Molecular Regulation of Direct Cardiac Reprogramming

A heart attack (also known as myocardial infarction, MI) happens when the flow of blood to the heart is blocked. A massive heart attack can kill billions of cardiomyocytes. The heart has limited regenerative potential because adult mammalian cardiomyocytes cannot proliferate, therefore lost cardiomyocytes cannot be replaced. This causes permanent heart damage and results in decreased contraction properties to a large portion of the heart muscle. Therapeutic treatments for heart attack patients have improved dramatically over the past decades. However, due to the inability to replenish lost cardiomyocytes, heart failure is still the primary cause of death in the world. Cardiac fibroblasts (CFs) constitute ~50% of the cells in the heart and form scar tissue following heart injury. Reprogramming CFs to induced-cardiomyocytes (iCMs) by forced expression of cardiac specific transcription factors holds promise for enhancing cardiac repair by reducing scar tissue while simultaneously generating new cardiomyocytes. However, low efficiency as well as a lack of understanding of molecular mechanism of the reprogramming process have significantly hampered its clinical application. The two goals of my PhD study were 1) to optimize the cardiac reprogramming protocol by increasing the efficiency; and 2) to decipher molecular mechanisms of cardiac reprogramming using the information obtained from the optimization process. To improve the efficiency of reprogramming fibroblasts to iCMs by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of the reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 weeks of induction by AKT1 plus GHMT (AGHMT). Furthermore, AGHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of AKT1 whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3a (Foxo3a) acted downstream of AKT1 to influence fibroblast-to-cardiomyocyte reprogramming. Addition of AGHMT converted 50% of mouse embryo fibroblasts to beating cardiomyocytes. However, only 1% of adult fibroblasts displayed spontaneous beating after three weeks of induction by AGHMT. This indicates that there are "barriers" in adult fibroblasts that hinder cardiac reprogramming. We continued to optimize methods for reprogramming fibroblasts to cardiomyocytes in vitro and in vivo. To identify additional regulators of this reprogramming process, we carried out an unbiased screen of ~1,100 open reading frames (ORFs) encoding transcription factors and cytokines for the ability to enhance reprogramming by AGHMT in adult tail-tip fibroblasts (ATTFs). One of the strongest activators of cardiac reprogramming was Krüppel-Type Zinc-Finger Transcription Factor 281 (ZNF281). Adding ZNF281 in AGHMT converted ~30% of ATTFs to iCMs which is comparable to AGHMT reprogrammed MEFs. We showed that ZNF281 enhanced cardiac reprogramming by associating with GATA4 on cardiac enhancers and by inhibiting inflammatory signaling, which antagonizes cardiac reprogramming. Our findings not only identify AKT1 and ZNF281 as robust and efficient activators of adult cardiac reprogramming, but also provide new insights into the molecular mechanisms underlying direct cardiac reprogramming.