements in fabrication and culture techniques for these constructs have rendered them increasingly vital for cardiotoxicity prediction and drug efficacy evaluations. There is an escalating demand for standardized methodologies encompassing uniform fabrication, accurate disease modeling, and multidimensional phenotype assessments to facilitate a comprehensive understanding of these constructs. This review systematically examines hEHC, highlighting recent advancements in their cellular composition and functional characteristics, while stressing the necessity for thorough evaluations of significant heart disease phenotype, particularly in arrhythmia. Here, we propose a novel modular classification of cardiac model development based on specific modeling parameters and categorize existing research on in vitro functional assessment into various quantitative metrics. This classification framework provides researchers with innovative insights and strategies for personalized model design and evaluation.
... The appropriate stem cell response to electrical stimulation, particularly in differentiating into cardiomyocytes, has been demonstrated [11]. Given the role of pulsatile signals in the development of the cardiac syncytium [12], it has been shown that applying pulsatile electrical signals in cardiac tissue engineering can enhance the functional coupling of cells and promote the formation of synchronously contractile tissue constructs, as reported by different research groups [12][13][14]. ...
To enhance the differentiation and maturation of cardiomyocytes derived from human induced pluripotent stem cells, we developed a bioreactor system that simultaneously imposes biophysical and biochemical stimuli on these committed cardiomyocytes. The cells were cultured within biohydrogels composed of the extracellular matrix extracted from goat ventricles and purchased rat-origin collagen, which were housed in the elastic PDMS culture chambers of the bioreactor. Elastic and flexible electrodes composed of PEDOT/PSS, latex, and graphene flakes were embedded in the hydrogels and chamber walls, allowing cyclic stretch and electrical pulses to be simultaneously and coordinately applied to the cultured cells. Furthermore, a dynamic analysis method employing the transverse forced oscillation theory of a cantilever was used to analyze and discriminate the subtype-specific beating behavior of the cardiomyocytes. It was found that myosin light chain 2v (MLC2v), a ventricular cell marker, was primarily upregulated in cells aggregated on the (+) electrode side, while cardiomyocytes with faint MLC2v but strong cardiac troponin T (cTNT) expression aggregated at the ground electrode (GND) side. mRNA analysis using rtPCR and the gel beating dynamics further suggested a subtype deviation on the different electrode sides. This study demonstrated the potential of our bioreactor system in enhancing cardiac differentiation and maturation, and it showed an intriguing phenomenon of cardiomyocyte subtype aggregation on different electrodes, which may be developed into a new method to enhance the maturation and separation of cardiomyocyte subtypes.
... An engineered platform for heteropolar heart organoids has also been developed to respond to chamber-targeting drugs. 65 Human stem cells have been shown to differentiate into norepinephrine-secreting sympathetic neurons when coculture with cardiomyocytes, regulating heart rate in ventricular cardiomyocytes. 66 Although cardiomyocytes assist in neuronal maturation, the impact of neurocardiac co-culture on cardiac organoids and sympathetic CVDs remains to be further explored. ...
Cardiovascular diseases cause significant morbidity and mortality worldwide. Engineered cardiac organoids are being developed and used to replicate cardiac tissues supporting cardiac morphogenesis and development. These organoids have applications in drug screening, cardiac disease models and regenerative medicine. Therefore, a thorough understanding of cardiac organoids and a comprehensive overview of their development are essential for cardiac tissue engineering. This review summarises different types of cardiac organoids used to explore cardiac function, including those based on co-culture, aggregation, scaffolds, and geometries. The self-assembly of monolayers, multilayers and aggravated cardiomyocytes forms biofunctional cell aggregates in cardiac organoids, elucidating the formation mechanism of scaffold-free cardiac organoids. In contrast, scaffolds such as decellularised extracellular matrices, three-dimensional hydrogels and bioprinting techniques provide a supportive framework for cardiac organoids, playing a crucial role in cardiac development. Different geometries are engineered to create cardiac organoids, facilitating the investigation of intrinsic communication between cardiac organoids and biomechanical pathways. Additionally, this review emphasises the relationship between cardiac organoids and the cardiac system, and evaluates their clinical applications. This review aims to provide valuable insights into the study of three-dimensional cardiac organoids and their clinical potential.
... Electrical-pacing-induced contraction training has been demonstrated to strengthen the electromechanical properties of hiPSC-CMs [113]. The development of the "Biowire" platform employs electrical stimulation to significantly improve the maturation of hiPSC-CMs [125,126]. Increasing stimulation frequency from 1 Hz to 6 Hz resulted in more refined sarcomere structures, mature electrophysiological properties, and enhanced Ca 2+ handling capabilities in regulating cardiac muscle contraction. Then, Radisic et al. fabricated a scalable "Biowire II" platform by an automated workflow [127]. ...
Cardiac organoids offer sophisticated 3D structures that emulate key aspects of human heart development and function. This review traces the evolution of cardiac organoid technology, from early stem cell differentiation protocols to advanced bioengineering approaches. We discuss the methodologies for creating cardiac organoids, including self-organization techniques, biomaterial-based scaffolds, 3D bioprinting, and organ-on-chip platforms, which have significantly enhanced the structural complexity and physiological relevance of in vitro cardiac models. We examine their applications in fundamental research and medical innovations, highlighting their potential to transform our understanding of cardiac biology and pathology. The integration of multiple cell types, vascularization strategies, and maturation protocols has led to more faithful representations of the adult human heart. However, challenges remain in achieving full functional maturity and scalability. We critically assess the current limitations and outline future directions for advancing cardiac organoid technology. By providing a comprehensive analysis of the field, this review aims to catalyze further innovation in cardiac tissue engineering and facilitate its translation to clinical applications.
... Having seen excellent interactions between iCMs and iMacs in a 2D setting, our next focus was to study the effect of iMacs addition on ECTs. Although majority of conventional ECT models only focused on cardiomyocytes, or a combination of cardiomyocytes and cardiac fibroblasts [58,87,88], newer studies underline the immense benefits of adding iMacs into ECTs in terms of improving tissue function [23] and vascularisation [89]. In line with these findings, addition of iMacs significantly reduced the variability in the beat rate of the tissues, improved cell alignment and iCM elongation, as well as remarkably increased tissue compaction. ...
Cardiovascular disease stands as the leading cause of death globally, claiming approximately 19 million lives in 2020. On the contrary, the development of cardiovascular drugs is experiencing a decline, largely due to the bottleneck in understanding the pathophysiology of various heart diseases and assessing the effects of drugs on healthy human hearts. The development of induced pluripotent stem cell (iPSC) technology and the availability of cardiac cell types in vitro, has resulted in a surge in efforts to fabricate human cardiac models for disease modelling and drug discovery applications. Although numerous attempts evidence successful fabrication of 3 dimensional (3D) engineered heart tissues, the innate immune cell population of the myocardium ;particularly cardiac macrophages, was until recently, overlooked. With increasing appreciation of the interactions between cardiomyocytes and macrophages in the myocardium, in this work, isogenic populations of cardiac resident-like macrophages and cardiomyocytes were generated using iPSCs, to understand the interactions between the two cell types in both 2D and 3D settings, and subjected to electric stimulation. After characterizing iPSC-derived macrophages (iMacs) and iPSC-derived cardiomyocytes (iCMs) in depth, the conditioning of iMacs to align to a cardiac resident macrophage-like phenotype in the presence of iCMs in 2D culture was explored. In coculture with iCMs, iMacs upregulated known genes expressed by cardiac resident macrophages. Additionally, in co-culture with iMacs, iCMs displayed an elongated morphology, improved calcium function and an increase in known maturation genes such as the ratio between MYH7 and MYH6 as well as SERCA2. In a 2D setting, iMacs showed the ability to electrically couple with iCMs and facilitate synchronous beating in iCM cultures. The 2D characterisation was translated into an engineered cardiac tissue model, wherein, improvement in tissue characteristics in the presence of iMacs was demonstrated in terms of increased cell alignment, enhanced cardiomyocyte elongation, physiologically relevant beat rates and improved tissue compaction. Taken together, these findings may open new avenues to use iMacs in engineered cardiac tissue models, not only as an innate immune cell source, but also as a support cell type to improve cardiomyocyte function and maturation.
... In this way, this group was able to model left ventricular hypertrophy with 8-month electrical conditioning. 22 Recently, there has been a lot of research on organoid modeling of heart disease. The generation of these models will give us information about the formation and progression of different diseases and even their treatments. ...
Three-dimensional (3D) myocardial tissues for studying human heart biology, physiology and pharmacology have recently received lots of attention. Organoids as 3D mini-organs are created from multiple cell types (i.e. induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs)) with other supporting co-cultured cells such as endothelial cells or fibroblasts. Cardiac organoid culture technologies are bringing about significant advances in organ research and allows for the establishment of tissue regeneration and disease modeling. The present review provides an overview of the recent advances in human cardiac organoid platforms in disease biology and for cardiovascular regenerative medicine.
... As a result, conduction velocity trended upward in recently published work (Fig. 5c). Multiple models achieved average conduction velocities greater than 30 cm s −1 , including models with dynamic mechanical culture 22 , small molecules targeting signaling pathways 31 , conductive biomaterials 75 , decellularized ECM 153,154 , human perinatal stem cell-derived ECM 40 , ventricular-specific tissues 155 and 3D-printed tricultures 156 . ...
Recent innovations in differentiating cardiomyocytes from human induced pluripotent stem cells (hiPSCs) have unlocked a viable path to creating in vitro cardiac models. Currently, hiPSC-derived cardiomyocytes (hiPSC-CMs) remain immature, leading many in the field to explore approaches to enhance cell and tissue maturation. Here, we systematically analyzed 300 studies using hiPSC-CM models to determine common fabrication, maturation and assessment techniques used to evaluate cardiomyocyte functionality and maturity and compiled the data into an open-access database. Based on this analysis, we present the diversity of, and current trends in, in vitro models and highlight the most common and promising practices for functional assessments. We further analyzed outputs spanning structural maturity, contractile function, electrophysiology and gene expression and note field-wide improvements over time. Finally, we discuss opportunities to collectively pursue the shared goal of hiPSC-CM model development, maturation and assessment that we believe are critical for engineering mature cardiac tissue.
... In recent years, to replicate the inflammatory microenvironment, heart-on-a-chip models have integrated iPSC-derived cardiomyocytes, 13,[57][58][59]84 cardiac fibroblasts, 39,57,58 macrophages, 83,85 peripheral blood mononuclear cells (PBMCs), 13,59 and monocytes. 86 A vascularized heart-on-a-chip system, termed Integrated Vasculature for Assessing Dynamic Events (InVADE), ...
