Transient Cell Cycle Induction in Cardiomyocytes to Treat Ischemic Heart Failure

Abstract

The regenerative capacity of the heart after myocardial infarction (MI) is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 and Cdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in 15-20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after MI. Here, we aim to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Also, we aim to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure. Temporal bulk and single-cell RNAseq and further biochemical validations of mature hiPS-CMs treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population after 48 h post-infection with 4F, which was associated with sarcomere disassembly and metabolic reprogramming. Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic non-integrating lentivirus (NIL) encoding the 4F; each is driven by a TNNT2 promoter (TNNT2-4F-NIL). TNNT2-4F-NIL or control virus was injected intramyocardially one week after MI in rats or pigs. TNNT2-4F-NIL treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus treated animals four weeks post-injection. In conclusion, the present study provides a mechanistic demonstration of the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors using a novel transient and cardiomyocyte-specific viral construct.

Induced Cardiomyocyte Proliferation: A Promising Approach to Cure Heart Failure

“…In a trial for preclinical application of 4F, Abouleisa et al cloned a polycistronic non-integrating lentivirus encoding 4F in which each factor was driven by TNNT2 promotor (TNNT2-4F-NIL) to induce cell cycling transiently and specifically in cardiomyocytes. Intramyocardial injection of TNNT2-4F-NIL improved systolic functions and reduced the scar size after ischemic reperfusion in rats and pigs compared to control-treated rats and pigs [ 36 ].…”

“…Therefore, several recent studies have aimed to develop or use existing transgenic lineage tracing models to distinguish between activation of the cell cycle and true cytokinesis in vitro and in vivo. Mosaic analysis of the dual marker mouse model (MADM) [ 121 ] has become the gold standard to demonstrate true cytokinesis in cardiomyocytes in vivo, as demonstrated by several recent studies [ 35 , 36 , 60 , 109 , 122 ]. Several other interesting models have been recently generated to demonstrate the progress of cardiomyocytes through the cell cycle in vivo, such as the double transgenic Myh6-eGFP-anillin/Myh6-H2BmCh mice [ 123 ].…”

“…Interestingly, a recent study generated a Ki67-based lineage and traced mice to track the cell cycle activation in cardiomyocytes [ 129 ]. Finally, a recent preprint described the generation of an Aurora kinase B-based reporter to be used in identification of cardiomyocyte cell cycle activation in vitro and in vivo [ 36 ]. These new tools will enrich the field of cardiomyocyte proliferation research and enable the identification of new pathways that could modulate cardiomyocyte cell cycle progression.…”

“…That is essential to avoid the possibility of an unnecessary increase in cardiac mass and the development of diastolic heart failure [ 64 , 112 , 130 , 131 ]. A recent preprint tried to address some of these limitations and demonstrated that transient and cardiomyocyte expression of cell cycle induction is a promising tool to safely induce cardiomyocyte proliferation, with no obvious toxicity in rodents and large animals [ 36 ].…”