Protective Effects Of Heat Shock Protein A12B On Myocardial Ischemia/Reperfusion Injury In Mice

Background and Purpose:Coronary heart disease(CHD)has been one of the main causes of death in the whole world.The prevalence of this disease and morbidity is increasing.Moreover,CHD is affecting more young individuals.So far,the reperfusion therapy has already been the leading strategy in CHD therapeutic process.Restoring the blood flow after the ischemia has been proved to limit the size of infarction,to improve the long-term prognosis and to reduce the death of cardiac attack.However,at the early stage of reperfusion,additional damage to myocardium could appear,including the increasement of infracted area,the arrhythmia or even the sudden death.This phenomenon is known as ischemia/reperfusion(I/R)injury.The decrease of the damage from I/R is the way to preserve the benefit of the reperfusion therapy.The pathogenesis of myocardial I/R Injury is still unclear.Data shows it could be related to endothelial dysfunction,oxidative stress and calcium overload.Apart from these,I/R could induce myocardium apoptosis through changing the level of pro-apoptosis protein and anti-apoptosis protein or activating the death receptor.The result of apoptosis is the loss of volume of myocardium cells and the impairment of cardiac function.From this view,myocardium apoptosis is one of the essential pathogenesis of I/R injury and the reduction of apoptosis means a lot to myocardial protection.According to recent studies,heat shock protein A12B(HSPA12B),cloned in atherosclerotic lesions,protected the cerebrum from I/R injury by keeping the integrity of blood brain barrier and reducing the apoptosis of brain cell and also attenuated the remodeling after myocardial infarction by increasing the density of capillary.However,the biological function of HSPA12B in myocardial I/R injury is still unclear.In this study,mice that overexpressing HSPA12B were used to explore the role of HSPA12B in myocardial I/R injury and the underlying mechanism.Methods:1.Creation of hspal2b transgenic mice:We research team cooperated with Nanjing University Model Animal Research Institute.The animal model was created through introducing the human hspal2b transgene into the pronuclei of C57BL/6 mouse fertilized eggs by microinjection and the verification was tested by immunoblot and immunofluorescent.2.Creation of myocardial I/R model:After inhaling of 1.5-2%isoflurane,the heart was exposed and the left anterior descending coronary artery was ligated for 45 min.Then the artery was reperfused by releasing the knot of suture.In the sham group,the same procedure was performed except for LAD ligation.3.Measurement of HSPA12B:The mRNA expression of HSPA12B was measured by real-time PCR.4.Evaluation of cardiac function:The cardiac function was evaluated by echocardiogram.5.Measurement of the infarction area:The infarction area was demonstrated by TTC staining.6.Assessment of myocardium apoptosis:Apoptosis in cardiomyocyte was detected using the TUNEL assay and the protein associated with apoptosis was detected by immunoblot.7.Exploration of signaling pathway:the protein around the PI3K/Akt/mTOR signaling pathway was detected by immuoblotting.The cardiac function was assessed again after treating the mice with the inhibitor of PI3K.8.Statistical analysis:Measurement data is expressed in mean differences ±standard deviation(?± SD).The data was handled with t-test,ANOVA and Tukey post hoc test by GraphPad Prism6,and P<0.05 was significant.Results:1.Myocardial I/R increased the mRNA expression of HSPA12B expression.2.Overexpression of HSPA12B improved the systolic function.3.Overexpression of HSPA12B limited the infarction area.4.Overexpression of HSPA12B reduced myocardium apoptosis.5.Overexpression of HSPA12B activated the PI3K/Akt/mTOR signaling pathway.6.The protection of HSPA12B against I/R induced myocardial disfunction disappeared after the inhibition of the PI3K/Akt/mTOR signaling pathway.Conclusions:Myocardial I/R injury increased the expression of HSPA12B and the overexpression of HSPA12B is demonstrated to improve the cardiac function,to limit the infracted area and to reduce the myocardium apoptosis.These cardiac protections were depended on the activation of the PI3K/Akt/mTOR signaling pathway.The research results suggested that HSPA12B could be an alternative approach to the treatment of I/R injury.