Cathepsin K inhibition promotes efficient differentiation of human embryonic stem cells to mature cardiomyocytes by mediating glucolipid metabolism and cellular energy homeostasis.

Background and aim: Generation of functional cardiomyocytes from human pluripotent stem cells (hPSCs) offers promising applications for cardiac regenerative medicine. Proper control of pluripotency and differentiation is vital for generating high-quality cardiomyocytes and repairing damaged myocardium. Cathepsin K, a lysosomal cysteine protease, is a potential target for cardiovascular disease treatment; however, its role in cardiomyocyte differentiation and regeneration is unclear. This study aims to investigate the effects and mechanisms of cathepsin K inhibition on the differentiation of human embryonic stem cell-induced cardiomyocytes (hESC-CMs) and myocardial generation.

Methods: We cultured H9-hESCs in CDM3 medium to induce myocardial differentiation, adding cathepsin K inhibitor II (1 μM) on days 2, 5 and 8, respectively. Cells were observed and collected 48 h after each treatment. The morphology and contractile clusters of H9-hESCs were tracked with microscopy and video recording. Pluripotency and cardiac markers were assessed at each stage of differentiation. We also examined glucose and lipid metabolism, mitochondrion-related markers, apoptosis and autophagy.

Results: CDM3 medium effectively differentiated high-density H9-hESCs into mature, spontaneously contracting cardiomyocytes. Cathepsin K inhibition accelerates the differentiation of H9-hESCs into cardiac mesoderm and cardiac precursor cells (CPCs) by reducing apoptosis, decreasing glycolysis and fatty acid metabolism at the early and middle stages, and subsequently facilitate the development and differentiation of cardiomyocytes by enhancing glucolipid metabolism and oxidative phosphorylation at the late stage. Meanwhile, cathepsin K inhibition enhanced mitochondrial function and lysosome-related gene transcription during the differentiation process.

Conclusion: Our study highlights the potential of cathepsin K inhibition for renewable cardiomyocytes and suggests exploring metabolic pathways and signaling to improve cardiac regeneration and organoid development.