Cardiovascular diseases are the leading cause of morbidity and mortality worldwide with numerous inflammatory cell etiologies associated with impaired cardiac function and heart failure. Inflammatory cardiomyopathy, also known as myocarditis, is an acquired cardiomyopathy characterized by inflammatory cell infiltration into the myocardium with a high risk of progression to deteriorated cardiac function. Recently, amidst the ongoing COVID-19 pandemic, the emergence of acute myocarditis as a complication of SARS-CoV-2 has garnered significant concern. Given its mechanisms remain elusive in conjunction with the recent withdrawal of previously FDA-approved antiviral therapeutics and prophylactics due to unexpected cardiotoxicity, there is a pressing need for human-mimetic platforms to investigate disease pathogenesis, model dysfunctional features, and support pre-clinical drug screening. Traditional in vitro models for studying cardiovascular diseases have inherent limitations in recapitulating the complexity of the in vivo microenvironment. Heart-on-a-chip technologies, combining microfabrication, microfluidics, and tissue engineering techniques, have emerged as a promising approach for modeling inflammatory cardiac diseases like myocarditis. This review outlines the established and emerging conditions of inflamed myocardium, identifying key features essential for recapitulating inflamed myocardial structure and functions in heart-on-a-chip models, highlighting recent advancements, including the integration of anisotropic contractile geometry, cardiomyocyte maturity, electromechanical functions, vascularization, circulating immunity, and patient/sex specificity. Finally, we discuss the limitations and future perspectives necessary for the clinical translation of these advanced technologies.
... Zhang et al proposed an anisotropic and conductive scaffold in combination with ES that mediated electrical signal transmittance and promoted myotube maturation [30]. Zhao et al built a scalable tissue-cultivation platform that enables the creation of electrophysiologically distinct atrial and ventricular tissues [31]. However, there are still few reports on developing a straightforward biofabrication method that incorporates multiscale anisotropic structural features to provide a biomimetic 3D mechanical environment. ...
Mimicking the multilayered, anisotropic, elastic structure of cardiac tissues for controlled guidiance of 3D cellular orientation is essential in designing bionic scaffolds for cardiac tissue biofabrication. Here, a hierarchically organized, anisotropic, wavy and conductive polycaprolactone/Au scaffold was created in a facile fashion based on mechanical memory during fabrication. The bionic 3D scaffold shows good biocompatibility, excellent biomimetic mechanical properties that guide myoblast alignment, support the hyperelastic behavior observed in native cardiac muscle tissue, and promote myotube maturation, which holds potential for cardiac muscle engineering and the establishment of an in vitro culture platform for drug screening.
... We recently reported the use of the Biowire II platform for generating functional 3D engineered cardiac tissues that display several hallmarks of the adult human myocardium, as well as expected responses to known compounds Zhao et al., 2019). Both cardiac and skeletal muscle have been demonstrated to require electromechanical stimulations to mature. ...
Therapeutic development for skeletal muscle diseases is challenged by a lack of ex vivo models that recapitulate human muscle physiology. Here, we engineered 3D human skeletal muscle tissue in the Biowire II platform that could be maintained and electrically stimulated long‐term. Increasing differentiation time enhanced myotube formation, modulated myogenic gene expression, and increased twitch and tetanic forces. When we mimicked exercise training by applying chronic electrical stimulation, the “exercised” skeletal muscle tissues showed increased myotube size and a contractility profile, fatigue resistance, and gene expression changes comparable to in vivo models of exercise training. Additionally, tissues also responded with expected physiological changes to known pharmacological treatment. To our knowledge, this is the first evidence of a human engineered 3D skeletal muscle tissue that recapitulates in vivo models of exercise. By recapitulating key features of human skeletal muscle, we demonstrated that the Biowire II platform may be used by the pharmaceutical industry as a model for identifying and optimizing therapeutic drug candidates that modulate skeletal muscle function.
... Metabolic strategies and the impact on the maturation of hiPSC-cardiomyocytes Multiple interventions have been explored to promote the maturation of hiPSCcardiomyocytes, such as prolonging the duration of culture [34,35], culturing the cells in 2D [36] or 3D platforms [15,37], exposing immature cardiomyocytes to physical [38], electrical [39], and mechanical stimuli [40][41][42], and including neurohormonal factors [43] and micro-RNAs (miRNAs) [44] in the culture media. The heart is an electrically and mechanically specialized organ. ...
Acute coronary syndromes, such as myocardial infarction (MI), lack effective therapies beyond heart transplantation, which is often hindered by donor scarcity and postoperative complications. Human induced pluripotent stem cells (hiPSCs) offer the possibility of myocardial regeneration by differentiating into cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-cardiomyocytes) exhibit fetal-like calcium flux and energy metabolism, which inhibits their engraftment. Several strategies have been explored to improve the therapeutic efficacy of hiPSC-cardiomyocytes, such as selectively enhancing energy substrate utilization and improving the transplantation environment. In this review, we have discussed the impact of altered mitochondrial biogenesis and metabolic switching on the maturation of hiPSC-cardiomyocytes. Additionally, we have discussed the limitations inherent in current methodologies for assessing metabolism in hiPSC-cardiomyocytes, and the challenges in achieving sufficient metabolic flexibility akin to that in the healthy adult heart.
... In addition, all phenotypes evaluated in this study were weighted equally to maintain an unbiased approach; however, it should be noted that the phenotypes evaluated herein may be of different clinical importance and that alternative analyses with increased emphasis on certain endpoints may be needed. Studies in other in vitro models that probe effects on cardiomyocyte contractility [91,92] or (micro)vasculature [93,94] are needed but may require triaging of some PFAS for such testing because of the low-throughput. Second, the library of PFAS tested and the number of iPSC-derived cardiomyocyte donors available were both limited. ...
Per- and poly-fluoroalkyl substances (PFAS) are emerging contaminants of concern because of their wide use, persistence, and potential to be hazardous to both humans and the environment. Several PFAS have been designated as substances of concern; however, most PFAS in commerce lack toxicology and exposure data to evaluate their potential hazards and risks. Cardiotoxicity has been identified as a likely human health concern, and cell-based assays are the most sensible approach for screening and prioritization of PFAS. Human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a widely used method to test for cardiotoxicity, and recent studies showed that many PFAS affect these cells. Because iPSC-derived cardiomyocytes are available from different donors, they also can be used to quantify human variability in responses to PFAS. The primary objective of this study was to characterize potential human cardiotoxic hazard, risk, and inter-individual variability in responses to PFAS. A total of 56 PFAS from different subclasses were tested in concentration-response using human iPSC-derived cardiomyocytes from 16 donors without known heart disease. Kinetic calcium flux and high-content imaging were used to evaluate biologically-relevant phenotypes such as beat frequency, repolarization, and cytotoxicity. Of the tested PFAS, 46 showed concentration-response effects in at least one phenotype and donor; however, a wide range of sensitivities were observed across donors. Inter-individual variability in the effects could be quantified for 19 PFAS, and risk characterization could be performed for 20 PFAS based on available exposure information. For most tested PFAS, toxicodynamic variability was within a factor of 10 and the margins of exposure were above 100. This study identified PFAS that may pose cardiotoxicity risk and have high inter-individual variability. It also demonstrated the feasibility of using a population-based human in vitro method to quantify population variability and identify cardiotoxicity risks of emerging contaminants.
Advances in human induced pluripotent stem cell (iPSC) technology have enabled the generation of robust, cardiac-specific, in vitro systems for cardiac disease modeling, drug screening, and cardiotoxicity studies. Human iPSCs can be differentiated into iPSC-derived cardiac
myocytes (iPSC-CMs), a model cell population able to recapitulate the phenotypes of complex cardiac diseases—mirroring both the observations made with in vivo animal models and
the clinical manifestations seen in human patients. To improve the physiological relevance of
iPSC-CM model systems, researchers are shifting from 2D to 3D models and incorporating
a variety of cardiac specific cell types including cardiac fibroblasts, and vascular endothelial
cells. In this paper, we will review the development of 2D and 3D iPSC-CM models, survey
the various disease contexts in which these models have been utilized, and discuss the benefits and limitations of each strategy.
Safety attrition due to drug-induced inotropic changes remains a significant risk factor for drug development. Mitigating these events during early screening remains challenging. Several in vitro predictive models have been developed to address these issues, with varying success in detecting drug-induced inotropic changes. In this study, we compared traditional two-dimensional human-induced pluripotent stem cell-derived cardiomyocytes (2D hiPSC-CMs) with three-dimensional engineered cardiac tissues (3D ECTs) to assess their ability to detect drug-induced inotropic changes in 17 drugs with known mechanisms of action. The models were exposed to various test compounds, and their responses were evaluated by measuring either the active force or maximum contraction speed. The 3D ECTs successfully detected all the tested positive inotropes, whereas the 2D hiPSC-CMs failed to detect the two compounds. Both models demonstrated high predictability for negative inotropy and showed similar results for detecting non-active compounds, except for higher concentrations of phentolamine, zimelidine, and tamsulosin. Irregular beating was less likely to occur in the 3D ECTs, suggesting that 3D ECTs provided superior detection of contractility compared to 2D hiPSC-CMs. Genetic analysis revealed a more mature phenotype for the 3D ECTs compared to the 2D hiPSC-CMs, and the compound-related target expression was comparable to that in the adult human heart tissues. The 3D ECTs captured inotropic changes more accurately and thus represented a more translatable model than the 2D hiPSC-CMs. Overall, contractility assessment using the 3D ECTs could be advantageous for profiling candidate compounds and mechanistic investigations of hemodynamic changes during in vivo or clinical studies.
Cardiovascular diseases (CVD), the leading cause of death worldwide, and their strong association with fibrosis highlight the pressing need for innovative antifibrotic therapies. In vitro models have emerged as valuable tools for replicating cardiac fibrosis ‘in a dish’, facilitating the study of disease mechanisms and serving as platforms for drug testing and development. These in vitro systems encompass 2D and 3D models, each with its own limitations and advantages. 2D models offer high reproducibility, cost-effectiveness, and high-throughput capabilities, but they oversimplify the complex fibrotic environment. On the other hand, 3D models provide greater biological relevance but are more complex, harder to reproduce, and less suited for high-throughput screening. The choice of model depends on the specific research question and the stage of drug development. Despite significant progress, challenges remain, including the integration of immune cells in cardiac fibrosis and optimizing the scalability and throughput of highly biomimetic systems. Herein, we review recent in vitro cardiac fibrosis models, with a focus on their shared characteristics and remaining challenges, and explore how in vitro fibrosis models of other organs could inspire novel approaches in cardiac research, showcasing potential strategies that could be adapted to refine myocardial fibrosis models.
