Heart tissue possesses complex structural organization on multiple scales, from macro- to nano-, but nanoscale control of cardiac function has not been extensively analyzed. Inspired by ultrastructural analysis of the native tissue, we constructed a scalable, nanotopographically controlled model of myocardium mimicking the in vivo ventricular organization. Guided by nanoscale mechanical cues provided by the underlying hydrogel, the tissue constructs displayed anisotropic action potential propagation and contractility characteristic of the native tissue. Surprisingly, cell geometry, action potential conduction velocity, and the expression of a cell–cell coupling protein were exquisitely sensitive to differences in the substratum nanoscale features of the surrounding extracellular matrix. We propose that controlling cell–material interactions on the nanoscale can stipulate structure and function on the tissue level and yield novel insights into in vivo tissue physiology, while providing materials for tissue repair.

BackgroundThe production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods.Methodology/Principal FindingsWe systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.Conclusion/SignificanceThis efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

Introduction: Inflammation is a prominent feature of arrhythmogenic cardiomyopathy (ACM), but whether it contributes to the disease phenotype is not known. To define the role of inflammation in the pathogenesis of ACM, we characterized effects of inhibition of inflammatory signaling in ACM models in vitro and in vivo, and in cardiac myocytes from patient induced pluripotent stem cells (hiPSCs). Results: Activation of NFκB signaling, indicated by increased expression and nuclear accumulation of phospho-RelA/p65, occurs in both an in vitro model of ACM (expression of JUP 2157del2 in neonatal rat ventricular myocytes), and in a robust murine model of ACM (homozygous knock-in of mutant desmoglein-2; Dsg2 mut/mut) that recapitulates the cardiac manifestations seen in ACM patients. Bay 11-7082, a small molecule inhibitor of NFκB signaling, prevented development of ACM disease features in vitro (abnormal redistribution of intercalated disk proteins, myocyte apoptosis, release of inflammatory cytokines) and in vivo (myocardial necrosis and fibrosis, LV contractile dysfunction, ECG abnormalities). Hearts of Dsg2 mut/mut mice expressed markedly increased levels of inflammatory cytokines and chemotactic molecules which were attenuated by Bay 11-7082. Salutary effects of Bay 11-7082 correlated with the extent to which production of selected cytokines had been blocked. NFκB signaling was also activated in cardiac myocytes derived from a patient with ACM. These cells produced and secreted abundant inflammatory cytokines under basal conditions, and this was also greatly reduced by Bay 11-7082. Conclusions: Inflammatory signaling is activated in ACM and it drives key features of the disease. Targeting inflammatory pathways may be an effective new mechanism-based therapy for ACM.

Abstract-Skeletal myoblasts are an attractive cell type for transplantation because they are autologous and resistant to ischemia. However, clinical trials of myoblast transplantation in heart failure have been plagued by ventricular tachyarrhythmias and sudden cardiac death. The pathogenesis of these arrhythmias is poorly understood, but may be related to the fact that skeletal muscle cells, unlike heart cells, are electrically isolated by the absence of gap junctions. Using a novel in vitro model of myoblast transplantation in cardiomyocyte monolayers, we investigated the mechanisms of transplant-associated arrhythmias. Cocultures of human skeletal myoblasts and rat cardiomyocytes resulted in reentrant arrhythmias (spiral waves) that reproduce the features of ventricular tachycardia seen in patients receiving myoblast transplants. These arrhythmias could be terminated by nitrendipine, an L-type calcium channel blocker, but not by the Na channel blocker lidocaine. Genetic modification of myoblasts to express the gap junction protein connexin43 decreased arrhythmogenicity in cocultures, suggesting a specific means for increasing the safety (and perhaps the efficacy) of myoblast transplantation in patients.