[Show abstract][Hide abstract] ABSTRACT: Human pluripotent stem cell derivatives show promise as an in vitro platform to study a range of human cardiovascular diseases. A better understanding of the biology of stem cells and their cardiovascular derivatives will help to understand the strengths and limitations of this new model system. G-protein coupled receptors (GPCRs) are key regulators of stem cell maintenance and differentiation and have an important role in cardiovascular cell signaling. In this review, we will therefore describe the state of knowledge concerning the regulatory role of GPCRs in both the generation and function of pluripotent stem cell derived-cardiomyocytes, -endothelial, and -vascular smooth muscle cells. We will consider how far the in vitro disease models recapitulate authentic GPCR signaling and provide a useful basis for discovery of disease mechanisms or design of therapeutic strategies.
Full-text · Article · Dec 2015 · Frontiers in Cell and Developmental Biology
[Show abstract][Hide abstract] ABSTRACT: Oblique plane microscopy (OPM) is a form of light sheet microscopy that uses a single high numerical aperture microscope objective for both fluorescence excitation and collection. In this paper, measurements of the relative collection efficiency of OPM are presented. An OPM system incorporating two sCMOS cameras is then introduced that enables single isolated cardiac myocytes to be studied continuously for 22 seconds in two dimensions at 667 frames per second with 960 × 200 pixels and for 30 seconds with 960 × 200 × 20 voxels at 25 volumes per second. In both cases OPM is able to record in two spectral channels, enabling intracellular calcium to be studied via the probe Fluo-4 AM simultaneously with the sarcolemma and transverse tubule network via the membrane dye Cellmask Orange. The OPM system was then applied to determine the spatial origin of spontaneous calcium waves for the first time and to measure the cell transverse tubule structure at their point of origin. Further results are presented to demonstrate that the OPM system can also be used to study calcium spark parameters depending on their relationship to the transverse tubule structure.
Full-text · Article · Oct 2015 · Journal of Biophotonics
[Show abstract][Hide abstract] ABSTRACT: Heart Failure is still a major cause of hospitalization and mortality in developed countries. Many clinical trials have tested the use of multipotent stem cells as a cardiac regenerative medicine. The benefit for the patients of this therapeutic intervention has remained limited. Herein, we review the pluripotent stem cells as a cell source for cardiac regeneration. We more specifically address the various challenges of this cell therapy approach. We question the cell delivery systems, the immune tolerance of allogenic cells, the potential pro-arrhythmic effects, various drug mediated interventions to facilitate cell grafting and, finally, we describe the pathological conditions that may benefit from such an innovative approach. As members of a transatlantic consortium of excellence of basic science researchers and clinicians, we propose some guidelines to be applied to cell types and modes of delivery in order to translate pluripotent stem cell cardiac derivatives into safe and effective clinical trials. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: We are developing an autofluorescence (AFL) lifetime-based technique to characterise the signatures associated with histological, metabolic and functional changes in myocardial disease. AFL spectroscopy exploits the properties of a number of endogenous molecules and offers the potential to avoid exogenous label use that could potentially alter the physiological environment. AFL measurements of molecules such as NADH and flavins may report energetic state changes, whilst signatures from matrix such as collagen inform of structural alterations. We investigated the application of a fibre-optic based single-point instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to a doxorubicin cardiomyopathy heart failure model (DOX-HF) in vivo.
Sprague-Dawley male rats received 1.25 mg/kg doxorubicin or vehicle (0.9% NaCl) via tail vein injection weekly for 8 weeks. AFL signals were measured in vivo in week 11, once heart failure phenotype was well established. DOX-HF(n = 8) LVEF 49.6% vs. 80.4% in controls (n = 4)(**p = 0.006). At 11 weeks, significant differences in AFL signals between DOX-HF(n = 6) and control (n = 3) in LV, RV and LV posterior wall were observed (*p < 0.05). A significantly increased proportion of collagen AFL was seen across all areas in DOX-HF (n = 6) vs. control (n = 3)(*p < 0.05). AFL-predicted collagen content was previously highly correlated with quantitative histology (r = 0.984,*p < 0.05).
