Contractility Drives Cardiomyocyte Maturation and the Response to Nanopatterns.

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 cTnC (cardiac troponin C) substitutions. This engineered cTnC cannot bind to calcium at site II, 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 imaging, metabolic assays, and calcium transient analysis. Proteomes were examined using 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 noncontractile cells.

RESULTS

Surprisingly, we found that sarcomeres formed in the D65A cTnC cardiomyocytes, though these sarcomeres were underdeveloped and disorganized. The D65A cardiomyocytes also exhibited significant proteomic maturation defects and abnormal calcium transients. Replating the noncontractile cardiomyocytes onto nanopatterns improved several structural and proteomic maturation metrics. In contrast, wildtype maturation did not benefit from the introduction of nanopatterns.

CONCLUSIONS

Calcium-activated contractility is dispensable for sarcomerogenesis but critical for cardiomyocyte maturation. In noncontractile, D65A cTnC cardiomyocytes, nanopatterns enhanced maturation, suggesting that external mechanical cues may partially compensate for defective contractility. However, nanopatterns did not facilitate wildtype maturation, suggesting that maturity may reduce the efficacy of nanopatterns. In addition to these novel findings, these mass spectrometry data sets cataloging iPSC-CM maturation represent a useful resource for the cardiovascular research community.