Organs‐on‐a‐chip (OOC) are an emergent technology that bridge the gap between current in vitro and in vivo models used to inform drug discovery and investigate disease pathophysiology. These systems offer improved bio‐relevance and controlled complexity through the integration of physical and/or chemical stimuli matched to physiologically relevant conditions. Although significant advancements have been made toward recreating organ‐specific physiology on chip, the methods available to study structure and function of the cell microenvironment are still limited. Established analysis approaches, including fluorescence microscopy, rely on laborious offline workflows that yield limited time‐point data. As the OOC field continues to evolve, there is a unique opportunity to engineer improved characterization methods into organ‐chip devices. This review provides an overview of current integrated sensing approaches that address current limitations and enable real‐time readout of relevant physiological parameters in OOC.
Cardiac tissue lacks regenerative capacity, making heart transplantation the primary treatment for end-stage heart failure. Engineered cardiac tissues developed through three-dimensional bioprinting (3DBP) offer a promising alternative. However, reproducing the native structure, cellular diversity, and functionality of cardiac tissue requires advanced cardiac bioinks. Major obstacles in CTE (cardiac tissue engineering) include accurately characterizing bioink properties, replicating the cardiac microenvironment, and achieving precise spatial organization. Optimizing bioink properties to closely mimic the extracellular matrix (ECM) is essential, as deviations may result in pathological effects. This review encompasses the rheological and electromechanical properties of bioinks and the function of the cardiac microenvironment in the design of functional cardiac constructs. Furthermore, it focuses on improving the rheological characteristics, printability, and functionality of bioinks, offering valuable perspectives for developing new bioinks especially designed for CTE.
Despite advancements in engineered heart tissue (EHT), challenges persist in achieving accurate dimensional accuracy of scaffolds and maturing human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs), a primary source of functional cardiac cells. Drawing inspiration from cardiac muscle fiber arrangement, a three‐dimensional (3D)‐printed multi‐layered microporous polycaprolactone (PCL) scaffold is created with interlayer angles set at 45° to replicate the precise structure of native cardiac tissue. Compared with the control group and 90° PCL scaffolds, the 45° PCL scaffolds exhibited superior biocompatibility for cell culture and improved hiPSC‐CM maturation in calcium handling. RNA sequencing demonstrated that the 45° PCL scaffold promotes the mature phenotype in hiPSC‐CMs by upregulating ion channel genes. Using the 45° PCL scaffold, a multi‐cellular EHT is successfully constructed, incorporating human cardiomyocytes, endothelial cells, and mesenchymal stem cells. These complex EHTs significantly enhanced hiPSC‐CM engraftment in vivo, attenuated ventricular remodeling, and improved cardiac function in mouse myocardial infarction. In summary, the myocardium‐specific structured EHT developed in this study represents a promising advancement in cardiovascular regenerative medicine.
Organs-on-chips (OoCs) have significantly advanced biomedical research by precisely reconstructing human microphysiological systems with biomimetic functions. However, achieving greater structural complexity of cell cultures on-chip for enhanced biological mimicry remains a challenge. To overcome these challenges, 3D bioprinting techniques can be used in directly building complex 3D cultures on chips, facilitating the in vitro engineering of organ-level models. Herein, we review the distinctive features of OoCs, along with the technical and biological challenges associated with replicating complex organ structures. We discuss recent bioprinting innovations that simplify the fabrication of OoCs while increasing their architectural complexity, leading to breakthroughs in the field and enabling the investigation of previously inaccessible biological problems. We highlight the challenges for the development of 3D bioprinted OoCs, concluding with a perspective on future directions aimed at facilitating their clinical translation.
Cardiovascular toxicity remains a primary concern in drug development, accounting for a significant portion of post‐market drug withdrawals due to adverse reactions such as arrhythmias. Traditional preclinical models, predominantly based on animal cells, often fail to replicate human cardiac physiology accurately, complicating the prediction of drug‐induced effects. Although human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) provide a more genetically relevant system, their use in 2D, static cultures does not sufficiently mimic the dynamic, 3D environment of the human heart. 3D cardiac organoids made from human iPSC‐CMs can potentially bridge this gap. However, most traditional electrophysiology assays, developed for single cells or 2D monolayers, are not readily adaptable to 3D organoids. This study uses optical calcium analysis of human organoids combined with miniaturized fluorescence microscopy (miniscope) and heart‐on‐a‐chip technology. This simple, inexpensive, and efficient platform provides robust on‐chip calcium imaging of human cardiac organoids. The versatility of the system is demonstrated through cardiotoxicity assay of drugs known to impact cardiac electrophysiology, including dofetilide, quinidine, and thapsigargin. The platform promises to advance drug testing by providing a more reliable and physiologically relevant assessment of cardiovascular toxicity, potentially reducing drug‐related adverse effects in clinical settings.
The heart, with its complex structural and functional characteristics, plays a critical role in sustaining life by pumping blood throughout the entire body to supply nutrients and oxygen. Engineered heart tissues have been introduced to reproduce heart functions to understand the pathophysiological properties of the heart and to test and develop potential therapeutics. Although numerous studies have been conducted in various fields to increase the functionality of heart tissue to be similar to reality, there are still many difficulties in reproducing the blood-pumping function of the heart. In this review, we discuss advancements in cells, biomaterials, and biofabrication in cardiac tissue engineering to achieve cardiac models that closely mimic the pumping function. Moreover, we provide insight into future directions by proposing future perspectives to overcome remaining challenges, such as scaling up and biomimetic patterning of blood vessels and nerves through bioprinting.
Current cardiac cell models for drug screening often face a trade-off between cellular maturity and achieving high throughput. While three-dimensional human induced pluripotent stem cell-based heart models typically exhibit more adult-like features, their application is hindered by the need for large cell numbers or complex equipment. Here, we developed cost-effective methods to scale up production of three-dimensional cardiac microtissues (cMTs) containing three cardiac cell types, and assess calcium transients and action potential metrics for high-throughput screening (HTS). Automating the procedure revealed reproducible drug responsiveness and predictive accuracy in a reference compound screen. Furthermore, an arrhythmic phenotype was reliably triggered in cMTs containing cardiomyocytes with a RYR2 mutation. A screen of FDA-approved drugs identified 17 drugs that rescued the arrhythmic phenotype. Our findings underscore the scalability of cMTs and their utility in disease modelling and HTS. The advanced "technology-readiness-level" of cMTs supports their regulatory uptake and acceptance within the pharmaceutical industry.
Replicating the mechanical tension of natural tissues is essential for maintaining organ function and stability, posing a central challenge in tissue engineering and regenerative medicine. Existing methods for constructing tension...
Hands-on training in tissue engineering is often associated with specialized labs and expensive equipment, such as CO2 incubators. To minimize the use of costly commercial incubators and provide a more vivid engineering experience in a biology lab, we present a hands-on project that introduces medium to large groups of undergraduate or graduate students to tissue engineering. This project involves the culture of breast cancer spheroids in a do-it-yourself (DiY) incubator.
Students were divided into teams (three to four members) and provided with a kit to assemble a DiY incubator for Eppendorf tubes. The students also received Eppendorf tubes containing spheroids derived from MCF7 breast cancer cells, fresh culture medium, and syringes. A perception survey was applied to the students before and after the activity. The students were able to incubate their spheroids at a constant temperature of 37 °C in their DiY incubators and keep them alive and metabolically active for several days, by changing the medium using the components of the kit (syringes and fresh culture medium) in a laminar hood.
The students successfully documented the progression of the culture by following glucose consumption with a portable commercial glucometer, the changes in size of the spheroids, or the changes in color of the culture medium (from pink to yellow) using a smartphone and image analysis applications. Students learned basic techniques associated with cell culture and tissue incubation. After participating in the cell culture experiment, students perceived tissue engineering as more appealing, simpler (easier or clearer), and better structured than before the hands-on activity.
This simple research-based project illustrates key aspects of tissue engineering (i.e., the need to control temperature, maintain sterility, and renew nutrients to culture live tissues), while exposing students to a real hands-on project that they can do in a typical biological laboratory by assembling and using DiY incubators instead of depending on multiple and costly commercial CO2 incubators.
Highlights
•hiPSC-CM offer an alternative to in vivo models for predicting cardiotoxicity.
•hiPSC-CM monolayers detect pro-arrhythmic effects; inotropic detection is less established.
•Cardiac spheroids and engineered tissue may suit chronic cardiotoxicity studies (>2 weeks).
•Cardiac assays with non-myocyte cells may be key to identifying some cardiotoxicity forms.
•hiPSC-CM technologies are well placed to develop patient-specific assays in the future.
Recent advancements in flexible electronics have highlighted their potential in biomedical applications, primarily due to their human-friendly nature. This study introduces a new flexible electronic system designed for motion sensing in a biomimetic three-dimensional (3D) environment. The system features a self-healing gel matrix (chitosan-based hydrogel) that effectively mimics the dynamics of the extracellular matrix (ECM), and is integrated with a highly sensitive thin-film resistive strain sensor, which is fabricated by incorporating a cross-linked gold nanoparticle (GNP) thin film as the active conductive layer onto a biocompatible microphase-separated polyurethane (PU) substrate through a clean, rapid, and high-precision contact printing method. The GNP-PU strain sensor demonstrates high sensitivity (a gauge factor of ∼50), good stability, and waterproofing properties. The feasibility of detecting small motion was evaluated by sensing the beating of human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte spheroids embedded in the gel matrix. The integration of these components exemplifies a proof-of-concept for using flexible electronics comprising self-healing hydrogel and thin-film nanogold in cardiac sensing and offers promising insights into the development of next-generation biomimetic flexible electronic devices.
Recent years have seen the creation and popularization of various complex in vitro models (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.
Microphysiological systems (MPSs) are cellular models that replicate aspects of organ and tissue functions in vitro. In contrast with conventional cell cultures, MPSs often provide physiological mechanical cues to cells, include fluid flow and can be interlinked (hence, they are often referred to as microfluidic tissue chips or organs-on-chips). Here, by means of examples of MPSs of the vascular system, intestine, brain and heart, we advocate for the development of standards that allow for comparisons of quantitative physiological features in MPSs and humans. Such standards should ensure that the in vivo relevance and predictive value of MPSs can be properly assessed as fit-for-purpose in specific applications, such as the assessment of drug toxicity, the identification of therapeutics or the understanding of human physiology or disease. Specifically, we distinguish designed features, which can be controlled via the design of the MPS, from emergent features, which describe cellular function, and propose methods for improving MPSs with readouts and sensors for the quantitative monitoring of complex physiology towards enabling wider end-user adoption and regulatory acceptance.
