Nicole Feric’s research while affiliated with University of Massachusetts Boston and other places

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.

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.

Introduction: The development of patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an opportunity to study genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM), one of the most common inherited cardiac diseases. However, immaturity of the iPSC-CMs and the lack of a multicellular composition pose concerns over its faithfulness in disease modeling and its utility in developing mechanism-specific treatment. Methods: The Biowire platform was used to generate 3D engineered cardiac tissues (ECTs) using HCM patient-derived iPSC-CMs carrying a β-myosin mutation (MYH7-R403Q) and its isogenic control (WT), withal ECTs contained healthy human cardiac fibroblasts. ECTs were subjected to electro-mechanical maturation for 6 weeks before being used in HCM phenotype studies. Results: Both WT and R403Q ECTs exhibited mature cardiac phenotypes, including a lack of automaticity and a ventricular-like action potential (AP) with a resting membrane potential < −75 mV. Compared to WT, R403Q ECTs demonstrated many HCM-associated pathological changes including increased tissue size and cell volume, shortened sarcomere length and disorganized sarcomere structure. In functional assays, R403Q ECTs showed increased twitch amplitude, slower contractile kinetics, a less pronounced force-frequency relationship, a smaller post-rest potentiation, prolonged AP durations, and slower Ca²⁺ transient decay time. Finally, we observed downregulation of calcium handling genes and upregulation of NPPB in R403Q vs. WT ECTs. In an HCM phenotype prevention experiment, ECTs were treated for 5-weeks with 250 nM mavacamten or a vehicle control. We found that chronic mavacamten treatment of R403Q ECTs: (i) shortened relaxation time, (ii) reduced APD90 prolongation, (iii) upregulated ADRB2, ATP2A2, RYR2, and CACNA1C, (iv) decreased B-type natriuretic peptide (BNP) mRNA and protein expression levels, and (v) increased sarcomere length and reduced sarcomere disarray. Discussion: Taken together, we demonstrated R403Q ECTs generated in the Biowire platform recapitulated many cardiac hypertrophy phenotypes and that chronic mavacamten treatment prevented much of the pathology. This demonstrates that the Biowire ECTs are well-suited to phenotypic-based drug discovery in a human-relevant disease model.

The role of C‐type natriuretic peptide (CNP) in the regulation of cardiac function in humans remains to be established as previous investigations have been confined to animal model systems. Here, we used well‐characterized engineered cardiac tissues (ECTs) generated from human stem cell‐derived cardiomyocytes and fibroblasts to study the acute effects of CNP on contractility. Application of CNP elicited a positive inotropic response as evidenced by increases in maximum twitch amplitude, maximum contraction slope and maximum calcium amplitude. This inotropic response was accompanied by a positive lusitropic response as demonstrated by reductions in time from peak contraction to 90% of relaxation and time from peak calcium transient to 90% of decay that paralleled increases in maximum contraction decay slope and maximum calcium decay slope. To establish translatability, CNP‐induced changes in contractility were also assessed in rat ex vivo (isolated heart) and in vivo models. Here, the effects on force kinetics observed in ECTs mirrored those observed in both the ex vivo and in vivo model systems, whereas the increase in maximal force generation with CNP application was only detected in ECTs. In conclusion, CNP induces a positive inotropic and lusitropic response in ECTs, thus supporting an important role for CNP in the regulation of human cardiac function. The high degree of translatability between ECTs, ex vivo and in vivo models further supports a regulatory role for CNP and expands the current understanding of the translational value of human ECTs.

Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical stimulations are delivered to the myocardium during the absolute refractory period to enhance cardiac function. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. In the present study, we sought to extend these results to human 3D microphysiological systems to develop a robust model to evaluate various clinical CCM pulse parameters in vitro. HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video (i.e., pixel) and force-based analysis. CCM pulses were assessed at varying electrical ‘doses’ using a commercial pulse generator. A robust CCM contractile response was observed for 3D ECTs. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed no contractile response. 3D ECTs displayed enhanced contractile properties including increased contraction amplitude (i.e., force), and accelerated contraction and relaxation slopes under standard acute CCM stimulation. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter-dependent manner by adjustment of CCM pulse delay, duration, amplitude, and number relative to baseline. The observed acute effects subsided when the CCM stimulation was stopped and gradually returned to baseline. These data represent the first study of CCM in 3D hiPSC-CM models and provide a nonclinical tool to assess various CCM device signals in 3D human cardiac tissues prior to in vivo animal studies. Moreover, this work provides a foundation to evaluate the effects of additional cardiac medical devices in 3D ECTs.