Our instrument has sensitivity to characterise in vivo changes in DOX-HF model without use of exogenous compounds. Incorporation into coronary catheters or pacing leads could offer additional diagnostic information and monitoring. Identification of earlier changes could permit development of a screening strategy to identify cancer patients developing cardiomyopathy to permit judicious adjustment of chemotherapy.
No preview · Article · Jun 2015 · Heart (British Cardiac Society)
[Show abstract][Hide abstract] ABSTRACT: Huntington disease (HD; OMIM 143100), a progressive neurodegenerative disorder, is caused by an expanded trinucleotide CAG (polyQ) motif in the HTT gene. Cardiovascular symptoms, often present in early stage HD patients, are, in general, ascribed to dysautonomia. However, cardio-specific expression of polyQ peptides caused pathological response in murine models, suggesting the presence of a nervous system-independent heart phenotype in HD patients. A positive correlation between the CAG repeat size and severity of symptoms observed in HD patients has also been observed in in vitro HD cellular models. Here, we test the suitability of human embryonic stem cell (hESC) lines carrying HD-specific mutation as in vitro models for understanding molecular mechanisms of cardiac pathology seen in HD patients. We have differentiated three HD-hESC lines into cardiomyocytes and investigated CAG stability up to 60 days after starting differentiation. To assess CAG stability in other tissues, the lines were also subjected to in vivo differentiation into teratomas for 10 weeks. Neither directed differentiation into cardiomyocytes in vitro nor in vivo differentiation into teratomas, rich in immature neuronal tissue, led to an increase in the number of CAG repeats. Although the CAG stability might be cell line-dependent, induced pluripotent stem cells generated from patients with larger numbers of CAG repeats could have an advantage as a research tool for understanding cardiac symptoms of HD patients.
[Show abstract][Hide abstract] ABSTRACT: Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT–PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα+ cells. Clonal progeny of single Sca1+ SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα− cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα+/CD31− cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα−/CD31+ cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1+ stem/progenitor cell.
Supplementary information available for this article at http://www.nature.com/ncomms/2015/150518/ncomms7930/suppinfo/ncomms7930_S1.html
Full-text · Article · May 2015 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: The use of anthracyclines such as doxorubicin (DOX) has improved outcome in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity is frequently irreversible and typically progresses to heart failure (HF) but our understanding of molecular mechanisms underlying this and essential for development of cardioprotective strategies remains largely obscure. As microRNAs (miRNAs) have been shown to play potent regulatory roles in both cardiovascular disease and cancer, we investigated miRNA changes in DOX-induced HF and the alteration of cellular processes downstream. Myocardial miRNA profiling was performed after DOX-induced injury, either via acute application to isolated cardiomyocytes or via chronic exposure in vivo, and compared with miRNA profiles from remodeled hearts following myocardial infarction. The miR-30 family was downregulated in all three models. We describe here that miR-30 act regulating the β-adrenergic pathway, where preferential β1- and β2-adrenoceptor (β1AR and β2AR) direct inhibition is combined with Giα-2 targeting for fine-tuning. Importantly, we show that miR-30 also target the pro-apoptotic gene BNIP3L/NIX. In aggregate, we demonstrate that high miR-30 levels are protective against DOX toxicity and correlate this in turn with lower reactive oxygen species generation. In addition, we identify GATA-6 as a mediator of DOX-associated reductions in miR-30 expression. In conclusion, we describe that DOX causes acute and sustained miR-30 downregulation in cardiomyocytes via GATA-6. miR-30 overexpression protects cardiac cells from DOX-induced apoptosis, and its maintenance represents a potential cardioprotective and anti-tumorigenic strategy for anthracyclines.
