UC Santa Barbara

Introduction: Heart function relies on the contractility of its muscle cells (cardiomyocytes). Human pluripotent stem cells (hPSC) can be differentiated into cardiomyocytes (hPSC-CMs). However, while their myogenic maturity increases with time, they do not resemble adult cardiomyocytes in their morphology, structural organization, mechanical output and electrophysiology. The low maturity of hPSC-CMs limits their potential to model, study and treat heart diseases. Hypothesis: Since the organization of sarcomeres regulates the mechanical output of cardiomyocytes, we hypothesized that enhanced sarcomere maturity correlates with the mechanical output of these cells and the physiology of matured cardiomyocytes. Methods: We cultured single hPSC-CMs on 2000 μm2 rectangular protein patterns on polyacrylamide substrates with a modulus of 10 kPa to resemble the morphology of ventricular cardiomyocytes and match healthy adult ventricular stiffness. We infected seeded cells with rAV CAG-LifeAct-Tag RFP to label actin and image sarcomeres in contracting live cells. We estimated forces generated during single-cell contractile events with traction force microscopy and calculated power (force x cell shortening velocity). We varied cell elongation by changing the cell aspect ratio (1:1 to 7:1) and maintaining constant area. We used substrates having stiffness of 6 kPa (embryonic myocardium), 10 kPa (adult) and 35 kPa (infarcted myocardium). Results: Optimized power output was observed in cells with a 7:1 aspect ratio. The size, organization and dynamics of sarcomeres in these elongated hPSC-CMs are more mature. Optimized power output was observed in cells with a 7:1 aspect ratio. The power generated by hPSC-CMs on 35 kPa substrates was significantly reduced, while reduced variations in the power output across different aspect ratios was observed in 6 kPa. Forces generated by patterned hPSC-CMs were related to the organization, dynamics and size of sarcomeres. Electrophysiological parameters closer to that of mature cardiomyocytes were also observed with patch-clamp measurements in these hPSC-CMs with mature sarcomere organization. Conclusions: Stiffness and shape contribute to the structural and electrophysiological maturation of hPSC-CMs.