Activin A has been linked to cardiac dysfunction in aging and disease, with elevated circulating levels found in patients with hypertension, atherosclerosis, and heart failure. Here, we investigated whether Activin A directly impairs cardiomyocyte (CM) contractile function and kinetics utilizing cell, tissue, and animal models. Hydrodynamic gene delivery-mediated overexpression of Activin A in wild-type mice was sufficient to impair cardiac function, and resulted in increased cardiac stress markers (N-terminal pro-atrial natriuretic peptide) and cardiac atrophy. In human-induced pluripotent stem cell-derived (hiPSC) CMs, Activin A caused increased phosphorylation of SMAD2/3 and significantly upregulated SERPINE1 and FSTL3 (markers of SMAD2/3 activation and activin signaling, respectively). Activin A signaling in hiPSC-CMs resulted in impaired contractility, prolonged relaxation kinetics, and spontaneous beating in a dose-dependent manner. To identify the cardiac cellular source of Activin A, inflammatory cytokines were applied to human cardiac fibroblasts. Interleukin -1β induced a strong upregulation of Activin A. Mechanistically, we observed that Activin A-treated hiPSC-CMs exhibited impaired diastolic calcium handling with reduced expression of calcium regulatory genes (SERCA2, RYR2, CACNB2). Importantly, when Activin A was inhibited with an anti-Activin A antibody, maladaptive calcium handling and CM contractile dysfunction were abrogated. Therefore, inflammatory cytokines may play a key role by acting on cardiac fibroblasts, causing local upregulation of Activin A that directly acts on CMs to impair contractility. These findings demonstrate that Activin A acts directly on CMs, which may contribute to the cardiac dysfunction seen in aging populations and in patients with heart failure.

Introduction: Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical simulations are delivered to the myocardium during the absolute refractory period. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. Hypothesis: In the present study, we sought to extend these results to 3D microphysiological systems to develop a human-based model to evaluate various clinical CCM pulse parameters in vitro. Methods: HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video-based analysis and custom force analysis. CCM pulses were assessed at varying clinical ‘doses’ using a commercial pulse generator. Robust response was observed at physiological Ca concentrations [1.8 mM] for 3D ECTs. Results: Under standard acute CCM stimulation 3D ECTs displayed enhanced contractile properties including increased peak contraction amplitude (i.e., force), and faster contraction and relaxation kinetics. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter dependent manner. The observed effects subsided when the acute CCM stimulation was stopped and gradually returned to baseline. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed neutral response. Conclusions: These data represent the first study of acute CCM stimulation in a 3D hiPSC-CM model and provides a preclinical model to assess various CCM signals in human cardiac tissues prior to in vivo animal studies.

MK-8722, a systemic pan-AMPK activator improves glucose homeostasis in animal models of diabetes, but causes cardiac hypertrophy # . Two- and 3-dimensional tissue (2D,3D) constructs of human induced pluripotent stem cell derived cardiomyocytes (CM) (iCells, Fujifilm/CDI) were used to probe the translatability of cardiac hypertrophy and the role of AMPK in cardiomyocyte function. Chronic 10-day exposure (2 μM and 10 μM MK-8722) of iCell cardiomyocyte 2D monolayers, probed via monitoring of cellular impedance (CardioECR, Agilent), revealed a decrease in beat amplitude (-13%,2μM;-49%,10μM) as well as an increase in excitability, based on the ability to follow higher pacing frequencies (+81%,2uM;+34%,10uM). Direct force measurements in 3D iCell CM tissues (Biowire II, TARA Biosystems) reported loss of contractility when stimulated at 1Hz (-74%,2μM;-90%,10 μM), as well as changes in passive resting tension (+18%,2μM;-75%,10 μM) over chronic six-week exposure. MK-8722 caused a marked increase in Biowire excitability as revealed by a large drop in the voltage threshold of entrainment of paced tissues (-36%,2μM;-43%,10 μM). The increased excitability of both 2D and 3D CM models suggests a cellular correlate of the increased electrocardiographic QRS amplitude observed in rhesus monkeys upon chronic dosing with MK-8722 # . Hypertrophy of the Biowires could be clearly observed at the end of the 6-week incubation and presented as increased microtissue widths (+5%,2μM; +10%,10μM) and thickened regions of attachment to the chamber force sensors at each end of the Biowire. Volume estimation revealed increases (+22.3%, 2μM; +31.4%, 10 μM) in Biowire tissue volumes that were comparable to heart hypertrophic changes in preclinical test species # (mouse, rat, and rhesus monkey). The effect of 10μM MK-8722 was more pronounced when treatment began before tissues had completed a multi-week electrical stimulation maturation protocol, rather than after. The functional effects in both 2D and 3D CM models, and the contractile and structural readouts of the 3D Biowire model were highly informative in the interpretation of the clinical applicability of the pleiotropic cardiac effects of MK-8722 observed in preclinical testing. # Myers, et al. Science 357, p 507-511 (2017)