Heat shock protein 70 gene transfection protects rat myocardium cell against anoxia-reoxygeneration injury.

BACKGROUND: A number of studies suggest that the expression of heat shock protein 70 (HSP(70)) induced by heat stress are associated with protection against ischemia-reperfusion injury. But the protective effects may be contaminated by other factors in the same stress. This study was conducted to explore the protective role of HSP(70) expression in acute myocardial anoxia/reoxygeneration (A/R) injury with a liposome-mediated gene transfer technique for the introduction of pCDNA HSP(70) into the neonatal rat myocardial cells. In addition, heat shock stress cytoprotection was also investigated for comparison.
METHODS: The cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and the HS-treated rat myocardiocyte model were used. Three-day cultured myocardiocytes were randomly divided into four groups (n = 8): control group, A/R group, HS + A/R group and pCDNA HSP(70) + A/R group. A liposome-coated HSP(70) pCDNA plasmid was transfected into the primary neonatal rat myocardiocytes; HSP(70) mRNA and its protein were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell viability was assayed by monotetrazolium (MTT) and the lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activity of cells during incubation and the changes in cells ultrastructure were examined. NF-kappaB activity in the primary neonatal rat myocardiocytes was measured with flow cytometry.
RESULTS: Compared with viability in the A/R group ((35.4 +/- 6.9)%) the cell viability in the HS + A/R group ((72.8 +/- 11.6)%) and the pCDNA HSP(70) + A/R group ((76.3 +/- 12.2)%) was improved significantly (P < 0.05). The activity of LDH and CPK was significantly elevated in the A/R group. However, in the HS + A/R group and pCDNA HSP(70) + A/R group, significant decreases in activity were observed. The cell ultrastructure of the A/R group cells was abnormal, whereas nearly normal ultrastructure was observed in HS + A/R group and pCDNA HSP(70) + A/R group. HSP(70) mRNA and protein were slightly expressed in the myocardiocytes of the A/R group. However, obvious overexpression was observed in the HS + A/R group and in the pCDNA HSP(70) + A/R group (P < 0.01). And there was a significant difference between the HS + A/R group and the pCDNA HSP(70) + A/R group in the expression of HSP(70) mRNA and protein (P < 0.01). A high activity of NF-kappaB (5.76 +/- 0.64) was detected in the A/R group. But in the HS + A/R group there was a statistically significant decrease in the activity of NF-kappaB compared with the A/R group (3.11 +/- 0.52 vs 5.76 +/- 0.64, P < 0.01). The same statistically significant difference was also observed in the pCDNA HSP(70) + A/R group and A/R group (2.83 +/- 0.49 vs 5.76 +/- 0.64, P < 0.01).
CONCLUSIONS: Overexpression of HSP(70) alone by gene transfection leads to protection for cardiac myocyte against anoxia-reoxygeneration. These cardioprotective effects were related to the reduction in activation of NF-kappaB.