Norepinephrine inhibits cell cycle re‐entry of neonatal rat ventricular cardiomyocytes characterized by the absence of de novo nestin expression

Figures

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  FIGURE 1. Hypertrophic response of 1‐day‐old neonatal rat ventricular cardiomyocytes (NNVMs). (a) The surface area of individual mononucleated NNVMs (n = 4 independent neonatal rat litters) following a 24h treatment with NE (1 μM) or PDBu (100 nM) was significantly increased compared to untreated NNVMs and SB203580 (SB, 10 μM) treatment was without effect. Data were analyzed by a one‐way ANOVA followed by a Fisher LSD post hoc test and statistical difference was observed between PDBu versus Basal (A; p = 0.001), PDBu+SB versus SB (B; p = 0.001), NE versus basal (C; p = 0.001), and NE + SB versus SB (D; p = 0.001). (b) In parallel, norepinephrine (NE, 1 μM) and PDBu (100 nM) treatment of NNVMs (n = 4 independent neonatal rat litters) for 24 h significantly increased NPPA (atrial natriuretic peptide) mRNA levels and expression was potentiated following SB203580 treatment. mRNA data was normalized to GAPDH and HPRT‐1 mRNA levels (referred to as GEOMEAN). Data were analyzed by a one‐way ANOVA followed by a Fisher LSD post hoc test and statistical difference was observed between PDBu versus basal (A; p = 0.024), PDBu+SB versus PDBu (B; p = 2e‐6), NE versus basal (C; p = 0.008), and NE + SB…

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  FIGURE 2. The transcript profile of skeletal muscle genes. The 24h treatment of NNVMs (n = 4 independent neonatal rat litters) with PDBu (100 nM) significantly increased skeletal myosin light chain 1 (MYL1) (a; p = 7e‐4 vs. basal) and musculoskeletal embryonic nuclear protein 1 (Mustn1) mRNA levels (a; p = 3e‐5 vs. basal) whereas the mRNA expression of skeletal α‐Actin (ACTA1) (p = 0.06), skeletal muscle myosin heavy chain 2 (Myh2) (p = 0.84), and skeletal muscle‐specific GTP binding protein (Gem1) (p = 0.87) remained unchanged as compared to untreated (basal) NNVMs. PDBu/SB203580 (SB, 10 μM) treatment significantly inhibited the mRNA upregulation of MYL1 (b; p = 0.012 vs. PDBu) whereas Mustn1 mRNA levels remained unchanged as compared to PDBu (p = 0.60 vs. PDBu) but was significantly increased versus untreated or SB203580‐treated NNVMs (a; p = 1e‐5 vs. basal/SB‐treated). Moreover, PDBu/SB203580 treatment of NNVMs significantly increased ACTA1 (b; p = 0.013 vs. SB‐treated), Myh2 (b; p = 6e‐5 vs. SB‐treated), and Gem1 mRNA levels (b; p = 1e‐5 vs. SB‐treated) as compared to SB203580‐treated NNVMs. Norepinephrine (NE, 1 μM) stimulation of NNVMs for 24h significantly increased ACTA1 (c; p =…

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  FIGURE 3. Quantitative assessment of cell cycle re‐entry of 1‐day‐old neonatal rat ventricular cardiomyocytes (NNVMs) and recruitment of p38α/β MAPK signaling in response to sympathetic stimulation. (a) Nuclear incorporation of the nucleotide analog 5‐bromo‐2′‐deoxyuridine (BrdU; n = 8 independent neonatal rat litters) was similar between untreated (basal) and SB203580‐treated (SB, 10 μM) NNVMs. PDBu (100 nM) treatment NNVMs for 24 h significantly reduced 5‐bromo‐2′‐deoxyuridine (A; p = 5e‐4 vs. basal). In the presence of SB203580, PDBu‐mediated inhibition of 5‐bromo‐2′‐deoxyuridine incorporation was significantly reversed (B; p = 9e‐4 vs. PDBu). PDBu/SB203580 treatment led to the de novo nestin expression (C; p = 9e‐4 vs. PDBu) and increased the density that incorporated BrdU (D; p = 4e‐9 vs. PDBu). (b) Norepinephrine (NE, 1 μM) treatment NNVMs for 24 h significantly reduced the nuclear incorporation of 5‐bromo‐2′‐deoxyuridine (A; p = 2e‐4 vs. basal). In the presence of SB203580, NE‐mediated inhibition of 5‐bromo‐2′‐deoxyuridine incorporation was significantly reversed (B; p = 5e‐4 vs. PDBu). By contrast, in the presence of NE/SB203580, the cell cycle re‐entry of the predominant…

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  FIGURE 4. Qualitative assessment of cell cycle re‐entry of 1‐day‐old neonatal rat ventricular cardiomyocytes (a) In untreated NNVMs, 5‐bromo‐2′‐deoxyuridine incorporation (gray fluorescence) was identified in cardiac troponin‐T(+)‐NNVMs (TROP‐T, green fluorescence; indicated by arrow). Nestin (red fluorescence) staining was absent in cardiac troponin‐T(+)‐NNVMs whereas immunoreactivity was detected in ventricular fibroblasts lacking expression of the cardiomyocyte marker. (b) In norepinephrine (NE, 1 μM)‐treated NNVMs, the density of cardiac troponin‐T(+)‐NNVMs (green fluorescence; indicated by arrow) that incorporated 5‐bromo‐2′‐deoxyuridine (gray fluorescence) was significantly lower versus untreated NNVMs. (c) In NE/SB203580‐treated NNVMs, the density of cardiac troponin‐T(+)‐NNVMs (green fluorescence; indicated by arrow) that incorporated 5‐bromo‐2′‐deoxyuridine (gray fluorescence) was significantly increased versus NE‐treated NNVMs. Nestin (red fluorescence) immunoreactivity was absent in NE‐treated NNVMs but staining was detected in ventricular fibroblasts. (d) In PDBu (100 nM)‐treated NNVMs, the density of cardiac troponin‐T(+)‐NNVMs (green fluorescence; indicated by arrow) that…