Preview · Article · May 2015 · Cell Death & Disease
[Show abstract][Hide abstract] ABSTRACT: Gene therapy has been applied to cardiovascular disease for over 20 years but it is the application to heart failure which has generated recent interest in clinical trials. There is laboratory, and early clinical evidence that delivery of SERCA2a gene therapy is beneficial for heart failure and this therapy could become the first positive inotrope with anti-arrhythmic properties. In this review we will discuss the rationale for SERCA2a gene therapy as a viable strategy in heart failure, review the published data and discuss the on-going clinical trials, before concluding with comments on the future challenges and potential for this therapy.
No preview · Article · Apr 2015 · Human gene therapy
[Show abstract][Hide abstract] ABSTRACT: Stem cells have emerged as a justifiable and promising medical therapy for patients with heart failure. Many cell types have been contemplated during the last years, and there is promising preclinical and clinical data on their efficacy and safety in the setting of acute myocardial infarction and chronic heart failure. However, there are still many hurdles to be overcome for the routine clinical application of these cells. A better understanding of the biology of stem cells and their cardiovascular derivatives will help in the design of future strategies for cardiac regeneration.
[Show abstract][Hide abstract] ABSTRACT: Rationale Cardiomyocyte cell death plays a causal role in heart failure therefore its inhibition poses a promising therapeutic approach. However, investment is undermined due to poor translation and cardiotoxicity. Here, we describe validation of a potential target; mitogen activated protein 4 kinase kinase kinase kinase (MAP4K4).
Methodology/Results 4 weeks pressure overload (TAC; transverse aortic constriction) in alphaMyHC-MAP4K4 mice (cardiomyocyte-specific over-expression) induced apoptosis (3.60 %+1.01 TUNEL-positive cardiomyocytes, n=5, p<0.05 to wildtype (WT) littermates; 0.53 %+0.19, n=5). Peri-vascular fibrosis and upregulation of autophagy markers Beclin1 and LC3 (associated with heart failure) was evident only in TG-TAC mice (p<0.05 to WT-TAC). Systolic function assessed by pressure-volume loops (shams n=6, TAC-TG n=9, TAC-WT n=10) showed unaffected contractility and compliance in WT-TAC mice although relaxation was impaired (Tau, p<0.05). In contrast, TG-TAC mice had significantly impaired compliance (Emax, p<0.01), contractility (Ees-ESPVR, p<0.01) and relaxation (Tau, p<0.01).
Inhibition of MAP4K4, by siRNA-mediated knockdown or pharmacologically (selective in-house novel compound; 5 out of 141 kinases inhibited) was cardioprotective in rat neonatal and human iPSC-derived cardiomyocytes subjected to oxidative stress (H202) or C2-ceramide (measured by loss of membrane integrity, Caspase 3 activity, hypodiploid DNA).
Conclusions MAP4K4 drives cardiomyocyte cell death and dysfunction, thus poses an attractive target in heart failure. Our novel compound, validated in human and mouse models, represents a start-point for further development with improved translational potential.
No preview · Article · Dec 2014 · Heart (British Cardiac Society)
[Show abstract][Hide abstract] ABSTRACT: Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the ?-adrenoceptor (?AR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal ?1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.
Full-text · Article · Nov 2014 · Stem Cell Reports
[Show abstract][Hide abstract] ABSTRACT: Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet. We studied PI3K-FOXO1A pathways during differentiation of hESC towards endothelial lineage and on proliferation, maturation and cell death of hESC-derived endothelial cells (hESC-EC). During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodelling-related genes. PI3K inhibitor LY294002 activated FOXO1A and induced formation of CD31+ hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by siRNA showed lower mRNA levels of CD31 and angiopoietin2. LY294002 decreased proliferative activity of purified hESC-EC, whilst FOXO1A siRNA increased their proliferation. LY294002 inhibits migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. After in vivo conditioning of cells in athymic nude rats, cells retain their low FOXO1A expression levels. PI3K/FOXO1A pathway is important for function and survival of hESC-EC as well as in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs.
