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.

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.

Background-Mesenchymal stem cells (MSCs) are bone marrow stromal cells that are in phase 1 clinical studies of cellular cardiomyoplasty. However, the electrophysiological effects of MSC transplantation have not been studied. Although improvement of ventricular function would represent a positive outcome of MSC transplantation, focal application of stem cells has the potential downside of creating inhomogeneities that may predispose the heart to reentrant arrhythmias. In the present study we use an MSC and neonatal rat ventricular myocyte (NRVM) coculture system to investigate potential proarrhythmic consequences of MSC transplantation into the heart. Methods and Results-Human MSCs were cocultured with NRVMs in ratios of 1:99, 1:9, and 1:4 and optically mapped.We found that conduction velocity was decreased in cocultures compared with controls, but action potential duration (APD 80 ) was not affected. Reentrant arrhythmias were induced in 86% of cocultures containing 10% and 20% MSCs (nϭ36) but not in controls (nϭ7) or cocultures containing only 1% MSCs (nϭ4). Immunostaining, Western blot, and dye transfer revealed the presence of functional gap junctions involving MSCs. Conclusions-Our results suggest that mixtures of MSCs and NRVMs can produce an arrhythmogenic substrate. The mechanism of reentry is probably increased tissue heterogeneity resulting from electric coupling of inexcitable MSCs with myocytes.

Abstract-Structural and functional cardiac anisotropy varies with the development, location, and pathophysiology in the heart. The goal of this study was to design a cell culture model system in which the degree, change in fiber direction, and discontinuity of anisotropy can be controlled over centimeter-size length scales. Neonatal rat ventricular myocytes were cultured on fibronectin on 20-mm diameter circular cover slips. Structure-function relationships were assessed using immunostaining and optical mapping. Cell culture on microabraded cover slips yielded cell elongation and coalignment in the direction of abrasion, and uniform, macroscopically continuous, elliptical propagation with point stimulation. Coarser microabrasion (wider and deeper abrasion grooves) increased longitudinal (23.5 to 37.2 cm/s; rϭ0.66) and decreased transverse conduction velocity (18.1 to 9.2 cm/s; rϭϪ0.84), which resulted in increased longitudinal-to-transverse velocity anisotropy ratios (1.3 to 3.7, nϭ61). A thin transition zone between adjacent uniformly anisotropic areas with 45°or 90°difference in fiber orientation acted as a secondary source during 2ϫ threshold field stimulus. Cell culture on cover slips micropatterned with 12-or 25-m wide fibronectin lines and previously coated with decreasing concentrations of background fibronectin yielded transition from continuous to discontinuous anisotropic architecture with longitudinally oriented intercellular clefts, decreased transverse velocity (16.9 to 2.6 cm/s; rϭϪ0.95), increased velocity anisotropy ratios (1.6 to 5.6, nϭ70), and decreased longitudinal velocity (36.4 to 14.6 cm/s; rϭϪ0.85) for anisotropy ratios Ͼ3.5. Cultures of cardiac myocytes with controlled degree, uniformity and continuity of structural, and functional anisotropy may enable systematic 2-dimensional in vitro studies of macroscopic structure-related mechanisms of reentrant arrhythmias.

We have developed a novel method to deliver stem cells using 3D bioprinted cardiac patches, free of biomaterials. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), fibroblasts (FB) and endothelial cells (EC) were aggregated to create mixed cell spheroids. Cardiac patches were created from spheroids (CM:FB:EC = 70:15:15, 70:0:30, 45:40:15) using a 3D bioprinter. Cardiac patches were analyzed with light and video microscopy, immunohistochemistry, immunofluorescence, cell viability assays and optical electrical mapping. Cardiac tissue patches of all cell ratios beat spontaneously after 3D bioprinting. Patches exhibited ventricular-like action potential waveforms and uniform electrical conduction throughout the patch. Conduction velocities were higher and action potential durations were significantly longer in patches containing a lower percentage of FBs. Immunohistochemistry revealed staining for CM, FB and EC markers, with rudimentary CD31+ blood vessel formation. Immunofluorescence revealed the presence of Cx43, the main cardiac gap junction protein, localized to cell-cell borders. In vivo implantation suggests vascularization of 3D bioprinted cardiac patches with engraftment into native rat myocardium. This constitutes a significant step towards a new generation of stem cell-based treatment for heart failure.

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.

Background Following cardiac injury, activated cardiac myofibroblasts can influence tissue electrophysiology. Because mechanical coupling through adherens junctions provides a route for intercellular communication, we tested the hypothesis that myofibroblasts exert tonic contractile forces on the cardiomyocytes and affect electrical propagation via a process of mechanoelectric feedback. Methods and Results The role of mechanoelectric feedback was examined in transforming growth factor-beta (TGF-β) treated monolayers of co-cultured myofibroblasts and neonatal rat ventricular cells by inhibiting myofibroblast contraction and blocking mechanosensitive channels (MSCs). Untreated (control) and TGF-β treated (fibrotic) anisotropic monolayers were optically mapped for electrophysiological comparison. Longitudinal conduction velocity (LCV), transverse conduction velocity (TCV), and normalized action potential upstroke velocity (dV/dtmax) significantly decreased in fibrotic monolayers (14.4±0.7 (SEM) cm/s, 4.1±0.3 cm/s, n=53, and 3.1±0.2, n=14, respectively) compared with control monolayers (27.2±0.8 cm/s, 8.5±0.4 cm/s, n=40, and 4.9±0.1, n=12, respectively). Application of the excitation-contraction uncoupler, blebbistatin, or the MSC blocker, gadolinium or streptomycin, dramatically increased LCV, TCV and dV/dtmax in fibrotic monolayers (36±1 cm/s, 10±0.3 cm/s, n=17, and 4.5±0.1, n=14, respectively). Similar results were observed with Cx43-silenced cardiac myofibroblasts. Spiral wave induction in fibrotic monolayers also decreased following the aforementioned treatments. Finally, traction force measurements of individual myofibroblasts showed a significant increase with TGF-β, decrease with blebbistatin, and no change with MSC blockers. Conclusions These observations suggest that myofibroblast-myocyte mechanical interactions develop during cardiac injury, and that cardiac conduction may be impaired as a result of increased MSC activation owing to tension that is applied to the myocyte by the myofibroblast.