Mechanism Research Of CaMEK Gene Targeting Transduction In Reducing The Aging Myocardial Ischemia-reperfusion Injury

Objective: This study was designed to investigate the effect of ageing on myocardial protection of ischemic postconditioning and to detect the molecular mechanisms of disappearance of myocardial protection of ischemic postconditioning caused by ageing. We used cytomegalovirus promoter(CMV) and chicken β-actin promoter(CBA) recombinant AAV9 vector to validate tissue-specific expression, and screened to obtain better AAV9 vectors which can be used in gene therapy of heart disease. We used Ca MEK(Constitutively active MEK1, Ca MEK) gene recombinant AAV9 vector for the targeting transduction in aging mouse heart, to investigate whether exogenously introduced Ca MEK can be used in gene therapy, it can lead to targeted activation of ERK1/2, reduction of myocardial infarction, inhibition of myocardial apoptosis, prevention of myocardial ischemia-reperfusion injury. The study included: ①Application of mouse Langendorff isolated heart perfusion system, to compare the myocardial protection differences of postconditioning in young and aging mice, to clarify the heterogeneity and molecular mechanisms of ageing in ischemic myocardial protection. ②Via in vivo transduction experiments, to compare baculovirus vector system packaged recombinant vector AAV9 of CBA and CMV promoter, to study the GFP-mediated gene expression and specific time points of expression in mice tissues, to evaluate the safty of the recombinant AAV9 vector which were more suitable for gene therapy of heart disease. ③Targeted Ca MEK gene transduction of ageing mice myocardium, via in vitro and in vivo myocardial ischemia-reperfusion model, to investigate the mechanism of Ca MEK gene overexpression to reduce aging myocardial ischemia-reperfusion injury.Methods: Part I: To select male C57BL/6J mice with age of 3 to 4-month and 15 to 18-month, to establish Langendorff in vitro ischemic postconditioning model, isolated reperfusion(IR) heart undergo 45 minof global ischemia and 60 minof reperfusion, immediately after reperfusion, postconditioning(IPost) was performed as three cycles 10 s ischemia + 10 s reperfusion IPost treatment, young mice were administered with PD98059(ERK1/2 inhibitor). The myocardial infarct size, myocardial apoptosis, p-ERK1/2, and the expression of p-P70S6 K of p-AKT were observed. Part II: AAV9-CMV-e GFP and AAV9-CBA-e GFP vector with the dose of 1 × 1011 vg were injected into C57BL/6J mice via the tail vein, respectively. At 0 week, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 8 weeks of viral vector transduction, the laser confocal and Western Blot were used to detect GFP protein expression in heart, liver, spleen, lung, kidney, brain and skeletal muscle. AAV9 vector copy number was detected in different tissues using q-PCR, distribution, expressiom time of AAV9 viral particles were detected in vivo study, effects of r AAV9 vector transfection on were evaluated. The biochemical analyzer was used to detect serum cardiac enzymes LDH and CK, liver function AST and ALT,renal function Cr and BUN levels were also detected. We evaluated the targeting effects of CBA and CMV promoter recombinant AAV9 vectors. At the cellular level, primary cultured neonatal rat cardiomyocytes and BRL-3A rat liver cell line were used, they were transfected via AAV9-CMV-e GFP and AAV9-CBA-e GFP vector with different infection multiplicity(MOI=105, 106, 107).The fluorescence microscopy and cells loss machine were used to measure transfection efficiency of AAV9 vectors in vitro, TUNEL staining was used to detect apoptosis of cardiomyocytes and hepatocytes. Part III: 15 to 18 month aging male C57BL/6J mice were selected, saline, AAV9-CMV-e GFP and AAV9-CMV-Ca MEK were injected via the tail, respectively. 5 weeks later, Western Blot assay was used to detect myocardium MEK, p-ERK1/2 expression and p-P90 RSK. 5 weeks after in viral transduction, Langendorff isolated heart perfusion experiments and in vivo ischemia-reperfusion injury mice model were used, in vitro treatment was the same with the Part I. In vivo hearts were subjected to 60 min of ischemia and 24 h of reperfusion. The myocardial infarct size, apoptosis, the expression of cardiac enzymes CK, CK-MB, LDH, Bcl-2 and Bax were detected,Result: Part I: ① To compared with direct ischemia-reperfusion, ischemic postconditioningcan significantly decrease ischemia and myocardial infarction area in young mice,(39.7 ±4.6% vs 24.9±2.9%, P < 0.01); and cardiac myocyte apoptosis also significantly decreased(12.2±1.8% vs 7.7±1.3%, P<0.01). ②Ischemic postconditioning showed no myocardial ischemia protective effect in aging mice, infarct area in IR group and myocardial IPost group were(49.5 ± 7.5% vs 47.5 ± 7.3%, P>0.05), the apoptosis rate were(13.5 ± 2.3% vs 13.7 ± 2.4%, P>0.05), myocardial infarct size in aging mice was significantly higher than that in young mice group(P<0.01).