Christopher Kane

Action Potential Morphology of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Does Not Predict Cardiac Chamber Specificity and Is Dependent on Cell Density

Previous studies investigating human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have proposed the distinction of heart chamber-specific (atrial, ventricular, pacemaker) electrophysiological phenotypes based on action potential (AP) morphology. This suggestion has been based on data acquired using techniques that allow measurements from only a small number of cells and at low seeding densities. It has also been observed that density of culture affects the properties of iPSC-CMs. Here we systematically analyze AP morphology from iPSC-CMs at two seeding densities: 60,000 cells/well (confluent monolayer) and 15,000 cells/well (sparsely-seeded) using a noninvasive optical method. The confluent cells (n = 360) demonstrate a series of AP morphologies on a normally distributed spectrum with no evidence for specific subpopulations. The AP morphologies of sparsely seeded cells (n = 32) displayed a significantly different distribution, but even in this case there is no clear evidence of chamber-specificity. Reduction in gap junction conductance using carbenoxolone only minimally affected APD distribution in confluent cells. These data suggest that iPSC-CMs possess a sui generis AP morphology, and when observed in different seeding densities may encompass any shape including those resembling chamber-specific subtypes. These results may be explained by different functional maturation due to culture conditions.

Functional crosstalk between cardiac fibroblasts and adult cardiomyocytes by soluble mediators

AIMSnCrosstalk between cardiomyocytes and fibroblasts in physiological conditions and during disease remains poorly defined. Previous studies have shown that fibroblasts and myocytes interact via paracrine communication, but several experimental confounding factors, including the use of immature myocytes and the induction of alpha-smooth muscle actin (α-SMA) expression in fibroblasts by prolonged culture, have hindered our understanding of this phenomenon. We hypothesize that fibroblasts and myofibroblasts differentially affect cardiomyocytes viability, volume, and Ca(2+) handling via soluble mediators. More specifically here: (i) we compare the effects of freshly isolated fibroblasts and cultured fibroblasts from normal rat hearts on adult cardiomyocytes; (ii) we compare the effects of (freshly isolated) normal fibroblasts and myofibroblasts from pressure-overloaded hearts; and (iii) we study the contribution of TGF-β and the importance of the crosstalk between the two cell types.nnnMETHODS AND RESULTSnWe used co-culture methods and conditioned medium to investigate paracrine interaction between fibroblasts and cardiomyocytes. All fibroblast types reduce cardiomyocyte viability and increase cardiomyocyte volume but α-SMA-negative fibroblasts increase cardiomyocyte Ca(2+) transient amplitude, whereas cultured fibroblasts and myofibroblasts from pressure-overloaded hearts decrease Ca(2+) transient amplitude. In turn, cardiomyocytes release soluble mediators that affect fibroblast proliferation. Using SB431542 to block TGF-β type 1 receptors, we determined that TGF-β directly causes cardiomyocyte hypertrophy and participates in bi-directional regulatory signalling between fibroblasts and cardiomyocytes.nnnCONCLUSIONSnFibroblasts have different roles during physiology and disease in regulating myocardial function via soluble mediators. A crosstalk between fibroblasts and cardiomyocytes, controlled by TGF-β, is crucial in this interaction.

Excitation–contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold enormous potential in many fields of cardiovascular research. Overcoming many of the limitations of their embryonic counterparts, the application of iPSC-CMs ranges from facilitating investigation of familial cardiac disease and pharmacological toxicity screening to personalized medicine and autologous cardiac cell therapies. The main factor preventing the full realization of this potential is the limited maturity of iPSC-CMs, which display a number of substantial differences in comparison to adult cardiomyocytes. Excitation–contraction (EC) coupling, a fundamental property of cardiomyocytes, is often described in iPSC-CMs as being more analogous to neonatal than adult cardiomyocytes. With Ca2+ handling linked, directly or indirectly, to almost all other properties of cardiomyocytes, a solid understanding of this process will be crucial to fully realizing the potential of this technology. Here, we discuss the implications of differences in EC coupling when considering the potential applications of human iPSC-CMs in a number of areas as well as detailing the current understanding of this fundamental process in these cells.

