Distinct Protein Kinase C isoforms drive the cell cycle re-entry of two separate populations of neonatal rat ventricular cardiomyocytes

by Selleckchem | Apr 06 2023

The present study tested the hypothesis that PKC-α recruitment in the presence of the p38α/β MAPK inhibitor SB203580 facilitated the appearance and cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes (NNVMs) and induced a transcript profile delineating a proliferative phenotype. Phorbol 12,13-dibutyrate (PDBu) treatment did not induce de novo nestin expression or increase the cell cycle re-entry of 1-day old NNVMs but significantly increased Runx1 and CDKN2a mRNA levels and downregulated ECT2 mRNA expression. SB203580 administration to PDBu-treated NNVMs induced de novo nestin expression, preferentially increased the density (normalized to 500 NNVMs) of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu, 1.4±3 versus PDBu/SB203580, 128±34; n=5 independent litters), significantly inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation. PDBu treatment of NNVMs reduced PKC-α, PKC-δ and PKC-ε protein levels and GF109203X (conventional PKC isoform inhibitor) selectively attenuated PKC-α protein downregulation. GF109203X administration to PDBu/SB203580-treated NNVMs significantly reduced the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 40±46; n=5). Moreover, GF109203X/PDBu/SB203580 treatment unmasked the predominant appearance of a separate NNVM population that incorporated 5-bromo-2'-deoxyuridine (PDBu/SB203580/GF109203X, 192±42; n=5) delineated by the absence of de novo nestin expression. Sotrastaurin (conventional/novel PKC isoform inhibitor) administration to PDBu/SB203580-treated NNVMs significantly attenuated the density of nestin(+)-NNVMs (PDBu/SB203580/sotrastaurin, 8±10; n=4) and nestin(-)-NNVMs (PDBu/SB203580/sotrastaurin, 64±30; n=4) that incorporated 5-bromo-2'-deoxyuridine. These data reveal that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle and the preferential appearance of nestin(+)- or nestin(-)-NNVMs is driven by distinct PKC isoforms in the presence of SB203580.

Comments：

The study aimed to test the hypothesis that the recruitment of PKC-α in the presence of the p38α/β MAPK inhibitor SB203580 would induce the appearance and cell cycle re-entry of nestin(+)-neonatal rat ventricular cardiomyocytes (NNVMs) and initiate a transcript profile indicative of a proliferative phenotype. The researchers treated 1-day old NNVMs with phorbol 12,13-dibutyrate (PDBu) and observed that it did not induce the de novo expression of nestin or increase the cell cycle re-entry of NNVMs. However, PDBu treatment significantly increased Runx1 and CDKN2a mRNA levels while downregulating ECT2 mRNA expression.

When SB203580 was administered to PDBu-treated NNVMs, de novo nestin expression was induced, and there was a significant increase in the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine compared to PDBu treatment alone. Additionally, SB203580 inhibited CDKN2a and Runx1 mRNA upregulation and reversed ECT2 mRNA downregulation.

The researchers observed that PDBu treatment of NNVMs resulted in reduced protein levels of PKC-α, PKC-δ, and PKC-ε. Treatment with GF109203X, a conventional PKC isoform inhibitor, selectively attenuated PKC-α protein downregulation. When GF109203X was administered to PDBu/SB203580-treated NNVMs, there was a significant reduction in the density of nestin(+)-NNVMs that incorporated 5-bromo-2'-deoxyuridine. Additionally, a separate population of NNVMs that incorporated 5-bromo-2'-deoxyuridine was observed, which was delineated by the absence of de novo nestin expression.

Furthermore, when Sotrastaurin, a conventional/novel PKC isoform inhibitor, was administered to PDBu/SB203580-treated NNVMs, there was a significant attenuation in the density of nestin(+)-NNVMs and nestin(-)-NNVMs that incorporated 5-bromo-2'-deoxyuridine.

Overall, these findings suggest that the neonatal rat heart contains at least two separate populations of NNVMs that re-enter the cell cycle, and the preferential appearance of nestin(+)- or nestin(-)-NNVMs is driven by distinct PKC isoforms in the presence of SB203580.