Induction of Cardiomyocyte Proliferation via Transient Expression of Cell Cycle Factors as a Promising Therapy for Heart Failure

Baylor College Of Medicine, Houston TX

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Abstract

The high prevalence of heart disease is mainly due to the inability of the adult human heart to self-repair as the terminally differentiated CMs (CMs) undergo a complete cell cycle arrest. Understanding the mechanism(s) of CM proliferation in non-proliferating mature CMs and manipulating the process accordingly is key for cardiac regeneration. Recently, we showed that viral delivery of a combination of cell-cycle factors (CDK1, CDK4, cyclin B1, and cyclin D1 (4F)) induces robust CM proliferation and cardiac repair in mice, rats, and pigs. In addition to its translational potential, direct, robust induction of CM cell division in the post-mitotic adult CMs opened new opportunities to understand the barriers to adult CMs cell cycle induction. During the previous 3.5 years of our R01, we demonstrated the efficacy of the transient overexpression of the 4F, specifically in CMs, using a polycistronic non-integrating lentivirus (NIL) encoding the 4F, driven by a TNNT2 promoter (TNNT2-4F-NIL) in treating the ischemic heart failure in preclinical rat and pig models. In this R01 renewal proposal, we will continue our efforts to get the 4F-cell cycle induction in CMs closer to clinical translation using a CM-specific modified mRNA translational system (CM SMRTs) that allows exclusive gene expression in CM but not in non-CM. In addition, we will be injecting the 4F CM SMRTs into the heart muscle using a novel trans-endocardial injection catheter recently developed by Tenaya Therapeutics (NovoStar Endomyocardial Injection Catheter). Mechanistically, Following small molecule screening, we found that inhibition of L-Type calcium channel (LTCC) using Nifedipine improved CM proliferation in mature hiPS-CMs microtissues; nonetheless, small molecules often have some off-target effects. Therefore, to specifically inhibit LTCC, we overexpressed Ras-Related Associated with Diabetes (RRAD), an endogenous regulator of LTCC. Exogenous RRAD overexpression inhibits ICa,L in adult and embryonic ventricular myocytes. Our preliminary data suggest that RRAD overexpression promotes cell cycle induction, increases CM number, and upregulates cell cycle genes in primary neonatal and adult CMs in vivo in mouse hearts and ex vivo in human heart slices. Mechanistically, we demonstrated that RRAD overexpression reduces serine/threonine phosphatase calcineurin activity, as indicated by a significant reduction in calcineurin phosphatase activity and the expression levels of the calcineurin downstream effectors such as Rcan1 expression as well as translocation of Hoxb13 to the CM nucleus. Here, we will develop a new understanding at the molecular level of how LTCC inhibition enhances CM proliferation. Our central hypothesis is that 4F-CM-SMRTs is an efficient and safe treatment for ischemic heart failure, and the inhibition of LTCC increases the efficiency of the cell cycle induction in CMs and could be an additional therapeutic target for combination therapy. We expect that the knowledge gained from this project will lay the foundation for developing a new clinically applicable and efficient treatment for ischemic heart failure.