Laboratory studies of the heart use cell and tissue cultures to dissect heart function yet rely on animal models to measure pressure and volume dynamics. Here, we report tissue-engineered scale models of the human left ventricle, made of nanofibrous scaffolds that promote native-like anisotropic myocardial tissue genesis and chamber-level contractile function. Incorporating neonatal rat ventricular myocytes or cardiomyocytes derived from human induced pluripotent stem cells, the tissue-engineered ventricles have a diastolic chamber volume of ~500 µl (comparable to that of the native rat ventricle and approximately 1/250 the size of the human ventricle), and ejection fractions and contractile work 50–250 times smaller and 10⁴–10⁸ times smaller than the corresponding values for rodent and human ventricles, respectively. We also measured tissue coverage and alignment, calcium-transient propagation and pressure–volume loops in the presence or absence of test compounds. Moreover, we describe an instrumented bioreactor with ventricular-assist capabilities, and provide a proof-of-concept disease model of structural arrhythmia. The model ventricles can be evaluated with the same assays used in animal models and in clinical settings.
Generation of homogeneous populations of subtype-specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) and their comprehensive phenotyping is crucial for a better understanding of the subtype-related disease mechanisms and as tools for the development of chamber-specific drugs. The goals of this study were to apply a simple and efficient method for differentiation of iPSCs into defined functional CM subtypes in feeder-free conditions and to obtain a comprehensive understanding of the molecular, cell biological, and functional properties of atrial and ventricular iPSC-CMs on both the single-cell and engineered heart muscle (EHM) level. By a stage-specific activation of retinoic acid signaling in monolayer-based and well-defined culture, we showed that cardiac progenitors can be directed towards a highly homogeneous population of atrial CMs. By combining the transcriptome and proteome profiling of the iPSC-CM subtypes with functional characterizations via optical action potential and calcium imaging, and with contractile analyses in EHM, we demonstrated that atrial and ventricular iPSC-CMs and -EHM highly correspond to the atrial and ventricular heart muscle, respectively. This study provides a comprehensive understanding of the molecular and functional identities characteristic of atrial and ventricular iPSC-CMs and -EHM and supports their suitability in disease modeling and chamber-specific drug screening.
Due to the unique physicochemical properties exhibited by materials with nanoscale dimensions, there is currently a continuous increase in the number of engineered nanomaterials (ENMs) used in consumer goods. However, several reports associate ENM exposure to negative health outcomes such as cardiovascular diseases. Therefore, understanding the pathological consequences of ENM exposure represents an important challenge, requiring model systems that can provide mechanistic insights across different levels of ENM-based toxicity. To achieve this, we developed a mussel-inspired 3D microphysiological system (MPS) to measure cardiac contractility in the presence of ENMs. While multiple cardiac MPS have been reported as alternatives to in vivo testing, most systems only partially recapitulate the native extracellular matrix (ECM) structure. Here, we show how adhesive and aligned polydopamine (PDA)/polycaprolactone (PCL) nanofiber can be used to emulate the 3D native ECM environment of the myocardium. Such nanofiber scaffolds can support the formation of anisotropic and contractile muscular tissues. By integrating these fibers in a cardiac MPS, we assessed the effects of TiO2 and Ag nanoparticles on the contractile function of cardiac tissues. We found that these ENMs decrease the contractile function of cardiac tissues through structural damage to tissue architecture. Furthermore, the MPS with embedded sensors herein presents a way to non-invasively monitor the effects of ENM on cardiac tissue contractility at different time points. These results demonstrate the utility of our MPS as an analytical platform for understanding the functional impacts of ENMs while providing a biomimetic microenvironment to in vitro cardiac tissue samples.
Cardiac tissues generated from human induced pluripotent stem cells (iPSCs) can serve as platforms for patient-specific studies of physiology and disease1-6. However, the predictive power of these models is presently limited by the immature state of the cells1, 2, 5, 6. Here we show that this fundamental limitation can be overcome if cardiac tissues are formed from early-stage iPSC-derived cardiomyocytes soon after the initiation of spontaneous contractions and are subjected to physical conditioning with increasing intensity over time. After only four weeks of culture, for all iPSC lines studied, such tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiological sarcomere length (2.2 µm) and density of mitochondria (30%), the presence of transverse tubules, oxidative metabolism, a positive force-frequency relationship and functional calcium handling. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiological responses to isoproterenol and recapitulating pathological hypertrophy, supporting the utility of this tissue model for studies of cardiac development and disease.
Microphysiological systems and organs-on-chips promise to accelerate biomedical and pharmaceutical research by providing accurate in vitro replicas of human tissue. Aside from addressing the physiological accuracy of the model tissues, there is a pressing need for improving the throughput of these platforms. To do so, scalable data acquisition strategies must be introduced. To this end, we here present an instrumented 24-well plate platform for higher-throughput studies of engineered human stem cell-derived cardiac muscle tissues that recapitulate the laminar structure of the native ventricle. In each well of the platform, an embedded flexible strain gauge provides continuous and non-invasive readout of the contractile stress and beat rate of an engineered cardiac tissue. The sensors are based on micro-cracked titanium-gold thin films, which ensure that the sensors are highly compliant and robust. We demonstrate the value of the platform for toxicology and drug-testing purposes by performing 12 complete dose-response studies of cardiac and cardiotoxic drugs. Additionally, we showcase the ability to couple the cardiac tissues with endothelial barriers. In these studies, which mimic the passage of drugs through the blood vessels to the musculature of the heart, we regulate the temporal onset of cardiac drug responses by modulating endothelial barrier permeability in vitro.
Blatter discusses the initiation and spread of Ca release, Ca store depletion, and release termination in atrial myocytes.
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for drug testing and modelling genetic disorders. Abnormally low upstroke velocity is a current limitation. Here we investigated the use of 3D engineered heart tissue (EHT) as a culture method with greater resemblance to human heart tissue in comparison to standard technique of 2D monolayer (ML) format. INa was measured in ML or EHT using the standard patch-clamp technique. INa density was ~1.8 fold larger in EHT (−18.5 ± 1.9 pA/pF; n = 17) than in ML (−10.3 ± 1.2 pA/pF; n = 23; p < 0.001), approaching densities reported for human CM. Inactivation kinetics, voltage dependency of steady-state inactivation and activation of INa did not differ between EHT and ML and were similar to previously reported values for human CM. Action potential recordings with sharp microelectrodes showed similar upstroke velocities in EHT (219 ± 15 V/s, n = 13) and human left ventricle tissue (LV, 253 ± 7 V/s, n = 25). EHT showed a greater resemblance to LV in CM morphology and subcellular NaV1.5 distribution. INa in hiPSC-CM showed similar biophysical properties as in human CM. The EHT format promotes INa density and action potential upstroke velocity of hiPSC-CM towards adult values, indicating its usefulness as a model for excitability of human cardiac tissue.
Background:
-Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modelling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) towards an adult phenotype under defined conditions.
Methods:
-We systematically investigated cell composition, matrix and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We employed morphological, functional, and transcriptome analyses to benchmark maturation of EHM.
Results:
-EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M-bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency-response; (4) inotropic responses to β-adrenergic stimulation mediated via canonical ⓵- and ⓶-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and NT-proBNP release; all are classical hallmarks of heart failure. Additionally, we demonstrate scalability of EHM according to anticipated clinical demands for cardiac repair.
Conclusions:
-We provide proof-of-concept for a universally applicable technology for the engineering of macro-scale human myocardium for disease modelling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.
Biomedical research has relied on animal studies and conventional cell cultures for decades. Recently, microphysiological systems (MPS), also known as organs-on-chips, that recapitulate the structure and function of native tissues in vitro, have emerged as a promising alternative. However, current MPS typically lack integrated sensors and their fabrication requires multi-step lithographic processes. Here, we introduce a facile route for fabricating a new class of instrumented cardiac microphysiological devices via multimaterial three-dimensional (3D) printing. Specifically, we designed six functional inks, based on piezo-resistive, high-conductance, and biocompatible soft materials that enable integration of soft strain gauge sensors within micro-architectures that guide the self-assembly of physio-mimetic laminar cardiac tissues. We validated that these embedded sensors provide non-invasive, electronic readouts of tissue contractile stresses inside cell incubator environments. We further applied these devices to study drug responses, as well as the contractile development of human stem cell-derived laminar cardiac tissues over four weeks.
Background
Recent findings suggest that atrial fibrillation is associated with sudden cardiac death (SCD). We examined the incidence, characteristics, and factors associated with SCD in patients with atrial fibrillation.
Methods and Results
SCD was defined as witnessed death ≤60 minutes from the onset of new symptoms or unwitnessed death 1 to 24 hours after being observed alive, without another known cause of death. Predictors of SCD were examined using multivariate competing risks models. Over 2.8 years (median), 2349 patients died (40.5 per 1000 patient‐years), of which 1668 (71%) were cardiovascular deaths. SCD was the most common cause of cardiovascular death (n=749; median age 73 years; 70.6% male). Most SCD events occurred out of hospital (92.8%) and without prior symptoms (66.0%). Predictors of SCD included low ejection fraction, heart failure, and prior myocardial infarction (P<0.001 for each). Additional significant baseline predictors of SCD, but not of other causes of death, included male sex, electrocardiographic left ventricular hypertrophy, higher heart rate, nonuse of beta blockers, and use of digitalis. The latter was associated with SCD in patients with or without heart failure (adjusted hazard ratio 1.55 [95% CI 1.29–1.86] and 1.56 [95% CI 1.14–2.11], respectively; P interaction=0.73). The rate of SCD was numerically but not statistically lower with edoxaban (1.20% per year with lower dose edoxaban; 1.28% per year with higher dose edoxaban) compared with warfarin (1.40% per year).
Conclusion
SCD is the most common cause of cardiovascular death in patients with atrial fibrillation and has several distinct predictors, some of which are modifiable. These findings may be considered in planning research and treatment strategies for patients with atrial fibrillation.
Clinical Trial Registration
URL: https://www.clinicaltrials.gov. Unique identifier: NCT00781391.
Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane- and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling.
Tissue engineering approaches have the potential to increase the physiologic relevance of human iPSderived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires <1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (μ HM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.
We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimetre-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted with direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion.
