Effects of increasing intranuclear calcium levels via MCU inhibition on iPSC-derived cardiomyocyte differentiation and maturation

ABSTRCT Background: Cardiovascular diseases remain the leading cause of death worldwide, and the limited efficiency of human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation hampers its potential in disease modeling and regenerative therapy. Calcium signaling plays a central role in cardiac maturation, and proper regulation of intracellular calcium dynamics is essential for activating transcriptional programs that drive cardiomyocyte differentiation. Recent studies have suggested that closure of the mitochondrial permeability transition pore (mPTP) enhances cardiomyocyte differentiation by modulating calcium homeostasis and reducing reactive oxygen species (ROS). Building on this concept, we investigated the effects of 7-aminoindole (7-AI), a novel compound that inhibits mitochondrial calcium influx via the mitochondrial calcium uniporter (MCU), on cardiomyocyte differentiation. Methods: Using both mouse embryonic stem cells and hiPSCs, we treated cells undergoing differentiation with 7-AI at the cardiac progenitor stage (day 4) to inhibit MCU activity. We assessed differentiation efficiency by measuring nuclear and cytosolic calcium levels, activation of Ca²⁺/calmodulin-dependent protein kinase (CaMK), and phosphorylation status of the transcription factor cAMP response element-binding protein (CREB). Cardiac-specific gene expression was evaluated by quantifying cardiac cTnT, α-SA, and MYH6/7. Structural and functional maturation of the derived cardiomyocytes was determined using immunostaining and contractility assays. Results: Treatment with 7-AI significantly increased nuclear calcium levels and activated both CaMK and CREB, leading to the enhanced expression of cardiac-specific genes. Both mouse embryonic and hiPSC-derived cardiomyocytes displayed improved structural organization and contractile properties after 7-AI treatment. Comparative analysis between wild-type and CREB-deficient cells confirmed that CREB is essential for proper cardiomyocyte maturation because CREB deficiency leads to reduced cardiac marker expression and impaired myofibrillar organization. Conclusions: Our study demonstrated that 7-AI enhanced cardiomyocyte differentiation by inhibiting MCU, thereby redistributing calcium from the mitochondria to the nucleus. This redistribution activates CaMK and CREB, which in turn upregulate cardiac-specific gene expression, ultimately promoting the structural and functional maturation of cardiomyocytes. Targeting calcium dynamics during the cardiac progenitor stage represents a novel strategy for improving the efficiency of cardiac differentiation. These findings provide valuable insights into the molecular mechanisms governing cardiac maturation and offer a promising approach to generate functional cardiomyocytes for therapeutic applications.