Cell Shape and Maturation Impacts α-Actinin-2 Tension in iPSC-derived Cardiomyocytes

The contractile activity of cardiomyocytes (CMs) critical to heart function emerges from the collective shortening of sarcomeres. However, how these sarcomeric forces are transmitted within CMs during this process remains poorly understood. Traction force microscopy (TFM) has been used to measure overall forces exerted by CMs, but it falls short in providing insights into which specific proteins within sarcomeres transmit and whether cell shape influences forces within each sarcomere. Here, we aimed to characterize force generation on α-actinin-2, a z-disc protein that crosslinks anti-parallel actin filaments from adjacent sarcomeres and transmits force within a cell. By incorporating a Förster Resonance Energy Transfer (FRET)-based molecular tension sensor in α-actinin-2, we measured contraction-induced deformation of the α-actinin-2 sensor in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cultured on rectangular and circular adhesive patterns. We observed α-actinin-2 localized within sarcomeres, and actinin-2 loading correlated with sarcomere maturation and organization. α-actinin-2 tension increased in contracting rectangular-shaped cells, but not in circular cells. Moreover, the increase in tension was only observed in rectangular cardiomyocytes that were cultured for 5 days in culture, and not after 24h. Interestingly, the spread of FRET index values was increased in both rectangular and circular cells after 5 days in culture, compared to cells that were kept for 24h in culture. Together, these data suggest that cell shape and maturation modulates tension on a load-bearing sarcomeric protein, α-actinin-2, and highlights the importance of characterizing tension across sarcomeric structures to understand cardiomyocyte contractile activity.