A subpopulation of 7-day old neonatal rat ventricular cardiomyocytes retains the inherent ability to re-enter the cell cycle delineated by the de novo nestin expression

Abstract

Introduction

Studies have reported that injury to the 7-day old neonatal rodent heart failed to elicit a regenerative response. It remains presently unknown whether the latter paradigm is attributed in part to an inherent loss of ventricular cardiomyocytes to re-enter the cell cycle.

Objective

To examine whether 7-day old neonatal rat ventricular cardiomyocytes can re-enter the cell cycle and express a pattern of gene expression associated with a potential cardiac regenerative response.

Method

Ventricular cardiomyocytes (NNVMs) were isolated from 7-day old neonatal Sprague-Dawley rats and treated for 3 days with the phorbol ester phorbol 12,13 dibutyrate (PDBu; 100 nM) in the absence or presence of the p38a/b MAPK inhibitor SB203580 (10 mM). Cell cycle re-entry and gene expression was determined by QPCR and Bulk RNA Seq Analysis. Partial apex resection of the 7-day old neonatal rat heart was performed and pups sacrificed 3 days later.

Results

80% of the population of untreated 7-day old NNVMs were mononucleated. PDBu treatment for 3-days failed to promote cell cycle re-entry but induced a hypertrophic response delineated by a significant upregulation of Nppa and Nppb mRNAs. SB203580 administration to PDBu treated NNVMs translated to cell cycle re-entry characterized by nuclear 5-bromo-2′-deoxyuridine incorporation and nuclear staining of phosphorylated serine10 of histone 3 of a subpopulation characterized by de novo expression of the intermediate filament protein nestin. The latter paradigm was confirmed by Western blot and QPCR. The appearance/cell cycle re-entry of nestin (+)-NNVMs was associated with a significant mRNA upregulation of a panel of glycolytic enzymes (PDK3, PFKP, PKM, Slc2a1) and concomitant reduction of enzymes implicated in fatty acid oxidation (Cpt1b, Sdha, Acsl1). Three days following partial apex-resected 7-day old neonatal rat heart, the appearance of a subpopulation of NNVMs associated with de novo nestin expression was identified adjacent to the damaged region and significantly re-entered the S-phase and G2-M phase of the cell cycle.

Conclusion

A subpopulation of 7-day old neonatal rat ventricular cardiomyocytes possess the inherent ability to re-enter the cell cycle in vitro and in vivo characterized by the selective expression of the intermediate filament protein nestin.