No preview · Article · Nov 2014 · Stem Cells and Development
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells form a highly specialised lining of all blood vessels where they provide an anti-thrombotic surface on the luminal side and protect the underlying vascular smooth muscle on the abluminal side. Specialised functions of endothelial cells include their unique ability to release vasoactive hormones and to morphologically adapt to complex shear stress. Stem cell derived-endothelial cells have a growing number of applications and will be critical in any organ regeneration programme. Generally endothelial cells are identified in stem cell studies by well-recognised markers such as CD31. However, the ability of stem cell-derived endothelial cells to release vasoactive hormones and align with shear stress has not been studied extensively. With this in mind, we have compared directly the ability of endothelial cells derived from a range of stem cell sources, including embryonic stem cells (hESC-EC) and adult progenitors in blood (blood out growth endothelial cells, BOEC) with those cultured from mature vessels, to release the vasoconstrictor peptide endothelin (ET)-1, the cardioprotective hormone prostacyclin, and to respond morphologically to conditions of complex shear stress. All endothelial cell types, except hESC-EC, released high and comparable levels of ET-1 and prostacyclin. Under static culture conditions all endothelial cell types, except for hESC-EC, had the typical cobblestone morphology whilst hESC-EC had an elongated phenotype. When cells were grown under shear stress endothelial cells from vessels (human aorta) or BOEC elongated and aligned in the direction of shear. By contrast hESC-EC did not align in the direction of shear stress. These observations show key differences in endothelial cells derived from embryonic stem cells versus those from blood progenitor cells, and that BOEC are more similar than hESC-EC to endothelial cells from vessels. This may be advantageous in some settings particularly where an in vitro test bed is required. However, for other applications, because of low ET-1 release hESC-EC may prove to be protected from vascular inflammation.
No preview · Article · Nov 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Aims
During cardiac hypertrophy, cardiomyocytes (CMs) increase in the size and expression of cytoskeletal proteins while reactivating a foetal gene programme. The process is proposed to be dependent on increased nuclear export and, since nuclear pore trafficking has limited capacity, a linked decrease in import. Our objective was to investigate the role of nuclear import and export in control of hypertrophy in rat and human heart failure (HF).
Methods and results
In myocardial tissue and isolated CMs from patients with dilated cardiomyopathy, nuclear size was increased; Nucleoporin p62, cytoplasmic RanBP1, and nuclear translocation of importins (α and β) were decreased while Exportin-1 was increased. CM from a rat HF model 16 weeks after myocardial infarction (MI) reproduced these nuclear changes. Nuclear import, determined by the rate of uptake of nuclear localization sequence (NLS)-tagged fluorescent substrate, was also decreased and this change was observed from 4 weeks after MI, before HF has developed. Treatment of isolated rat CMs with phenylephrine (PE) for 48 h produced similar cell and nuclear size increases, nuclear import and export protein rearrangement, and NLS substrate uptake decrease through p38 MAPK and HDAC-dependent pathways. The change in NLS substrate uptake occurred within 15 min of PE exposure. Inhibition of nuclear export with leptomycin B reversed established nuclear changes in PE-treated rat CMs and decreased NLS substrate uptake and cell/nuclear size in human CMs.
Nuclear transport changes related to increased export and decreased import are an early event in hypertrophic development. Hypertrophy can be prevented, or even reversed, by targeting import/export, which may open new therapeutic opportunities.
Full-text · Article · Oct 2014 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: In Takotsubo cardiomyopathy, the left ventricle shows apical ballooning combined with basal hypercontractility. Both clinical observations in humans and recent experimental work on isolated rat ventricular myocytes suggest the dominant mechanisms of this syndrome are related to acute catecholamine overload. However, relating observed differences in single cells to the capacity of such alterations to result in the extreme changes in ventricular shape seen in Takotsubo syndrome is difficult.
