The Chinese University of Hong Kong (CUHK), Hong Kong
Abstract: Adult human cardiomyocytes (CMs) have limited ability to regenerate. When they are lost after injury or disease, the heart often suffers permanent damage. Promoting cardiomyocyte proliferation is therefore an important avenue to repair the heart. Centrosomes are an established mediator of proliferation in many cell types but its role in CM proliferation is not well understood. We recently showed that verapamil, an L-type calcium channel blocker, could promote the proliferation of human pluripotent stem cell-(hPSC-) CMs. We aim to comprehensively evaluate its effect on CMs and dissect its mechanisms, particularly in relation to centrosomes. Our results showed that verapamil enhanced hPSC-CM proliferation, as measured by EdU incorporation and Ki67 staining, and increased nuclear and cell count. Both mononucleated and binucleated hPSC-CMs contributed to proliferation. Verapamil treated hPSC-CMs adopted a developmentally immature, embryonic-like phenotype, in terms of sarcomeric structure and mitochondrial function. The proliferative and immature phenotype induced by verapamil is reversible, and hPSC-CMs resumed maturation upon cessation of treatment. Although verapamil promoted an immaturity, treated cells retained cardiac identity, and did not express markers of pluripotency. Mechanistically, transcriptomic analysis of verapamil treated hPSC-CMs revealed a molecular profile consistent with increased cell cycle activity and reduced maturation. Bioinformatics analysis revealed upstream regulators governing these changes. Lastly, we demonstrated that hPSC-CMs underwent centrosome reassembly upon verapamil treatment, and this was positively associated with proliferation. Altered centrosome assembly could modulate hiPSC-CM proliferation. In conclusion, our data confirms that verapamil can induce cardiomyocyte proliferation, and that this is accompanied by the acquisition of an embryonic phenotype. The effect of verapamil is reversible, thus allowing controlled, regulatable proliferation. We further revealed centrosome assembly as a key contributor to hiPSC-CM proliferation induced by verapamil. Our study provides new understanding of about CM proliferation and centrosome assembly to aid in the development of new strategies to promote cardiac regeneration.