The Fallacy of Assigning Chamber Specificity to iPSC Cardiac Myocytes from Action Potential Morphology

The production and identification of chamber-specific, induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) is a crucial step in the application of this technology in physiology, pharmacology, and regenerative studies. The significance of toxicology tests would be strongly diminished if, for instance, drugs for atrial fibrillation were tested in ventricular myocytes or pathophysiological mechanisms involved in hypertrophic cardiomyopathy were studied in atrial myocytes. One could predict that the transplantation of nodal myocytes in the infarcted ventricle would have a strong arrhythmogenic potential and could be disastrous in the setting of myocardial repair strategies.

Concise Review: Criteria for Chamber‐Specific Categorization of Human Cardiac Myocytes Derived from Pluripotent Stem Cells

Human pluripotent stem cell‐derived cardiomyocytes (PSC‐CMs) have great potential application in almost all areas of cardiovascular research. A current major goal of the field is to build on the past success of differentiation strategies to produce CMs with the properties of those originating from the different chambers of the adult human heart. With no anatomical origin or developmental pathway to draw on, the question of how to judge the success of such approaches and assess the chamber specificity of PSC‐CMs has become increasingly important; commonly used methods have substantial limitations and are based on limited evidence to form such an assessment. In this article, we discuss the need for chamber‐specific PSC‐CMs in a number of areas as well as current approaches used to assess these cells on their likeness to those from different chambers of the heart. Furthermore, describing in detail the structural and functional features that distinguish the different chamber‐specific human adult cardiac myocytes, we propose an evidence‐based tool to aid investigators in the phenotypic characterization of differentiated PSC‐CMs. Stem Cells 2017;35:1881–1897

Investigation of cardiac fibroblasts using myocardial slices

Abstract Aims Cardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro, CFs undergo rapid phenotypic changes including increased expression of α-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices. Methods and results Unloading of myocardial slices induced CF proliferation without α-SMA expression up to 7u2009days in culture. CFs migrating onto the culture plastic support or cultured on glass expressed αSMA within 3u2009days. The cells on the slice remained αSMA(−) despite transforming growth factor-β (20u2009ng/ml) or angiotensin II (200u2009µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at Days 3 and 7 (P < 0.001). Conclusions Myocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential targets.

Criteria for chamber-specific categorisation of human cardiac myocytes derived from pluripotent stem cells

Human pluripotent stem cell‐derived cardiomyocytes (PSC‐CMs) have great potential application in almost all areas of cardiovascular research. A current major goal of the field is to build on the past success of differentiation strategies to produce CMs with the properties of those originating from the different chambers of the adult human heart. With no anatomical origin or developmental pathway to draw on, the question of how to judge the success of such approaches and assess the chamber specificity of PSC‐CMs has become increasingly important; commonly used methods have substantial limitations and are based on limited evidence to form such an assessment. In this article, we discuss the need for chamber‐specific PSC‐CMs in a number of areas as well as current approaches used to assess these cells on their likeness to those from different chambers of the heart. Furthermore, describing in detail the structural and functional features that distinguish the different chamber‐specific human adult cardiac myocytes, we propose an evidence‐based tool to aid investigators in the phenotypic characterization of differentiated PSC‐CMs. Stem Cells 2017;35:1881–1897

Human Cardiac Fibroblasts Engage the Sarcoplasmic Reticulum in Induced Pluripotent Stem Cell-Derived Cardiomyocyte Excitation–Contraction Coupling

Ca2+-induced Ca2+ release (CICR), the process whereby a small influx of Ca2+ across the sarcolemma triggers a significantly larger Ca2+ release from the sarcoplasmic reticulum (SR), is at the heart of efficient cardiac excitation–contraction coupling (ECC). This process is rudimentary in neonatal