Abstract
Reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a potentially promising strategy for regenerating a damaged heart. However, low fibroblast-cardiomyocyte conversion rates remain a major challenge in this reprogramming. To this end, here we conducted a chemical screen and identified four agents, insulin-like growth factor-1, Mll1 inhibitor MM589, transforming growth factor-β inhibitor A83-01, and Bmi1 inhibitor PTC-209, termed IMAP, which coordinately enhanced reprogramming efficiency. Using α-muscle heavy chain-GFP-tagged mouse embryo fibroblasts as a starting cell type, we observed that the IMAP treatment increases iCM formation 6-fold. IMAP stimulated higher cardiac troponin T and α-actinin expression and increased sarcomere formation, coinciding with up-regulated expression of many cardiac genes and down-regulated fibroblast gene expression. Furthermore, IMAP promoted higher spontaneous beating and calcium transient activities of iCMs derived from neonatal cardiac fibroblasts. Intriguingly, we also observed that the IMAP treatment repressed many genes involved in immune responses, particularly those in specific C-C chemokine signaling pathways. We therefore investigated the roles of C-C motif chemokine ligand 3 (CCL3), CCL6, and CCL17 in cardiac reprogramming and observed that they inhibited iCM formation, whereas inhibitors of C-C motif chemokine receptor 1 (CCR1), CCR4, and CCR5 had the opposite effect. These results indicated that the IMAP treatment directly suppresses specific C-C chemokine signaling pathways and thereby enhances cardiac reprogramming. In conclusion, a combination of four chemicals, named here IMAP, suppresses specific C-C chemokine signaling pathways and facilitates Mef2c/Gata4/Tbx5 (MGT)-induced cardiac reprogramming, providing a potential means for iCM formation in clinical applications.
Highlights
Reprogramming of fibroblasts into induced cardiomyocytes is a potentially promising strategy for regenerating a damaged heart
mouse embryonic fibroblasts (MEF) were treated with chemicals every 3 days after the infection of retrovirus expressing polycistronic Mef2c/Gata4/Tbx5 (MGT) [18]. 2 weeks later, the reprogramming efficiency was evaluated by percentage of green fluorescent protein (GFP)-positive cells and related gene expression (Fig. 1A)
Based on the above results, we examined C-C chemokine signaling pathways C motif chemokine ligand 6 (Ccl6)-chemokine receptor 1 (Ccr1), Ccl17-Ccr4 and Ccl3-Ccr5 in cardiac reprogramming, as they were significantly down-regulated in MGTϩIMAP–treated group
Summary
To identify small molecules that could enhance cardiac reprogramming, we used a screening platform reported previously [26, 28]. After 2 weeks of reprogramming with IMAP treatment, immunofluorescence staining showed 3- to 4-fold increases in the expression of cardiac troponin T (cTnT) and ␣-actinin as well as significantly increased cTnT/GFP– double positive cells (Fig. 2D and Fig. S1A). It is possible that potential CM contamination could contribute to the iCM population To address this potential issue, MEFs or NCFs without GMT infection and IMAP treatment were examined for GFP-positive signals from initial fibroblast isolation to 4 weeks after iCM induction. Indicating that iCM matured further under extended IMAP treatment These data indicated that iCMs were reprogrammed from originally isolated fibroblasts and showed much better CM functionality such as spontaneous beating and calcium transient in MGTϩIMAP groups than those obtained from MGTϩDMSO groups. Our research showed that IMAP enhanced cardiac reprogramming by eliminating iCM reprogramming barriers imposed by specific C-C chemokine pathways (Fig. 6I)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
