Enhancing Cardiac Reprogramming by Suppressing Specific C-C Chemokine Signaling Pathways

Reprogramming fibroblasts into induced cardiomyocytes (iCMs) is a potentially promising strategy for heart regeneration. Yet a major challenge is the low conversion rate. To address this challenge, we screened and identified four chemicals, insulin-like growth factor-1, Mll1 inhibitor MM589, transforming growth factor-β inhibitor A83-01, and Bmi1 inhibitor PTC-209, termed as IMAP, that coordinately enhanced reprogramming efficiency. Using α-muscle heavy chain-green fluorescent protein mouse embryo fibroblasts as the staring cell type, we observed a six-fold increase of iCM formation with IMAP treatment. IMAP stimulated higher cardiac troponin T and α-actinin expression and more sarcomere formation with up-regulation of many cardiac genes and down-regulation of fibroblast genes. Furthermore, IMAP promoted higher spontaneous beating and calcium transient activities of iCMs derived from neonatal cardiac fibroblasts. Intriguingly, we identified that many genes involved in immune responses, particularly those in specific C-C chemokine signaling pathways, were repressed with IMAP treatment. We next tested C-C chemokine ligands Ccl3, Ccl6, and Ccl17 in cardiac reprogramming and observed inhibitory effect on iCM formation, while corresponding inhibitors of Ccr1, Ccr4, and Ccr5 had the opposite effect. These results indicated that suppression of specific C-C chemokine signaling pathways was a direct down-stream event of IMAP treatment that enhanced cardiac reprogramming. In conclusion, we identified a combination of four chemicals IMAP in suppressing specific C-C chemokine signaling pathways and facilitating MGT-induced cardiac reprogramming. Our studies revealed the role of these specific C-C chemokine signaling pathways in cardiac reprogramming and provide potential targets in iCM formation and its clinical applications.