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Background A number of studies suggest that the expression of heat shock protein 70(HSP_(70))induced by heat stressare associated with protection against ischemia-reperfusion injury.But the protective effects may be contaminated byother factors in the same stress.This study was conducted to explore the protective role of HSP_(70)expression in acutemyocardial anoxia/reoxygeneration(A/R)injury with a liposome-mediated gene transfer technique for the introduction ofpCDNA HSP_(70)into the neonatal rat myocardial cells.In addition,heat shock stress cytoprotection was also investigatedfor comparison.Methods The cultured primary neonatal rat myocardiocytes with an acute myocardial A/R injury model and theHS-treated rat myocardiocyte model were used.Three-day cultured myocardiocytes were randomly divided into fourgroups(n=8):control group,A/R group,HS+A/R group and pCDNA HSP_(70)+A/R group.A liposome-coated HSP_(70)pCDNAplasmid was transfected into the primary neonatal rat myocardiocytes;HSPTo mRNA and its protein were confirmed byreverse transcriptase polymerase chain reaction(RT-PCR)and Western blotting.The cell viability was assayed bymonotetrazolium(MTT)and the lactate dehydrogenase(LDH)and creatine phosphokinase(CPK)activity of cells duringincubation and the changes in cells ultrastructure were examined.NF-κB activity in the primary neonatal ratmyocardiocytes 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 CPKwas significantly elevated in the A/R group.However,in the HS+A/R group and pCDNA HSP_(70)+A/R group,significantdecreases in activity were observed.The cell ultrastructure of the A/R group cells was abnormal,whereas nearly normalultrastructure was observed in HS+A/R group and pCDNA HSP_(70)+A/R group.HSP_(70)mRNA and protein were slightlyexpressed in the myocardiocytes of the A/R group.However,obvious overexpression was observed in the HS+A/R groupand in the pCDNA HSP_(70)+A/R group(P<0.01).And there was a significant difference between the HS+A/R group and thepCDNA HSP_(70)+A/R group in the expression of HSP_(70)mRNA and protein(P<0.01).A high activity of NF-κB(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 ofNF-κB compared with the A/R group(3.11±0.52 vs 5.76±0.64,P<0.01).The same statistically significant difference wasalso 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 againstanoxia-reoxygeneration.These cardioprotective effects were related to the reduction in activation of NF-κB.
Background A number of studies suggest that the expression of heat shock protein 70 (HSP 70) induced by heat stress associated with protection against ischemia-reperfusion injury. But the protective effects may be contaminated byother factors in the same stress.This study was conducted to explore the protective role of HSP_ (70) expression in acutemyocardial 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. Shock stress cytoprotection was also carriedfor comparisons.Methods The cultured primary neonatal rat myocardiocytes with an acute myocardial A / R injury model and theHS-treated rat myocardiocyte were were divided.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) pCDNAplasmid was transfected into the primary neonatal rat myocardiocytes; HSPTo mRNA and its proteins 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 duringincubation and the changes in cells ultrastructure were examined. NF-κB 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 significantly (P <0.05). The activity of LDH and CPK was significantly elevated in the A / R group . Significantly in the HS + A / R group and pCDNA HSP_ (70) + A / R group, significant enzymes in the activity were observed. The cell ultrastructure of the A / R group cells was abnormal, as nearly normalultrastructure was observed in HS + A / R group and pCDNA HSP_ (70) + A / R group.HSP_ (70) mRNA and protein were slightlyexpressed in the myoca rdioOf course, significant 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 high activity of NF-κB (5.76 ± 0.64) was detected in the A / R group and the pCDNA HSP_ (70) + A / R group in the expression of HSP_ (70) mRNA and protein R group.But in the HS + A / R group there was a significant displacement in the activity of NF-κB compared with the A / R group (3.11 ± 0.52 vs 5.76 ± 0.64, P <0.01) .the same statistically significant difference wasalso 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 (70) alone by gene transfection leads to protection for cardiac myocyte againstanoxia-reoxygeneration.These cardioprotective effects were related to the reduction in activation of NF-κB.