By using a computational model of the rat left ventricle, we investigate which mechanisms can give rise to the typical shape of the ventricle observed in this syndrome.
Three potential dominant mechanisms related to effects of beta-adrenergic stimulation were considered: apical-basal variation of calcium transients due to differences in L-type and SERCA activation, apical-basal variation of calcium sensitivity due to differences in troponin I phosphorylation, and apical-basal variation in maximal active tension due to e.g. the negative inotropic effects of p38 MAPK. Furthermore, we investigated the interaction of these spatial variations in the presence of a failing Frank-Starling mechanism.
We conclude that a large portion of the apex needs to be affected by severe changes in calcium regulation or contractile function to result in apical ballooning, and smooth linear variation from apex to base are unlikely to result in the typical ventricular shape observed in this syndrome. A failing Frank-Starling mechanism significantly increases apical ballooning at end-systole, and may be an important additional factor underpinning Takotsubo syndrome.
[Show abstract][Hide abstract] ABSTRACT: Cardiac progenitor/stem cells in adult hearts are a potential mode of self-repair, therapeutic product, and target for activation in situ, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT-PCR and clonal analyses, we define four sub-populations in adult mouse myocardium sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, platelet/endothelial cell adhesion molecule 1 (CD31), and platelet derived growth factor receptor-α (PDGFRα). SP status predicted clonogenicity plus cardiogenic genes' expression (Gata4/6, Hand2, Tbx5/20), properties segregating more specifically to PDGFRα+ cells. PDGFRα- cells were characterized, instead, by Kdr/Flk1, Cdh5, CD31 and lack of clone formation. Clonal progeny of single Sca1+ SP cells showed tri-lineage potential (cardiomyocyte, endothelial, smooth muscle) after cardiac grafting. By fate-mapping, PDGFRα+ CD31- cells derived from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα- CD31+ cells (Gata5, Wt1 low; Flk1, Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca-1+ stem cell.
Full-text · Article · Jul 2014 · Cardiovascular Research
[Show abstract][Hide abstract] ABSTRACT: Rationale: The use of anthracyclines such as doxorubicin (DOX) has improved mortality and morbidity in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity is frequently irreversible and typically progresses to heart failure (HF). However, our understanding of molecular mechanisms essential for development of cardioprotective strategies and to predict HF remain largely obscure.
Objective: As microRNAs (miRNAs) have been shown to play potent regulatory roles in both cardiovascular disease and cancer, we investigated miRNA changes in chemotherapy-induced HF and the alteration of cellular processes downstream.
Methods and results: Myocardial miRNA profiling was performed after DOX-induced injury, either via acute application to isolated cardiomyocytes in vitro or chronic exposure in vivo, and also compared with miRNA profiles from remodeled hearts following myocardial infarction; the miR-30 family was down-regulated in all three models. We confirmed β1- and β2-adrenoceptors (β1AR, β2AR), Gi alpha 2 (Giα-2) and the pro-apoptotic gene BNIP3L/NIX as miR-30 targets. We describe regulation of the β-adrenergic pathway by miR-30, where a preferential βAR inhibition results in a β-blocker like effect. We demonstrate that high miR-30 levels are protective against DOX insult and correlate with lower reactive oxygen species (ROS) generation. Moreover, we identify GATA-6 as a mediator of the DOX-associated reduction in miR-30 expression.
Conclusion: DOX causes acute and sustained miR-30 down-regulation in cardiomyocytes via GATA-6. miR-30 overexpression protected cardiac cells from DOX-induced apoptosis, and its maintenance represents a potential cardioprotective strategy for anthracycline cardiomyopathy. Additionally, intriguing synergic anti-cancer effects were observed for miR-30.
Preview · Article · Jul 2014 · Cardiovascular Research