Investigating The Role Of Contraction During Myofibril Formation Using Human Stem Cell-Derived Cardiomyocytes

Mechanical contraction is thought to play an essential role during cardiomyocyte development, including formation of contractile elements and cellular hypertrophy. However, the mechanisms by which contraction impacts sarcomere assembly and maturation of developing cardiomyocytes are unknown. To understand the role of contraction during myofibril development in cardiomyocytes, we used CRISPR Cas9 to generate a non-contractile human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model by introducing the point mutation D65A into the cardiac troponin C (cTnC D65A) gene. The D65A mutation prevents calcium binding to site II of cTnC, thereby preventing activation of sarcomere contraction. Previous studies have reported complete sarcomere disassembly when contraction was inhibited. Therefore, we hypothesized that these non-contracting cardiomyocytes would not form myofibrils or that myofibrils would be more like stress fibers. However, upon cardiomyocyte differentiation, immune-fluorescent staining of alpha actinin showed cTnC D65A-CMs form and maintain myofibrils (> 60 days) with the full complement of sarcomere proteins, including all three troponin subunits. cTnC D65A-CMs had spontaneous and stimulated calcium transients (in the absence of contraction) and matured at a similar timescale as wild type (WT) cells, as demonstrated by the isoform changes from myosin heavy chain 6 to 7. When cTnC D65A-CMs were transduced with adenoviral vectors (AVs) to express WT cTnC, contraction was initiated within a few days. Conversely, when WT hiPSC-CMs were transduced with AVs to express cTnC D65A, contraction was inhibited. hiPSC-CMs containing WT or D65A cTnC had elongated morphology when cultured on nanopatterned surfaces with improved myofibrillar alignment, directionality, and bundling. Inhibition of contraction by expression of D65A cTnC resulted in complete myofibrillar disarray, with the emergence of muscle stress fibers in the cellular periphery. Thus, cTnC D65A hiPSC-CMs are an exciting new tool to study cardiomyocyte development.