p38α MAPK inhibition translates to cell cycle re-entry of neonatal rat ventricular cardiomyocytes and de novo nestin expression in response to thrombin and after apex resection

Figures

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  Figure 1. Cell cycle re-entry and de novo nestin expression in neonatal rat ventricular cardiomyocytes. (A) Modest number of untreated neonatal rat ventricular cardiomyocytes (NNVMs) delineated by cardiac troponin-T staining (green fluorescence) re-entered the cell cycle as determined by bromodeoxyuridine (BrdU; grey fluorescence) incorporation. Furthermore, nestin staining (red fluorescence) of NNVMs was not observed. (B) Three day treatment with thrombin (1 U/ml) failed to increase cell cycle re-entry in NNVMs as depicted by BrdU incorporation and nestin staining was absent. By contrast, nestin staining detected in cells lacking troponin-T immunoreactivity were identified as neonatal rat ventricular fibroblasts (See Supplemental Fig. 2). (C) The co-treatment with the p38α/β MAPK inhibitor SB203580 (SB; 10 µM) and thrombin for three days promoted cycle re-entry and de novo nestin expression in NNVMs. DAPI staining identifies the nucleus (blue fluorescence).

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  Figure 2. Quantitative assessment of cell cycle re-entry and de novo nestin expression in NNVMs. (A) Graph represents a quantitative analysis of the density of cardiac troponin-T(+)-NNVMs that re-entered the cell cycle characterized by BrdU incorporation. (B) Graph represents a quantitative analysis of the density of cardiac troponin-T(+)-NNVMs that co-expressed nestin. (C) Graph represents a quantitative analysis of the density of cardiac troponin-T(+)-NNVMs that co-expressed nestin and incorporated BrdU. Experiments were performed on n = 5 independent preparations of neonatal ventricular cells and (*) denotes P < 0.05 versus thrombin or PDBu alone.

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  Figure 3. Thrombin stimulation of neonatal rat ventricular cells recruits p38α MAPK signaling. (A) The acute treatment of neonatal ventricular cells with thrombin (1 U/ml) increased phosphorylation of the threonine180/tyrosine182 residues of p38 MAPK (n = 2 independent experiments). (B) The acute treatment of neonatal ventricular cells with thrombin (1 U/ml) significantly increased phosphorylation of the serine82 residue of heat shock protein (HSP27; 27 kDa) (n = 3 independent experiments) at 20 and 30 minutes. Co-treatment with the p38α/β MAPK inhibitor SB203580 (SB; 10 µM) abrogated thrombin-mediated phosphorylation of HSP27 (n = 3 independent experiments). (*) Denotes P < 0.05 versus basal, (**) denotes P < 0.05 versus thrombin and p38 MAPK and HSP27 phosphorylation data was normalized to total p38α MAPK and HSP27 protein content.

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  Figure 4. Nestin expression in neonatal rat ventricular cells. (A) Thrombin (THR; 1 U/ml) treatment of neonatal rat ventricular cells for three days increased nestin protein levels and co-treatment with SB203580 (SB; 10 µM) non-significantly increased the response. The increased protein levels of nestin after thrombin and SB203580 co-treatment was attributed to the de novo expression of the intermediate filament protein in neonatal rat ventricular cardiomyocytes (See Fig. 1C). (B) Thrombin (1 U/ml) treatment of 1st passage neonatal rat ventricular fibroblasts for 24 hours increased nestin protein levels and co-treatment with SB203580 (SB; 10 µM) did not suppress expression. (C) Phorbol 12,13-dibutyrate (PDBu; 100 nM) treatment of neonatal rat ventricular cells for three days increased nestin protein levels and co-treatment with SB203580 (SB; 10 µM) potentiated the response. The increased protein levels of nestin after PDBu and SB203580 co-treatment was attributed to the de novo expression of the intermediate filament protein in neonatal rat ventricular cardiomyocytes (See Supplemental Fig. 1). In each Western blot performed, nestin (~220 kDa) and GAPDH (~37 kDa) data represent images…

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  Figure 5. Immunohistochemical analysis of apex-resected 1-day old neonatal rat pups. (A1,A2) Three days after apex resection of vehicle-treated neonatal rat pups (n = 5), Masson trichrome staining revealed the presence of a large fibrin clot sealing the exposed left ventricular chamber. (B1,B2) Following administration of the p38α/β MAPK inhibitor SB203580 (5 mg/kg) to apex-resected neonatal rat hearts (n = 6), the overall fibrin clot region was smaller but did not reach statistical significance. However, the fibrin clot length sealing the exposed left ventricular chamber appeared to be reduced. In the SB203580-treated neonatal rat pup depicted in panel B2, the exposed left ventricular chamber was completely sealed with myocardial tissue and a large fibrin clot persisted. The blue line demarcates the fibrin clot region.