Background: Congenital long QT syndrome type 2 (abnormal hERG potassium channel) patients can develop flat, asymmetric, and notched T waves. Similar observations have been made with a limited number of hERG-blocking drugs. However, it is not known how additional calcium or late sodium block, that can decrease torsade risk, affects T wave morphology.
Methods and Results: Twenty-two healthy subjects received a single dose of a pure hERG blocker (dofetilide) and 3 drugs that also block calcium or sodium (quinidine, ranolazine, and verapamil) as part of a 5-period, placebo-controlled cross-over trial. At pre-dose and 15 time-points postdose, ECGs and plasma drug concentration were assessed. Patch clamp experiments were performed to assess block of hERG, calcium (L-type) and late sodium currents for each drug. Pure hERG block (dofetilide) and strong hERG block with lesser calcium and late sodium block (quinidine) caused substantial T wave morphology changes (P<0.001). Strong late sodium current and hERG block (ranolazine) still caused T wave morphology changes (P<0.01). Strong calcium and hERG block (verapamil) did not cause T wave morphology changes. At equivalent QTc prolongation, multichannel blockers (quinidine and ranolazine) caused equal or greater T wave morphology changes compared with pure hERG block (dofetilide).
Conclusions: T wave morphology changes are directly related to amount of hERG block; however, with quinidine and ranolazine, multichannel block did not prevent T wave morphology changes. A combined approach of assessing multiple ion channels, along with ECG intervals and T wave morphology may provide the greatest insight into drug-ion channel interactions and torsade de pointes risk.
Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity. Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC50/EC50) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.
Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in
vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in
vitro data with human myocardial biopsies detected overlapping expression changes between the in
vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.
Study of monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combined patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene encoding tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS 'heart-on-chip' tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole-cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies and advances iPSC-based in vitro modeling of cardiomyopathy.
Study of monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combined patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene encoding tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS 'heart-on-chip' tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole-cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies and advances iPSC-based in vitro modeling of cardiomyopathy.
Polydimethylsiloxane (PDMS) has numerous desirable properties for fabricating microfluidic devices, including optical transparency, flexibility, biocompatibility, and fabrication by casting; however, partitioning of small hydrophobic molecules into the bulk of PDMS hinders industrial acceptance of PDMS microfluidic devices for chemical processing and drug development applications. Here we describe an attractive alternative material that is similar to PDMS in terms of optical transparency, flexibility and castability, but that is also resistant to absorption of small hydrophobic molecules.
Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca(2+) handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.
It is unknown whether atrial fibrillation (AF) is associated with an increased risk of sudden cardiac death (SCD) in the general population. This association was examined in 2 population-based cohorts.
In the Atherosclerosis Risk in Communities (ARIC) Study, we analyzed data from 15 439 participants (baseline age, 45-64 years; 55.2% women; and 26.6% black) from baseline (1987-1989) through December 31, 2001. In the Cardiovascular Health Study (CHS), we analyzed data from 5479 participants (baseline age, ≥65 years; 58.2% women; and 15.4% black) from baseline (first cohort, 1989-1990; second cohort, 1992-1993) through December 31, 2006. The main outcome was physician-adjudicated SCD, defined as death from a sudden, pulseless condition presumed to be due to a ventricular tachyarrhythmia. The secondary outcome was non-SCD (NSCD), defined as coronary heart disease death not meeting SCD criteria. We used Cox proportional hazards models to assess the association between AF and SCD/NSCD, adjusting for baseline demographic and cardiovascular risk factors.
In the ARIC Study, 894 AF, 269 SCD, and 233 NSCD events occurred during follow-up (median, 13.1 years). The crude incidence rates of SCD were 2.89 per 1000 person-years (with AF) and 1.30 per 1000 person-years (without AF). The multivariable hazard ratios (HRs) (95% CIs) of AF for SCD and NSCD were 3.26 (2.17-4.91) and 2.43 (1.60-3.71), respectively. In the CHS, 1458 AF, 292 SCD, and 581 NSCD events occurred during follow-up (median, 13.1 years). The crude incidence rates of SCD were 12.00 per 1000 person-years (with AF) and 3.82 per 1000 person-years (without AF). The multivariable HRs (95% CIs) of AF for SCD and NSCD were 2.14 (1.60-2.87) and 3.10 (2.58-3.72), respectively. The meta-analyzed HRs (95% CIs) of AF for SCD and NSCD were 2.47 (1.95-3.13) and 2.98 (2.52-3.53), respectively.
Incident AF is associated with an increased risk of SCD and NSCD in the general population. Additional research to identify predictors of SCD in patients with AF is warranted.
Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.
Cardiac tissue engineering aims at repairing the diseased heart and developing cardiac tissues for basic research and predictive toxicology applications. Since the first description of engineered heart tissue 15 years ago, major development steps were directed toward these three goals. Technical innovations led to improved three-dimensional cardiac tissue structure and near physiological contractile force development. Automation and standardization allow medium throughput screening. Larger constructs composed of many small engineered heart tissues or stacked cell sheet tissues were tested for cardiac repair and were associated with functional improvements in rats. Whether these approaches can be simply transferred to larger animals or the human patients remains to be tested. The availability of an unrestricted human cardiac myocyte cell source from human embryonic stem cells or human-induced pluripotent stem cells is a major breakthrough. This review summarizes current tissue engineering techniques with their strengths and limitations and possible future applications.
G. Y. Oudit, Z. Kassiri, R. Sah, R. J. Ramirez, C. Zobel and P. H. Backx. The Molecular Physiology of the Cardiac Transient Outward Potassium Current (Ito) in Normal and Diseased Myocardium. Journal of Molecular and Cellular Cardiology (2001) 33, 851–872. The Ca2+-independent transient outward potassium current (Ito) plays an important role in early repolarization of the cardiac action potential. Itohas been clearly demonstrated in myocytes from different cardiac regions and species. Two kinetic variants of cardiac Itohave been identified: fast Ito, called Ito,f, and slow Ito, called Ito,s. Recent findings suggest that Ito,fis formed by assembly of Kv4.2and/or Kv4.3alpha pore-forming voltage-gated subunits while Ito,sis comprised of Kv1.4and possibly Kv1.7subunits. In addition, several regulatory subunits and pathways modulating the level and biophysical properties of cardiac Itohave been identified. Experimental findings and data from computer modeling of cardiac action potentials have conclusively established an important physiological role of Itoin rodents, with its role in large mammals being less well defined due to complex interplay between a multitude of cardiac ionic currents. A central and consistent electrophysiological change in cardiac disease is the reduction in Itodensity with a loss of heterogeneity of Itoexpression and associated action potential prolongation. Alterations of Itoin rodent cardiac disease have been linked to repolarization abnormalities and alterations in intracellular Ca2+homeostasis, while in larger mammals the link with functional changes is far less certain. We review the current literature on the molecular basis for cardiac Itoand the functional consequences of changes in Itothat occur in cardiovascular disease.
Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30-40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5-10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research.
Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine on maximum rate of depolarization (V(max)), repolarization and spontaneous depolarization have not yet been reported in human isolated cardiac preparations. The same applies to large animals close to human in heart size and spontaneous frequency. Using microelectrode technique action potential characteristics and by applying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent (0.1-10 μM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneous rate of firing of action potentials and produced a concentration- and frequency-dependent V(max) block in dog Purkinje fibers while action potential duration measured at 50% of repolarization was shortened. In the presence of ivabradine, at 400 ms cycle length, V(max) block developed with an onset kinetic rate constant of 13.9 ± 3.2 beat(-1) in dog ventricular muscle. In addition to a fast recovery of V(max) from inactivation (τ=41-46 ms) observed in control, a second slow component for recovery of V(max) was expressed (offset kinetics of V(max) block) having a time constant of 8.76 ± 1.34 s. In dog after attenuation of the repolarization reserve ivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation of repolarization was only observed at 10 μM ivabradine. Ivabradine in addition to the Class V antiarrhythmic effect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment of patients with ischemic heart disease liable to disturbances of cardiac rhythm.
Cellular electrophysiology experiments, important for understanding cardiac arrhythmia mechanisms, are usually performed with channels expressed in non myocytes, or with non-human myocytes. Differences between cell types and species affect results. Thus, an accurate model for the undiseased human ventricular action potential (AP) which reproduces a broad range of physiological behaviors is needed. Such a model requires extensive experimental data, but essential elements have been unavailable. Here, we develop a human ventricular AP model using new undiseased human ventricular data: Ca(2+) versus voltage dependent inactivation of L-type Ca(2+) current (I(CaL)); kinetics for the transient outward, rapid delayed rectifier (I(Kr)), Na(+)/Ca(2+) exchange (I(NaCa)), and inward rectifier currents; AP recordings at all physiological cycle lengths; and rate dependence and restitution of AP duration (APD) with and without a variety of specific channel blockers. Simulated APs reproduced the experimental AP morphology, APD rate dependence, and restitution. Using undiseased human mRNA and protein data, models for different transmural cell types were developed. Experiments for rate dependence of Ca(2+) (including peak and decay) and intracellular sodium ([Na(+)](i)) in undiseased human myocytes were quantitatively reproduced by the model. Early afterdepolarizations were induced by I(Kr) block during slow pacing, and AP and Ca(2+) alternans appeared at rates >200 bpm, as observed in the nonfailing human ventricle. Ca(2+)/calmodulin-dependent protein kinase II (CaMK) modulated rate dependence of Ca(2+) cycling. I(NaCa) linked Ca(2+) alternation to AP alternans. CaMK suppression or SERCA upregulation eliminated alternans. Steady state APD rate dependence was caused primarily by changes in [Na(+)](i), via its modulation of the electrogenic Na(+)/K(+) ATPase current. At fast pacing rates, late Na(+) current and I(CaL) were also contributors. APD shortening during restitution was primarily dependent on reduced late Na(+) and I(CaL) currents due to inactivation at short diastolic intervals, with additional contribution from elevated I(Kr) due to incomplete deactivation.
