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Localization and expression of Hsp27 and ?B-crystallin in rat primary myocardial cells during heat stress in vitro.

ABSTRACT: Neonatal rat primary myocardial cells were subjected to heat stress in vitro, as a model for investigating the distribution and expression of Hsp27 and ?B-crystallin. After exposure to heat stress at 42°C for different durations, the activities of enzymes expressed during cell damage increased in the supernatant of the heat-stressed myocardial cells from 10 min, and the pathological lesions were characterized by karyopyknosis and acute degeneration. Thus, cell damage was induced at the onset of heat stress. Immunofluorescence analysis showed stronger positive signals for both Hsp27 and ?B-crystallin from 10 min to 240 min of exposure compared to the control cells. According to the Western blotting results, during the 480 min of heat stress, no significant variation was found in Hsp27 and ?B-crystallin expression; however, significant differences were found in the induction of their corresponding mRNAs. The expression of these small heat shock proteins (sHsps) was probably delayed or overtaxed due to the rapid consumption of sHsps in myocardial cells at the onset of heat stress. Our findings indicate that Hsp27 and ?B-crystallin do play a role in the response of cardiac cells to heat stress, but the details of their function remain to be investigated.

SUBMITTER: Tang S

PROVIDER: S-EPMC3716771 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Localization and expression of Hsp27 and αB-crystallin in rat primary myocardial cells during heat stress in vitro.

Tang Shu S Buriro Rehana R Liu Zhijun Z Zhang Miao M Ali Islam I Adam Abdelnasir A Hartung Jörg J Bao Endong E

PloS one 20130719 7

Neonatal rat primary myocardial cells were subjected to heat stress in vitro, as a model for investigating the distribution and expression of Hsp27 and αB-crystallin. After exposure to heat stress at 42°C for different durations, the activities of enzymes expressed during cell damage increased in the supernatant of the heat-stressed myocardial cells from 10 min, and the pathological lesions were characterized by karyopyknosis and acute degeneration. Thus, cell damage was induced at the onset of ...[more]

PMID: 23894407

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Project description:We previously demonstrated that myocardial p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) are phosphorylated following cardioplegic arrest in patients undergoing cardiac surgery and correlate with reduced cardiac function. The following studies were performed to determine whether inhibition of p38 MAPK and/or overexpression of nonphosphorylatable HSP27 improves cardiac function following cardioplegic arrest. Langendorff-perfused isolated rat hearts were subjected to 2 h of intermittent cold cardioplegia followed by 30 min of reperfusion. Hearts were treated with (CP+SB) or without (CP) the p38 MAPK inhibitor SB-203580 (5 μM) supplied in the cardioplegia. Sham-treated hearts served as controls. In separate experiments, isolated rat ventricular myocytes infected with either green fluorescent protein (GFP) or a nonphosphorylatable HSP27 mutant (3A-HSP27) were subjected to 3 h of cold hypoxic cardioplegia and simulated reperfusion (CP) followed by video microscopy and length change measurements. Baseline parameters of cardiac function were similar between groups [left ventricular developed pressure (LVDP), 119 ± 4.9 mmHg; positive and negative first derivatives of LV pressure (± dP/dt), 3,139 ± 245 and 2, 314 ± 110 mmHg/s]. CP resulted in reduced cardiac function (LVDP, 72.2 ± 5.8 mmHg; ± dP/dt, 2,076 ± 231 and -1,317 ± 156 mmHg/s) compared with baseline. Treatment with 5 μM SB-203580 significantly improved CP-induced cardiac function (LVDP, 101.9 ± 0 mmHg; ± dP/dt, 2,836 ± 163 and -2,108 ± 120 mmHg/s; P = 0.03, 0.01, and 0.04, CP+SB vs. CP). Inhibition of p38 MAPK significantly lowered CP-induced p38 MAPK, HSP27, and αB-crystallin (cryAB) phosphorylation. In vitro CP decreased myocyte length changes from 10.3 ± 1.5% (GFP) to 5.7 ± 0.8% (GFP+CP). Infection with 3A-HSP27 completely rescued CP-induced decreased myocyte contraction (11.1 ± 1.0%). However, infection with 3A-HSP27 did not block the endogenous HSP27 response. We conclude that inhibition of p38 MAPK and subsequent HSP27 and cryAB phosphorylation and/or overexpression of nonphosphorylatable HSP27 significantly improves cardiac performance following cardioplegic arrest. Modulation of HSP27 phosphorylation may improve myocardial stunning following cardiac surgery.

Project description:Cell shape and adhesion and their proper controls are fundamental for all biological systems. Mesenchymal cells migrate at an average rate of 6 to 60 μm/hr, depending on the extracellular matrix environment and cell signaling. Myotubes, fully differentiated muscle cells, are specialized for power-generation and therefore lose motility. Cell spreading and stabilities of focal adhesion are regulated by the critical protein vinculin from immature myoblast to mature costamere of differentiated myotubes where myofibril Z-band linked to sarcolemma. The Z-band is constituted from microtubules, intermediate filaments, cell adhesion molecules and other adapter proteins that communicate with the outer environment. Mesenchymal cells, including myoblast cells, convert actomyosin contraction forces to tension through mechano-responsive adhesion assembly complexes as Z-band equivalents. There is growing evidence that microtubule dynamics are involved in the generation of contractile forces; however, the roles of microtubules in cell adhesion dynamics are not well determined. Here, we show for the first time that αB-crystallin, a molecular chaperon for tubulin/microtubules, is involved in cell shape determination. Moreover, knockdown of this molecule caused myoblasts and glioma cells to lose their ability for adhesion as they tended to behave like migratory cells. Surprisingly, αB-crystallin knockdown in both C6 glial cells and L6 myoblast permitted cells to migrate more rapidly (2.7 times faster for C6 and 1.3 times faster for L6 cells) than dermal fibroblast. On the other hand, overexpression of αB-crystallin in cells led to an immortal phenotype because of persistent adhesion. Position of matured focal adhesion as visualized by vinculin immuno-staining, stress fiber direction, length, and density were clearly αB-crystallin dependent. These results indicate that the small HSP αB-crystallin has important roles for cell adhesion, and thus microtubule dynamics are necessary for persistent adhesion.

Dataset Information

Localization and expression of Hsp27 and ?B-crystallin in rat primary myocardial cells during heat stress in vitro.

Publications

Localization and expression of Hsp27 and αB-crystallin in rat primary myocardial cells during heat stress in vitro.

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