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  Figure 6. Morphology of the apex-resected neonatal rat heart. (A,B) As compared to sham rat pups (n = 5), left ventricular and septal wall thickness were significantly reduced after apex resection (APEX; n = 5). SB203580 (SB; 5 mg/kg) treatment of apex-resected neonatal rat pups (APEX + SB; n = 6) selectively restored left ventricular wall thickness. (C) Masson trichrome staining revealed that the fibrin clot area was reduced in SB203580-treated apex-resected neonatal rat pups, but did not reach statistical significance as compared to vehicle-treated apex-resected pups. (D) Immunofluorescence approach delineating ventricular cardiomyocytes by cardiac troponin-T staining revealed that SB203580 treatment of apex-resected hearts (n = 6) significantly shortened the fibrin clot length sealing the exposed left ventricular chamber as compared to vehicle-treated apex-resected hearts (n = 5). (*) Denotes P < 0.05 versus sham rats or vehicle-apex-resected hearts and (**) denotes P < 0.05 versus vehicle-treated apex-resected hearts.

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  Figure 7. Bromodeoxyurdine incorporation and phosphohistone-3 staining in the viable myocardium of the apex-resected region of untreated- and SB203580-treated rat hearts. (A) In vehicle-treated apex-resected neonatal hearts (n = 4), cardiac troponin-T staining (green fluorescence) of ventricular cardiomyocytes revealed the presence of a significantly large fibrin clot sealing the exposed left ventricular chamber. Phosphohistone-3 (PHH3; red fluorescence) and bromodeoxyuridine (BrdU; grey fluorescence) incorporation was detected adjacent to the fibrin clot and in the viable myocardium of the resected apex. (B) In the SB203580-treated apex-resected neonatal heart, cardiac troponin-T staining of ventricular cardiomyocytes revealed that the exposed left ventricular chamber was almost completely sealed. Phosphohistone-3 (PHH3) and bromodeoxyuridine (BrdU) incorporation was detected adjacent to the fibrin clot and in the viable myocardium of the resected apex. DAPI staining identifies the nucleus (blue fluorescence).

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  Figure 8. Quantitative assessment of cell cycle re-entry of NNVMs identified in the viable myocardium of the apex-resected region. The density of cardiac troponin-T (Trop-T) ventricular cardiomyocytes that incorporated (A) bromodeoxyuridine (Brdu) and (B) expressed phosphohistone-3 (PHH3) were significantly increased in SB203580-treated apex-resected neonatal hearts (APEX + SB; n = 5–6) compared to vehicle-treated apex-resected hearts (APEX; n = 4–5). (*) Denotes P < 0.05 versus untreated apex-resected hearts. (*) Denotes P < 0.05 versus vehicle-treated apex-resected hearts. (C) In SB203580-treated apex-resected neonatal hearts, marginalization of cardiac troponin-T to the periphery was detected in numerous ventricular cardiomyocytes concomitant with nuclear expression of the G2-M marker phosphohistone-3 (indicated by arrow). An analogous phenotype was identified in the vehicle-treated apex-resected neonatal rat heart. DAPI staining identifies the nucleus (blue fluorescence).

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  Figure 9. The predominant appearance of nestin(+)-cells along the border of the fibrin clot. (A) In a vehicle-treated apex-resected heart, a large fibrin clot sealing the exposed left ventricular chamber was evident. Furthermore, a population of nestin(+)-cells (red fluorescence) was detected bordering the entire length of the fibrin clot region. (B) In a SB203580-treated apex-resected heart, the resected left ventricle was completely sealed with myocardial tissue characterized by the appearance of cardiac troponin-T(+)-NNVMs. Furthermore, an important population of nestin(+)-cells (red fluorescence) was still prevalent juxtaposed to the fibrin clot. DAPI staining identifies the nucleus (blue fluorescence).

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  Figure 10. Nestin/cardiac troponin-T(+)-ventricular cardiomyocytes were detected exclusively adjacent to the fibrin clot region and a subpopulation re-entered the cell cycle. (A) The majority of nestin(+)-cells (red fluorescence) identified adjacent to the fibrin clot region were neonatal ventricular cardiomyocytes characterized by the co-expression of cardiac troponin-T (green fluorescence). (B) In SB203580-treated apex-resected hearts, a subpopulation of cardiac troponin-T/nestin(+)-neonatal ventricular cardiomyocytes bordering the fibrin clot region re-entered the cell cycle (indicated by arrow) characterized by bromodeoxyuridine (Brdu; grey fluorescence) incorporation. Cell cycle re-entry was also observed in cardiac troponin-T/nestin(+)-neonatal ventricular cardiomyocytes along the border of the vehicle-treated apex-resected heart (see Fig. 11). DAPI staining identified the nucleus (blue fluorescence).

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  Figure 11. Quantitative assessment of cell cycle re-entry of NNVMs and nestin(+)-NNVMs residing selectively adjacent to the fibrin clot region of the apex-resected heart. (A) The density of cardiac troponin-T(+)-ventricular cardiomyocytes bordering the fibrin clot region that incorporated bromodeoxyuridine was significantly greater in SB203580-treated apex-resected neonatal hearts (APEX + SB; n = 5) compared to vehicle-treated apex-resected hearts (APEX; n = 4). (B,C) The density of cardiac troponin-T(+)-ventricular cardiomyocytes adjacent to the fibrin clot that co-expressed nestin and concomitantly incorporated bromodeoxyuridine (BrdU) were significantly increased in SB203580-treated apex-resected neonatal hearts (APEX + SB; n = 5) compared to vehicle-treated apex-resected hearts (APEX; n = 4). (*) Denotes P < 0.05 versus vehicle-treated apex-resected hearts.