cMyBP-C ablation in human engineered cardiac tissue causes progressive Ca2+-handling abnormalities.

Truncation mutations in cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM). Heterozygous carriers present with classical HCM, while homozygous carriers present with early onset HCM that rapidly progress to heart failure. We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes derived from these isogenic lines were used to generate cardiac micropatterns and engineered cardiac tissue constructs (ECTs) that were characterized for contractile function, Ca2+-handling, and Ca2+-sensitivity. While heterozygous frame shifts did not alter cMyBP-C protein levels in 2-D cardiomyocytes, cMyBP-C+/- ECTs were haploinsufficient. cMyBP-C-/- cardiac micropatterns produced increased strain with normal Ca2+-handling. After 2 wk of culture in ECT, contractile function was similar between the three genotypes; however, Ca2+-release was slower in the setting of reduced or absent cMyBP-C. At 6 wk in ECT culture, the Ca2+-handling abnormalities became more pronounced in both cMyBP-C+/- and cMyBP-C-/- ECTs, and force production became severely depressed in cMyBP-C-/- ECTs. RNA-seq analysis revealed enrichment of differentially expressed hypertrophic, sarcomeric, Ca2+-handling, and metabolic genes in cMyBP-C+/- and cMyBP-C-/- ECTs. Our data suggest a progressive phenotype caused by cMyBP-C haploinsufficiency and ablation that initially is hypercontractile, but progresses to hypocontractility with impaired relaxation. The severity of the phenotype correlates with the amount of cMyBP-C present, with more severe earlier phenotypes observed in cMyBP-C-/- than cMyBP-C+/- ECTs. We propose that while the primary effect of cMyBP-C haploinsufficiency or ablation may relate to myosin crossbridge orientation, the observed contractile phenotype is Ca2+-mediated.

Micropatterned substrates with physiological stiffness promote cell maturation and Pompe disease phenotype in human induced pluripotent stem cell-derived skeletal myocytes.

Recent advances in bioengineering have enabled cell culture systems that more closely mimic the native cellular environment. Here, we demonstrated that human induced pluripotent stem cell (iPSC)-derived myogenic progenitors formed highly-aligned myotubes and contracted when seeded on two-dimensional micropatterned platforms. The differentiated cells showed clear nuclear alignment and formed elongated myotubes dependent on the width of the micropatterned lanes. Topographical cues from micropatterning and physiological substrate stiffness improved the formation of well-aligned and multinucleated myotubes similar to myofibers. These aligned myotubes exhibited spontaneous contractions specifically along the long axis of the pattern. Notably, the micropatterned platforms developed bundle-like myotubes using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II) and even enhanced the disease phenotype as shown through the specific pathology of abnormal lysosome accumulations. A highly-aligned formation of matured myotubes holds great potential in further understanding the process of human muscle development, as well as advancing in vitro pharmacological studies for skeletal muscle diseases.