Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for use in engineering human tissues for regenerative therapy and drug screening.However, differentiated cardiomyocytes are phenotypically immature, reducing assay reliability when translating in vitro results to clinical studies and precluding hiPSC-derived cardiac tissues from therapeutic use in vivo. To address this, we have developed hybrid hydrogels comprised of decellularized porcine myocardial extracellular matrix (dECM) and reduced graphene oxide (rGO) to provide a more instructive microenvironment for proper cellular and tissue development. A tissue-specific protein profile was preserved post-decellularization, and through the modulation of rGO content and degree of reduction, the mechanical and electrical properties of the hydrogels could be tuned. Engineered heart tissues (EHTs) generated using dECM-rGO hydrogel scaffolds and hiPSC-derived cardiomyocytes exhibited significantly increased twitch forces at 14 days of culture and had increased the expression of genes that regulate contractile function. Similar improvements in various aspects of electrophysiological function, such as calcium-handling, action potential duration, and conduction velocity, were also induced by the hybrid biomaterial.We also demonstrate that dECM-rGO hydrogels can be used as a bioink to print cardiac tissues in a high-throughput manner, and these tissues were utilized to assess the proarrhythmic potential of cisapride. Action potential prolongation and beat interval irregularities was observed in dECM-rGO tissues at clinical doses of cisapride, indicating that the enhanced maturation of these tissues corresponded well with a capability to produce physiologically relevant drug responses.

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX.  Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.

A lack of electrical conductivity and structural organization in currently available biomaterial scaffolds limits their utility for generating physiologically representative models of functional cardiac tissue. Here we report on the development of scalable, graphene-functionalized topographies with anisotropic electrical conductivity for engineering the structural and functional phenotypes of macroscopic cardiac tissue constructs. Guided by anisotropic electroconductive and topographic cues, the tissue constructs displayed structural property enhancement in myofibrils and sarcomeres, and exhibited significant increases in the expression of cell-cell coupling and calcium handling proteins, as well as in action potential duration and peak calcium release.