Current pharmacologic strategies for the management of Atrial fibrillation (AF) include use of 1) sodium channel blockers, which are contraindicated in patients with coronary artery or structural heart disease because of their potent effect to slow conduction in the ventricles, 2) potassium channel blockers, which predispose to acquired long QT and Torsade de Pointes arrhythmias because of their potent effect to prolong ventricular repolarization, and 3) mixed ion channel blockers such as amiodarone, which are associated with multi-organ toxicity. Accordingly, recent studies have focused on agents that selectively affect the atria but not the ventricles. Several Atrial-selective approaches have been proposed for the management of AF, including inhibition of the Atrial-specific ultra rapid delayed rectified potassium current (IKur), acetylcholine-regulated inward rectifying potassium current (IK-ACh), or connexin-40 (Cx40). All three are largely exclusive to atria. Recent studies have proposed that an Atrial-selective depression of sodium channel-dependent parameters with agents such as ranolazine may be an alternative approach capable of effectively suppressing AF without increasing susceptibility to ventricular arrhythmias. Clinical evidence for Cx40 modulation or IK-ACh inhibition are lacking at this time. The available data suggest that Atrial-selective approaches involving a combination of INa, IKur, IKr, and, perhaps, Ito block may be more effective in the management of AF than pure IKur or INa block. The anti-AF efficacy of the Atrial-selective/predominant agents appears to be similar to that of conventionally used anti-AF agents, with the major apparent difference being that the latter are associated with ventricular arrhythmogenesis and extra cardiac toxicity.
The cardiac transient outward current (Ito) has been shown in several species to consist of two components: 1) a 4-aminopyridine (4-AP)-sensitive component (Ito1) and 2) a 4-AP-resistant component (Ito2). In rabbits, Ito2 is a Ca(2+)-dependent Cl- current [ICl(Ca)]; similar mechanisms have been suggested to underlie Ito2 in human atrium. We used whole cell patch-clamp techniques to define the mechanism of Ito2 (defined as the component resistant to 5 mM 4-AP) in human atrial myocytes, with parallel experiments performed in rabbit atrial cells. In rabbit atrium, Ito2 activated more slowly than Ito1 and had a bell-shaped current-voltage of Ito with properties similar to Ito2 in the rabbit, and a similar component recorded with pipette K+ replaced by Cs+ was suppressed by the substitution of methanesulfonate for Cl- in the superfusate. In human cells, a 4-AP-resistant Ito2 was recorded at a depolarizing pulse frequency of 1 Hz, but not at 0.1 Hz. Ito2 activated rapidly and inactivated earlier than Ito1, whereas its I-V relation was linear like that of Ito1. Ryanodine had no effect on human atrial Ito. When K(+)-free pipette solutions were used, no Ito was recorded in 30 human atrial myocytes, and external Cl- replacement with methanesulfonate failed to reveal an Ito. In 13 human myocytes, isoproterenol increased ICa but failed to activate an Ito compatible with ICl(Ca). Whereas caffeine suppressed human atrial Ito, it also suppressed Ito1 [in the presence of 200 microM Cd2+ to block ICa and 5 mM intracellular ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to buffer intracellular Ca2+] in both human and rabbit atrium, indicating an action unrelated to Ca(2+)-triggered Ca2+ release. In conclusion, we were unable to demonstrate the presence of ICl(Ca) in human atrial myocytes, and the 4-AP-resistant component of Ito appeared to be due to 4-AP unblocking.
Background:
Cardiac repolarization abnormalities in drug-induced and genetic long-QT syndrome may lead to afterdepolarizations and life-threatening ventricular arrhythmias. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) should help to overcome the limitations of animal models based on species differences in repolarization reserve. Here, we compared head-to-head the contribution of IKs (long QT1) and IKr (long QT2) on action potentials (APs) in human left ventricular (LV) tissue and hiPSC-CM-derived engineered heart tissue (EHT).
Methods:
APs were measured with sharp microelectrodes in EHT from 3 different control hiPSC-CM lines and in tissue preparations from failing LV.
Results:
EHT from hiPSC-CMs showed spontaneous diastolic depolarization and AP generation that were sensitive to low concentrations of ivabradine. IKr block by E-4031 prolonged AP duration at 90% repolarization with similar half-effective concentration in EHT and LV but larger effect size in EHT (+281 versus +110 ms in LV). Although IKr block alone evoked early afterdepolarizations and triggered activity in 50% of EHTs, slow pacing, reduced extracellular K+, and blocking of IKr, IKs, and IK1 were necessary to induce early afterdepolarizations in LV. In accordance with their clinical safety, moxifloxacin and verapamil did not induce early afterdepolarizations in EHT. In both EHT and LV, IKs block by HMR-1556 prolonged AP duration at 90% repolarization slightly in the combined presence of E-4031 and isoprenaline.
Conclusions:
EHT from hiPSC-CMs shows a lower repolarization reserve than human LV working myocardium and could thereby serve as a sensitive and specific human-based model for repolarization studies and arrhythmia, similar to Purkinje fibers. In both human LV and EHT, IKs only contributed to repolarization under adrenergic stimulation.
The ability to direct the differentiation of human pluripotent stem cells (hPSCs) to the different cardiomyocyte subtypes is a prerequisite for modeling specific forms of cardiovascular disease in vitro and for developing novel therapies to treat them. Here we have investigated the development of the human atrial and ventricular lineages from hPSCs, and we show that retinoic acid signaling at the mesoderm stage of development is required for atrial specification. Analyses of early developmental stages revealed that ventricular and atrial cardiomyocytes derive from different mesoderm populations that can be distinguished based on CD235a and RALDH2 expression, respectively. Molecular and electrophysiological characterization of the derivative cardiomyocytes revealed that optimal specification of ventricular and atrial cells is dependent on induction of the appropriate mesoderm. Together these findings provide new insights into the development of the human atrial and ventricular lineages that enable the generation of highly enriched, functional cardiomyocyte populations for therapeutic applications.
We describe sleuth (http://pachterlab.github.io/sleuth), a method for the differential analysis of gene expression data that utilizes bootstrapping in conjunction with response error linear modeling to decouple biological variance from inferential variance. sleuth is implemented in an interactive shiny app that utilizes kallisto quantifications and bootstraps for fast and accurate analysis of data from RNA-seq experiments.
Statement of significance:
There is a growing interest in creating engineered heart tissue constructs for basic cardiac research, applied research in cardiac pharmacology, and repair of damaged hearts. We address an unmet need to characterize fully the performance of these tissues with our simple "I-Wire" assay that allows application of controlled forces to three-dimensional cardiac fiber constructs and measurement of both the electrical and mechanical properties of the construct. The advantage of I-Wire over other approaches is that the constructs being measured are truly three-dimensional, rather than a single layer of cells grown within a microfluidic device. We anticipate that the I-Wire will be extremely useful for the evaluation of myocardial constructs created using cardiomyocytes derived from human induced pluripotent stem cells.
Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering “cardiobundles”, 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm²) and conduction velocity (52.5 cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (∼2.5-fold) and conduction velocity (∼1.4-fold). The average specific force of 23.2 mN/mm² and conduction velocity of 25.8 cm/s approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function in vitro.
We present kallisto, an RNA-seq quantification program that is two orders of magnitude faster than previous approaches and achieves similar accuracy. Kallisto pseudoaligns reads to a reference, producing a list of transcripts that are compatible with each read while avoiding alignment of individual bases. We use kallisto to analyze 30 million unaligned paired-end RNA-seq reads in <10 min on a standard laptop computer. This removes a major computational bottleneck in RNA-seq analysis.
A giant disruption of the heart
Certain forms of heart failure originate from genetic mutations. Understanding how the culprit mutant proteins alter normal heart function could lead to more effective treatments. One candidate is the giant protein tintin, which is mutated in a subset of patients with dilated cardiomyopathy. Through a combination of patient-derived stem cells, tissue engineering, and gene editing, Hinson et al. found that disease-associated titin mutations disrupt the function of the contractile unit in heart muscle. As a result, the heart does not respond properly to mechanical and other forms of stress.
Science , this issue p. 982
Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocte (ESC-CM) transplantation, thereby potentially preventing dilative remodelling and progression to heart failure.
Assessment of transport stability, long term survival, structural organisation, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction (MI) model.
We constructed EHMs from ESC-CMs and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). After ischemia / reperfusion (I/R) injury, EHMs were implanted onto immunocompromised rat hearts at 1 month to simulate chronic ischemia. Bioluminescence imaging (BLI) showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving up to 25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs -6.7±1.4% vs control -10.9±1.5%, n>12, P=0.05), we observed no difference between EHMs containing viable or non-viable human cardiomyocytes in this chronic xenotransplantation model (n>12, P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring.
EHM transplantation led to high engraftment rates, long term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic MI model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
Objectives:
The present work was designed to provide an initial characterization of M cells in the normal human heart.
Background:
Recent studies have uncovered a unique population of cells in the midmyocardial region of the canine ventricle. These cells, named M cells, were found to possess electrophysiologic features and a pharmacologic responsiveness different from those of other myocardial cells. Although well characterized in the dog, their presence or absence in the human heart is unknown.
Methods:
Standard microelectrode techniques were used to map slices of ventricular free wall obtained from normal human hearts (n = 4). Preparations were paced at cycle lengths ranging from 1 to 10 s.
Results:
We identified three cell subtypes: endocardial, subepicardial (M cells) and epicardial cells. The principal features differentiating M cells from the other cell subtypes were their longer action potential duration, more accentuated action potential duration rate relations and greater maximal rate of increase in action potential upstroke (Vmax). Our findings suggest that M cells represent approximately 30% of the cellular mass of the left ventricular wall. Concordance between changes in their repolarization and changes in QTU interval provide support for the role of M cells in the generation of the electrocardiographic (ECG) U wave.
Conclusions:
This study provides evidence for the existence of M cells in the human heart that contribute to heterogeneity of repolarization within the ventricular wall. Our findings provide strong support for the hypothesis that M cells contribute importantly to the manifestation of the U wave on the ECG.
Aims:
We aimed at investigating contractile changes after hypoxia-reoxygenation and dobutamine challenge in superfused human atrial pectinate muscle to see whether high versus low stimulation rate during hypoxia might account for outcome differences compatible with the definition of an in vitro model of myocardial stunning and whether pretreatment with the dihydropyridine Ca2+ entry blocker felodipine might afford protection.
Methods:
Human right atrial trabeculae obtained from adult patients were superfused in an organ bath with oxygenated (O2 content 16 ml/l) and modified (NaHCO3 25.7 mmol/l) Tyrode's solution at 37 degrees C. Dobutamine (1 nmol/l to 10 micromol/l) was superfused in 10 oxygenated preparations to select the optimal drug concentration to be used in another 22 which were randomized. Group (A) consisted of time-related controls (Tyrodes's solution for 225 min at cycle length (CL) 1600 ms and no dobutamine). There were two test groups, respectively: (B) low (1600 ms CL) and (C) high (400 ms CL) stimulation rate. After 60 min of stabilization, in groups B and C, hypoxic superfusion (O2 content 5 ml/l) lasted 60 min, then reoxygenation (60 min) and dobutamine challenge (1 micromol/l, 15 min) were performed. Analysis of variance for repeated measures with the Greenhouse-Geisser correction, and a repeated measures model with structured covariance (preparation mass, length, width and time-varying time to peak tension) matrices were used whereby grouping (G), time (T) and G x T interaction were weighted. Force-frequency relationship and post-pausal potentiation were studied after each phase. Electrophysiology, histomorphometry and electron microscopy were carried out (n=6). Felodipine (0.1 micromol/l, n=5) pretreatment (15 min before hypoxia) was given in parallel experiments.
