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.

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.

In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-β treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275±36 ms and conduction velocity (CV) of 10.6±4.2 cm/s (n=3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4±0.9 cm/s, n=35 vs. 30.1±3.2 cm/s, n=20 in the longitudinal direction and 4.3±0.3 cm/s vs. 9.3±0.9 cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6±1.3 cm/s and 3.1± 0.5 cm/s, n=11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.