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  FIGURE 5. Distribution of 1‐day‐old neonatal rat ventricular cardiomyocytes (NNVMs) that re‐entered the cell cycle. (a) A modest population (~20%) of NNVMs re‐entered the S‐phase of the cell cycle following a 24h treatment with PDBu (100 nM) and SB203580 (SB, 10 μM) (A; p = 1e‐10 vs. NNVMs that did not incorporate BrdU). Within the population of NNVMs that re‐entered the S‐phase, the predominant subpopulation was distinguished by de novo nestin expression (B; p = 0.0088 vs. nestin(−)‐NNVMs) whereas the minor subpopulation lacked expression of the intermediate filament protein. (b) Norepinephrine (NE, 1 μM) and SB203580 (SB, 10 μM) treatment of NNVMs for 24 h induced 5‐bromo‐2′‐deoxyuridine incorporation in a modest subpopulation (~14%) (C; p = 7e‐16 vs. NNVMs that did not incorporate BrdU). Within the population that re‐entered the cell cycle, the predominant subpopulation lacked de novo nestin expression (D; p = 1e‐13 vs. nestin(+) NNVMs). Data were analyzed by a Students paired t‐test.

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  FIGURE 6. The transcript profile of cell cycle genes. The 24h treatment of neonatal ventricular cells (n = 4 independent neonatal rat litters) with PDBu (100 nM) modestly but nonsignificantly increased nestin mRNA levels (p = 0.28) whereas Runx1 (a; p = 2e‐06 vs. basal) and CDKN2a (a; p = 3e‐07 vs. basal) mRNA levels were upregulated. By contrast, PDBu stimulation significantly reduced Ect2 (a; p = 2e‐05 vs. basal) and Bub1 (a; p = 3e‐06 vs. basal) mRNA expression as compared to untreated (basal) ventricular cells. SB203580 (SB, 10 μM) administration to PDBu‐treated ventricular cells potentiated nestin mRNA expression (b; p = 2e‐05 vs. PDBu), partially and significantly reduced elevated Runx1 (b; p = 0.0046 vs. PDBu) mRNA levels whereas CDKN2a mRNA expression remained increased and significantly greater (a; p = 3e‐07 vs. basal/SB203580) compared to untreated and SB203580‐treated ventricular cells. SB203580 administration to PDBu‐treated ventricular cells partially and significantly reversed the mRNA downregulation of Bub1 (b; p = 0.02 vs. PDBu) whereas Ect2 mRNA levels remained significantly reduced versus untreated and SB203580‐treated ventricular cells (a; p = 2e‐05 vs. basal/SB). The…

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  FIGURE 7. PKC isoform regulation in neonatal rat ventricular in response to NE and PDBu. (a) PDBu (100 nM) stimulation of ventricular cells (n = 3 independent neonatal rat litters) for 24 h robustly downregulated PKC‐α (a; p = 6e‐4 vs. basal), PKC‐ε (a; p = 6e‐8 vs. basal), and PKC‐δ (data not shown) protein levels versus untreated (basal) ventricular cells. By contrast, norepinephrine (NE, 1 μM) stimulation (n = 5–6 independent neonatal rat litters) for 24 h downregulated PKC‐ε (b; p = 6e‐6 vs. basal) protein levels whereas PKC‐α (p = 0.52) and PKC‐δ (data not shown) expression remained unchanged versus untreated (basal) ventricular cells. Data was analyzed by a Students' unpaired t‐test. (b, c) A 24h treatment of ventricular cells with a siRNA (n = 4 independent neonatal rat litters) directed against PKC‐α significantly reduced protein levels (a; p = 3.8e‐4 vs. basal) as compared to untreated (basal) ventricular cells. By contrast, ventricular cells treated with a scrambled siRNA had no effect on PKC‐α protein expression (p = 0.91). Data were analyzed by a one‐way ANOVA followed by a Fisher LSD post hoc test and PKC‐α protein levels were normalized to β‐Actin. (d) As indicated in Figure…

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  FIGURE 8. Cell cycle re‐entry of distinct subpopulations of NNVMs. The treatment of 1‐day old NNVMs with phorbol 12,13‐dibutyrate (PDBu) in the presence of the p38α/β MAPK inhibitor SB203580 led to the cell cycle re‐entry of two distinct subpopulations distinguished by the absence or de novo expression of the intermediate filament protein nestin. A siRNA approach targeting PKC‐α revealed that protein downregulation of the serine/threonine kinase selectively suppressed PDBu/SB203580‐mediated de novo nestin expression and cell cycle re‐entry of a subpopulation of NNVMs. By contrast, data from a previously published paper (Kebbe et al., 2023) and findings of the present study indirectly support the premise that the cell cycle re‐entry of the nestin(−)‐subpopulation in response to norepinephrine/SB203580 and PDBu/SB203580 treatment was driven by the novel isoform PKC‐ε. Additional experiments are warranted to unequivocally confirm the latter premise.