Results:
Time-related controls showed approximately 10% per hour decrease of developed tension and the Paradise test provided approximately 80% of control values. In test groups (as compared to baseline values) contractility was decreased approximately 65% after hypoxia-reoxygenation and it increased approximately 25% after dobutamine (G, 0.0065<P<0.0155; T, P=0.00005; G x T, P=0.00005). High stimulation rate during hypoxia worsened hypoxia-reoxygenation contractile changes, whereas reversibility after dobutamine was less. In both B and C groups during hypoxia, contractility decreased quite rapidly, although by 10 min or so a plateau (approximately 50%) was reached in group B, whereas in group C contractility decreased to <20%. None of the covariates contributed significantly to predict the dependent variables investigated. Force-frequency relationship and post-pausal potentiation were repeatable, paralleled overall changes due to hypoxia, reoxygenation and dobutamine challenge and were useful to discriminate Ca2+-related diastolic processes thus helping index myocardial contractile reserve. Force-frequency relationship was negative at high stimulation rates, concomitant to an abrupt change of shape and duration of action potential with little time for Ca2+-related Ca2+ release and ensuing systolic processes. Felodipine pretreatment enabled an unblunted response to dobutamine. Histomorphometry showed an unexpected 'fibrotic core'. At electron microscopy, subendocardial and deep part of the same pectinate muscles showed identical degrees of degenerative lesions. Superfused samples showed, unexpectedly, less anoxic lesions than preparations fixed within 15 min from surgical explant, although lesions were higher than in samples fixed immediately after explant.
Conclusions:
This might be a relevant model, whereby pharmacological or physical interventions are tested. Native human atrial trabeculae might be used without dissection and/or preservatives. If high stimulation rate during hypoxia is used the power of hypothesis testing is maximized. Future studies with this material will be easier and comparatively smaller series might be investigated. Felo
Understanding atrial fibrillation (AF) requires integrated understanding of ionic currents and Ca2+ transport in remodeled human atrium, but appropriate models are limited.
To study AF, we developed a new human atrial action potential (AP) model, derived from atrial experimental results and our human ventricular myocyte model.
Atria versus ventricles have lower I(K1), resulting in more depolarized resting membrane potential (≈7 mV). We used higher I(to,fast) density in atrium, removed I(to,slow), and included an atrial-specific I(Kur). I(NCX) and I(NaK) densities were reduced in atrial versus ventricular myocytes according to experimental results. SERCA function was altered to reproduce human atrial myocyte Ca2+ transients. To simulate chronic AF, we reduced I(CaL), I(to), I(Kur) and SERCA, and increased I(K1),I(Ks) and I(NCX). We also investigated the link between Kv1.5 channelopathy, [Ca2+]i, and AF. The sinus rhythm model showed a typical human atrial AP morphology. Consistent with experiments, the model showed shorter APs and reduced AP duration shortening at increasing pacing frequencies in AF or when I(CaL) was partially blocked, suggesting a crucial role of Ca2+ and Na+ in this effect. This also explained blunted Ca2+ transient and rate-adaptation of [Ca2+]i and [Na+]i in chronic AF. Moreover, increasing [Na+]i and altered I(NaK) and I(NCX) causes rate-dependent atrial AP shortening. Blocking I(Kur) to mimic Kv1.5 loss-of-function increased [Ca2+]i and caused early afterdepolarizations under adrenergic stress, as observed experimentally.
Our study provides a novel tool and insights into ionic bases of atrioventricular AP differences, and shows how Na+ and Ca2+ homeostases critically mediate abnormal repolarization in AF.
The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood.
We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches.
Using human embryonic stem cell and human induced pluripotent stem cell-derived cardiomyocytes in a 3-dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ≈10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation.
Our results indicate that both mechanical load and vascular cell coculture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.
The effects of 5-hydroxytryptamine (5-HT) on force of contraction (FC), action potential (AP) and calcium current (ICa) were studied in human right atrial and left ventricular heart muscle. 5-HT exerted a concentration-dependent increase in FC in multicellular atrial preparations; the EC50 was approximately 3 x 10(-7) mol/l. Maximal increases in FC (252 +/- 58% of control values; mean +/- SEM, n = 6) were obtained at 5-HT 10(-5) mol/l. At this concentration, ICa was increased four- to sevenfold in enzymatically isolated atrial myocytes. In contrast, ventricular preparations did not respond to 5-HT; FC, AP and ICa remained unaffected. In the same preparations, FC was increased by isoprenaline three- to fourfold. These results confirm the observation that 5-HT induces a positive inotropic effect in the human atrium, possibly mediated by activation of the adenylyl cyclase - cyclic AMP system. Our study demonstrates, however, the complete lack of functional 5-HT receptors, with respect to changes in FC, in the human ventricle. Since the positive inotropic effect of 5-HT in the human heart is obviously restricted to the atrium, our findings question the concept of developing 5-HT receptor agonists for the treatment of heart failure.
The effects of 5-carboxamidotryptamine (5-CT) and the gastrokinetic benzamides renzapride and cisapride on contractile force were investigated using isolated paced right atrial appendages from patients treated with β-adrenoceptor blocking agents who were undergoing open heart surgery. These effects were compared to those of 5-hydroxytryptamine (5-HT). The effects of the drugs on atrial cyclic AMP levels and cyclic AMP-dependent protein kinase ratios were also investigated.
The drugs all increased contractile force rank order of potency was 5-HT > renzapride > cisapride > 5-CT. The maximum responses, expressed as a fraction of the response to 200 μmol/l (−)-isoprenaline, were 5-HT 0.6, 5-CT 0.6, renzapride 0.4 and cisapride ≥ 0.2, suggesting that the latter two are partial agonists. 5-HT, 5-CT and renzapride but not cisapride caused significant shortening of time to peak force. The effects of the four drugs were blocked by molar concentrations of ICS 205-930, suggesting an involvement of 5-HT4 receptors. As expected of partial agonists both renzapride and cisapride caused simple competitive antagonism of the positive inotropic effects of 5-HT The estimated equilibrium dissociation constants pKp
(−log mol/l Kp
) were 6.7 for renzapride and 6.2 for cisapride. 5-CT at concentrations up to 10 μmol/l did not antagonise the effects of 5-HT. In the presence of (±)-propranolol 0.4 μmol/I, 5-HT 10 μmol/l, 5-CT 100 μmol/I, renzapride 10 μmol/l and cisapride 40 μmol/I significantly increased cyclic AMP levels. 5-HT and renzapride also significantly increased cyclic AMP-dependent protein kinase activity, whereas 5-CT caused only marginal stimulation and cisapride was ineffective.
The results confirm the existence of a human right atrial 5-HT receptor that is similar in nature to, but not necessarily identical with, the 5-HT4 receptor of mouse embryonic colliculi neurones. The main difference is that in human right atrium the benzamides are less potent and efficacious than 5-HT and that cisapride is less potent and less efficacious than renzapride while in mouse embryonic colliculi these two benzamides are equipotent with and more efficacious agonists than 5-HT We designate the human right atrial 5-HT receptor 5-HT4-like. The human right atrial 5-HT4-like receptor greatly resembles porcine sinoatrial and left atrial 5-HT4-like receptors and also appears to be similar to 5-HT4-like receptors of guinea-pig ileum and rat oesophagus.
The effects of serotonin (5-hydroxytryptamine; 5-HT) on the cardiovascular system are complex. These effects, consisting of bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction are mediated by three main sets of receptors called 5-HT1-like, 5-HT2, and 5-HT3. In addition, recent findings suggest the participation of a putative 5-HT4 receptor. Though selective 5-HT1A receptor agonists can lower heart rate (and arterial blood pressure), 5-HT usually lowers heart rate by eliciting an initial short-lasting hypotension due to bradycardia (von Bezold-Jarisch-like reflex) via 5-HT3 receptors located on sensory vagal nerve endings in the heart. Once this bradycardia reflex is suppressed--for example, during deep anesthesia, vagotomy, or spinal section--5-HT can increase heart rate in different species by a variety of mechanisms. Myocardial 5-HT1-like, 5-HT2, and 5-HT4 receptors appear to be involved in the cat, rat, and pig, respectively. 5-HT-induced tachycardia in the dog and rabbit is due mainly to release of catecholamines and involves 5-HT2 receptors on the adrenal medulla and 5-HT3 receptors on postganglionic cardiac sympathetic nerve fibers. Recently, 5-HT3 receptors also have been implicated in the 5-HT-induced tachycardia in the conscious dog. The blood pressure response to 5-HT is usually triphasic and consists of a von Bezold-Jarisch-like reflex, a middle pressor phase, and a longer-lasting hypotension. The pressor response is a consequence of vasoconstriction mediated via 5-HT2 receptors; however, vasoconstriction in the dog saphenous vein and cephalic arteries and arteriovenous anastomoses is due to stimulation of 5-HT1-like receptors. The depressor response exclusively involves 5-HT1-like receptors located at four different sites: (a) central nervous system (decrease in sympathetic and increase in vagal nervous activity), (b) sympathetic nerve terminals (reduction of transmitter release), (c) vascular smooth muscle (vasodilatation), and (d) vascular endothelium (release of a relaxant factor, probably nitric oxide). Arteriolar dilatation, together with the constriction of arteriovenous anastomoses, leads to an increase in nutrient (tissue; capillary) blood flow. The 5-HT1-like receptors are heterogeneous in nature; however, apart from the resemblance of the central nervous system 5-HT1-like receptor causing hypotension and bradycardia to the 5-HT1A binding subtype, the relationship of the other 5-HT1-like receptors to 5-HT1 binding subtypes is still debatable.(ABSTRACT TRUNCATED AT 400 WORDS)
The blocking action of 4-aminopyridine (4-AP) on the transient outward K+ current (ITO) in isolated rat ventricular myocytes was studied using the whole-cell configuration of the patch-clamp technique. 4-AP inhibition of ITO was concentration-dependent with half-maximal block occurring at 0.2 mM. At high concentrations (> 1 mM), 4-AP appeared to slow both the activation and inactivation phases of ITO. This resulted in a crossover phenomenon where in the presence of 4-AP the outward current was less than control at the beginning of a depolarizing pulse but crossed over during the pulse to become greater than control. Inhibition of ITO by 4-AP was voltage-dependent. Steady-state block of ITO by 4-AP was greatest at or near resting membrane potentials (i.e., -70 mV) but decreased with membrane depolarization. The voltage-dependence of block was steep and was well described by a Boltzmann relationship with a slope factor of approximately 4 mV. The midpoint potential for block was dependent on the concentration of 4-AP, being -41.6 +/- 0.4 mV (n = 9), -40.7 +/- 1.3 mV (n = 6), -34.0 +/- 1.6 mV (n = 5) and -30.1 +/- 0.2 (n = 15) at 0.3, 1, 3 and 10 mM, respectively. The midpoint potential for activation was -12.6 mV and was -46.9 mV for inactivation. The concentration-dependence of the voltage-dependence of 4-AP block can be explained by assuming that the sequential closed states through which the channel passes during activation exhibit successively lower affinities for 4-AP. Onset of ITO block by 4-AP was slow. The association (kON) and dissociation. (kOFF) rate constants for binding at -70 mV were: kON = 207 M-1 s-1 and kOFF = 0.090 s-1. The time constant for unblock (tau UNBLOCK) of ITO at 0 mV was independent of 4-AP concentration indicating that there was no binding of 4-AP at this potential. kOFF (1/tau UNBLOCK) at 0 mV was 2.4 s-1 which is approximately 25-fold faster then at -70 mV. The results suggest that 4-AP binds most strongly to closed channels with the inactivation gate open. The conformational changes that occur during channel opening induce a decrease in affinity for 4-AP so that when the channel is in the open state, 4-AP binding is at its weakest. The processes of 4-AP block and inactivation appear to be mutually exclusive.
