Calcium-Activated Sarcomere Contractility Drives Cardiomyocyte Maturation and the Response to External Mechanical Cues but is Dispensable for Sarcomere Formation.

BACKGROUND

Understanding the mechanisms of cardiomyocyte development is critical for fulfilling the potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Although myocyte development is known to depend on internal and external mechanical cues, further investigation is required to understand the contributions of different signals and how they are integrated together to generate an adult cardiomyocyte. Here, we address this gap by examining the role of calcium-activated contractility in sarcomere formation and maturation and its influence on the iPSC-CM response to nanopatterns.

METHODS

We generated iPSCs with homozygous D65A cardiac troponin C (cTnC) mutations. This mutation prevents calcium binding to site II of cTnC, resulting in tropomyosin blocking strong myosin binding to the thin filament and inhibiting sarcomere contraction. The iPSCs were differentiated into cardiomyocytes and matured in culture over 60 days. Cells were characterized via fluorescence imaging and calcium transient analysis. WT and mutant proteomes were examined via mass spectrometry throughout differentiation and maturation. We also replated partially matured cardiomyocytes onto nanopatterned surfaces to investigate how external mechanical signals affect maturation in contractile versus non-contractile cells.

RESULTS

Surprisingly, we found that sarcomeres formed in the cTnC D65A cardiomyocytes, though these sarcomeres were underdeveloped and disorganized. Mutant cardiomyocytes also exhibited significant proteomic maturation defects and abnormal calcium transients. Plating D65A cardiomyocytes on nanopatterns improved structural and proteomic maturation. However, plating WT cardiomyocytes on nanopatterns led to a reduction in sarcomeric and oxidative phosphorylation protein content.

CONCLUSIONS

Calcium-activated contractility is dispensable for sarcomerogenesis but critical for cardiomyocyte maturation. In non-contractile, mutant cardiomyocytes, nanopatterns enhance maturation, suggesting that external mechanical cues may partially compensate for defective contractility. However, nanopatterns did not facilitate WT maturation and may have hindered it. In addition to these novel findings, these large mass spectrometry datasets cataloging iPSC-CM maturation represent a useful resource for the cardiovascular community.