Abstract
Direct conversion of cardiac fibroblasts into induced cardiomyocytes (iCMs) by forced expression of defined factors holds great potential for regenerative medicine by offering an alternative strategy for treatment of heart disease. Successful iCM conversion can be achieved by minimally using three transcription factors, Mef2c (M), Gata4(G), and Tbx5 (T). Despite increasing interest in iCM mechanistic studies using MGT(polycistronic construct with optimal expression of M,G and T), the reprogramming efficiency varies among different laboratories. Two main Mef2c isoforms (isoform2, Mi2 and isoform4, Mi4) are present in heart and are used separately by different labs, for iCM reprogramming. It is currently unknown if differently spliced isoform of Mef2c contributes to varied reprogramming efficiency. Here, we used Mi2 and Mi4 together with Gata4 and Tbx5 in separate vectors or polycistronic vector, to convert fibroblasts to iCMs. We found that Mi2 can induce higher reprogramming efficiency than Mi4 in MEFs. Addition of Hand2 to MGT retroviral cocktail or polycistronic Mi2-GT retroviruses further enhanced the iCM conversion. Overall, this study demonstrated the isoform specific effects of Mef2c, during iCM reprogramming, clarified some discrepancy about varied efficiency among labs and might lead to future research into the role of alternative splicing and the consequent variants in cell fate determination.
Highlights
Ischemic heart disease is the leading cause of mortality worldwide, accounting for 8.93 million deaths per year [1]
We evaluated the expression of Mef2c variants by qPCR analysis, using exon-specific primers in murine primary cardiomyocytes (CMs) and different types of fibroblasts, including freshly isolated cardiac fibroblasts, explanted cardiac fibroblasts (ExCFs), explanted tail tip fibroblasts (ExTTFs), and mouse embryonic fibroblasts (MEFs)
Our results showed that the delivery of Mef2C isoform2 in a polycistronic construct can further enhance the efficiency of direct induced cardiomyocytes (iCMs) reprogramming with the addition of Hand2
Summary
Ischemic heart disease is the leading cause of mortality worldwide, accounting for 8.93 million deaths per year [1]. After myocardial infarction (MI), massive and permanent loss of cardiomyocytes (CMs) leads to cardiac malfunctions and eventually heart failure. Developing new strategies to replenish lost CMs to restore heart function remains a big challenge to be addressed. Recent advances in direct cardiac reprogramming provide a promising approach by converting fibroblast into induced cardiomyocytes (iCMs), for regenerating damaged heart tissue in situ. Ectopic expression of cardiac-lineage transcription factors (TFs) could directly reprogram fibroblasts into iCMs, without going through the pluripotent stage [2,3,4,5,6,7]. Delivery of minimal three TFs Mef2c (M), Gata (G), and Tbx (T) has been reported to successfully generate murine iCMs from fibroblasts, both in vitro
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