We aimed at investigating frequency-related changes of human atrial action potential (AP) in vitro to see whether baseline AP shape might account for different responses to increasing stimulation rates. Human right atrial trabeculae (n = 48) obtained from adult (n = 38, mean age 59 +/- 8, range 45-72 years) consecutive patients (approximately equal to 30% of those operated upon by a single surgeon; 1.26 preparations per patient, range 1-2) were superfused in an organ bath with oxygenated (O2 content 16 ml/l) and modified (NaHCO3 25.7 mmol/l) Tyrode's solution at 31 degrees C. Baseline electrophysiology (pacing: 1 ms duration, 2-4 mA current intensity) at cycle length (CL) of 1000 ms was recorded in 90% (43 out of 48) of the preparations. The frequency-related protocol (CL from 1600 to 300 ms) was, however, undertaken in 23 (48%) preparations because 20 (42%) became pacing unresponsive immediately after baseline recordings. No statistical differences were seen when baseline electrophysiological parameters (mean +/- SD) were grouped according to late pacing responsiveness (n = 43 vs. n = 23): respectively, resting membrane potential (RMP) was -74 +/- 6 vs. -75 +/- 4 mV, maximal upstroke velocity (Vmax) 172 +/- 60 vs. 173 +/- 39 V/s, AP amplitude (APA) 89 +/- 11 vs. 91 +/- 8 mV and AP durations were at 30% (APD30%) 10 +/- 13 vs. 13 +/- 18 ms, 50% (APD50%) 45 +/- 79 vs. 62 +/- 91 ms and 90% (APD90%) 383 +/- 103 vs. 407 +/- 108 ms. To classify baseline AP shape, two criteria were adopted: criterion 1 ("objective"), based on APA (cut-off 90 mV) and APD90% (cut-off 500 ms) computed values and criterion 2 ("visual") derived from the literature. These criteria enabled us to differentiate three AP shape types: type 1 (spike and dome), type 3 (no dome) and type 4 (extremely prolonged). At baseline, the two criteria diagnosed different proportions of AP shape types. There were, however, no intra-type statistical differences among electrophysiological parameters. By criterion 1, analysis of variance (ANOVA) showed significant inter-type differences of RMP,Vmax, APA, APD50 and 90% and by criterion 2 of APA, APD30, 50 and 90%, respectively. To facilitate comparisons with previous published data, criterion 2 was selected to analyse frequency-related changes of AP shape types. At low stimulation rate, ANOVA for repeated measures (with Greenhouse-Geisser epsilon correction) showed inter-type differences for APD30, 50 and 90% (P = 0.00005). RMP, Vmax, APA and APD90% were overall frequency-related (P = 0.00005). Inter-type frequency-related differences were however seen only for APD90%. Human atrial AP durations (30, 50 and 90%) enable differentiation among AP shape types (1, 3 and 4). By a standardized use-dependent protocol overall RMP, Vmax, APA and APD90% are frequency-related. AP shape accounts for frequency-related changes of APD90% only. A type 4 AP shape with much prolonged AP duration had a flat frequency dependence. At high stimulation rates, adult type 1 and 3 AP shapes are indistinguishable. Use-dependent and pharmacological investigations in human atrial myocytes need to take AP shape into account.
There has been debate regarding the level of sarcoplasmic reticulum (SR) Ca2+ ATPase protein in heart failure. We have used the SR Ca2+ ATPase inhibitor thapsigargin to investigate the functional contribution of this uptake system to contraction and relaxation in myocytes from failing and non-failing human ventricle.
Myocytes were isolated from 28 failing and 18 non-failing ventricles and stimulated at 0.2 Hz, 32 degrees C in Krebs-Henseleit solution. Contraction amplitude and speed were compared before and after treatment with 1 mumol/l thapsigargin for 20 min to deplete SR Ca2+ stores.
Initial beat duration was longer in myocytes from failing hearts. Addition of thapsigargin significantly prolonged the beat in myocytes from both groups, but the increase was greater in non-failing hearts (beat duration increased by 0.79 +/- 0.12 s in myocytes from non-failing hearts compared with 0.37 +/- 0.12 s in those from failing, P < 0.02). The contraction amplitude increased at high stimulation frequencies in myocytes from non-failing hearts (from 2.6% shortening at 0.1 Hz to 4.6% at 1 Hz, P < 0.001, n = 9), but not in those from failing hearts (1.8% at 0.1 Hz compared with 1.7% at 1 Hz, n = 5). Thapsigargin abolished the positive staircase in the non-failing, but had no effect in the failing group. Contraction amplitude following a rest interval was significantly depressed relative to steady-state levels in myocytes from the non-failing hearts (44.8 +/- 10.3% at 3 min), but not in failing (102 +/- 18%, P < 0.01 compared to non-failing at 3 min). Following thapsigargin treatment, there were no longer significant differences between failing and non-failing myocytes in the time course of the beat, frequency response or post-rest behaviour.
The differences between myocytes from failing and non-failing hearts were reduced by inhibition of SR function. These results are consistent with the hypothesis that the initial differences had been due to decreased SR Ca2+ uptake.
The response of the heart to altered hemodynamic loading is growth or remodeling of myocytes and the extracellular matrix. In order to describe and mathematically model this dynamic and complex system of growing and resorbing tissue, the stimulating factor for tissue growth must be found, and up to now is not known. Most evidence, both in tissue and at the cellular level, points to a mechanical factor as the stimulus, and most likely a deformation signal is transduced to initiate protein synthesis. At the cellular level mechanotransduction likely takes place at the cellular membrane, although multiple biochemical and mechanical pathways have been proposed which induce transcription in the nucleus and eventual protein upregulation. The results of a recent mathematical analysis based on experimental data suggest that end-diastolic fiber strain at the tissue level may be the stimulus to one mode of tissue growth: volume-overload hypertrophy. This is the only mechanical factor that we found to be normalized after volume overload hypertrophy. But other studies do not agree with this result, and other modes of hypertrophy may be regulated by different factors or combinations of factors.
Hypertension is a common precursor of serious disorders including stroke, myocardial infarction, congestive heart failure, and renal failure in whites and to a greater extent in African Americans. Large genetic-epidemiological studies of hypertension are needed to gain information that will improve future methods for diagnosis, treatment, and prevention of hypertension, a major contributor to cardiovascular morbidity and mortality.
We report successful implementation of a new structure of research collaboration involving four NHLBI "Networks," coordinated under the Family Blood Pressure Program. The Hypertension Genetic Epidemiology Network (HyperGEN) involves scientists from six universities and the NHLBI who seek to identify and characterize genes promoting hypertension. Blood samples and clinical data were projected to be collected from a sample of 2244 hypertensive siblings diagnosed before age 60 from 960 sibships (half African-American) with two or more affected persons. Nonparametric sibship linkage analysis of over one million genotype determinations (20 candidate loci and 387 anonymous marker loci) was projected to have sufficient power for detecting genetic loci promoting hypertension. For loci showing evidence for linkage in this study and for loci reported linked or associated with hypertension by other groups, genotypes are compared in hypertensive cases versus population-based controls to identify or confirm genetic variants associated with hypertension. For some of these genetic variants associated with hypertension, detailed physiological and biochemical characterization of untreated adult offspring carriers versus non-carriers may help elucidate the pathophysiological mechanisms that promote hypertension.
The projected sample size of 2244 hypertensive participants was surpassed, as 2407 hypertensive individuals (1262 African-Americans and 1145 whites) from 917 sibships were examined. Detailed consent forms were designed to offer participants several options for DNA testing; 94% of participants gave permission for DNA testing now or in the future for any confidential medical research, with only 6% requesting restrictions for tests performed on their DNA. Since this is a family study, participants also are asked to list all first degree relatives (along with names, addresses, and phone numbers) and to indicate for each relative whether they were willing to allow study staff to make a contact. Seventy percent gave permission to contact some relatives; about 30% gave permission to contact all first degree relatives; and less than 1% asked that no relatives be contacted. Successes after the first four years of this study include: 1) productive collaboration of eight centers from six different locations; 2) early achievement of recruitment goals for study participants including African-Americans; 3) an encouraging rate of consent for DNA testing (including future testing) and relative contacting; 4) completed analyses of genetic linkage and association for several candidate gene markers and polymorphisms; 5) completed genotyping of random markers for over half of the full sample; and 6) early sharing of results among the four Family Blood Pressure Program networks for candidate and genome search analyses.
Experience after four years of this five-year program (1995-2000) suggests that the newly initiated NHLBI Network Program mechanism is fulfilling many of the expectations for which it was designed. It may serve as a paradigm for future genetic research that can benefit from large sample sizes, frequent sharing of ideas among laboratories, and prompt independent confirmation of early findings, which are required in the search for common genes with relatively small effects such as those that predispose to human hypertension.