③Ischemic postconditioning induced p-ERK1/2 and p-P70S6 K expression increase in early reperfusion of young myocardial. But this effect was not seen in ageing mice cardiac effects. ERK1/2 inhibitor PD98059 can make protective effect of ischemic postconditioning completely disappeare in young myocardial, at the same time, ischemic postconditioning can’t induce increased p-AKT expression in both young and ageing mice myocardial. Part II: ①Confocal laser scanning and Western Blot showed that at 5 weeks of CMV and CBA recombinant AAV9 vector transduction, GFP protein was mainly expressed in cardiac and liver, there was almost no expression in spleen, lung, kidney, brain and skeletal muscle. ②At each detection time point, CMV promoter induced GFP protein expression in the myocardium was significantly higher than that in the liver. 1 week after AAV9 vectors transduction, myocardial and liver began to express GFP protein, with the extension of time, the expression gradually increased, and transduction reached the peak at first 5 weeks. Its heart and liver transfection efficiency were(84.0 ± 3.2% vs 23.6 ± 3.4%, P<0.01) and(75.0 ± 3.5% vs 2.2 ± 0.3%, P<0.01) at 8 weeks. It indicated that AAV9-CMV-e GFP vector showed strong target affinity for the myocardial, it can be stably, efficiently expressed in the myocardium for long-term. However CMV promoter activity can be easily silenced in the liver. ③ AAV9-CBA-e GFP vector has a strong hepatotropism, at each observation point, its expression in the liver was significantly higher than that in the myocardium, the expression was enhanced with time and it reached the peak at 5 weeks. At this point, the liver and heart transfection efficiency were(88.0 ± 3.9% vs 7.7± 0.7%, P<0.01) and(84.4 ± 2.4% vs 6.4 ± 0.4%, P<0.01) at the 8 weeks. ④At each detection point, q-PCR detection showed that CMV promoter AAV9 vector distribution was significantly higher than the CBA promoter in the myocardium(P<0.01). It further confirmed myocardial tropism of AAV9-CMV-e GFP vector and hepatotropism of AAV9-CBA-e GFP vector. ⑤The serum cardiac enzymes LDH and CK, liver function AST and ALT,renal function Cr and BUN levels showed no significant changes between r AAV9 groups and saline group(P>0.05). ⑥Flow cytometry showed that CMV and CBApromoter AAV9 vectors both can efficiently transfect cardiomyocytes and hepatocytes. With the increase of infectious dose and it gradually increased. However, when compared at the same infection dose, the transfection efficiency of two promoter showed no significant difference in cardiac myocytes and hepatocytes(P>0.05). ⑦Compared to non-transfected cells, high doses of CMV and the CBA AAV9 vector transfected cells did not result in significant cardiac and liver cell apoptosis. Part III: ①Ca MEK gene transduction can efficiently activate aging cardiac ERK1 / 2 signaling pathway. When compared between the control group and the empty virus group, the expression of MEK, p-ERK1/2, p-P70S6 K and p-P90 RSK significantly increased(P<0.01). ② For in vitro heart research, Ca MEK gene overexpression can lead to aging myocardial protective effects. The myocardial infarct size in empty virus group, Ca MEK gene transduction IR and IPost was 48.0 ± 7.7% vs 44.0 ± 8.0%, 31.3 ± 5.3% vs 28.5 ± 6.5%, the myocyte apoptosis rate was 14.3 ± 2.1% vs 13.6 ± 1.9%, 5.0 ± 1.3% vs 4.9 ± 1.2%. The infarct size and apoptosis rate was statistically significant different when compared with the empty virus group(P<0.01). It indicated that ERK1/2 signaling pathway can effectively protect the ageing ischemic myocardium after activation. ③For in vivo heart research, Ca MEK gene overexpression can equally and effectively reduce infarct size and aging cardiac cell apoptosis. At 1h after aging myocardial ischemia, 24 h after reperfusion, infarct size in control, empty virus and Ca MEK groups were 44.2 ± 7.4% vs 43.4 ± 6.0% vs 27.5 ± 4.7%. The myocyte apoptosis rate was 16.1 ± 1.9 % vs 16.4 ± 2.0 % vs 5.5 ± 2.3%, when compared with the control and empty virus(P<0.01). The apoptosis related protein Bcl-2 / Bax significantly increased, when compared with empty virus group(P<0.01). ④ In Ca MEK group, cardiac enzymes of CK, CK-MB, LDH was also significantly reduced, the difference was statistically significant.Conclusion: ①ERK1/2 signaling pathway is involved in ischemic postconditioning and mediated myocardial protection as age heterogeneity key facor. Postconditioning in early reperfusion alleviated myocardial ischemia-reperfusion injury via activating ERK1/2 signaling pathway in young mice, the AKT signaling pathway is not involved in the cardioprotective effect of ischemic postconditioning. ② Myocardial ischemia postconditioning did not show the protective effect in aging myocardial, it was associated with valid activation of ERK1/2 signaling pathway, and the sensitivity to ischemia-reperfusion injury significant increased in the aging myocardial, the infarct area and apoptosissignificant increased after myocardial infarction and reperfusion. ③CMV promoter recombinant AAV9 vectors can be expressed in cardiac efficiently and stably for a long-term. However, the CMV promoter activity can easily be silent in the liver, AAV9-CMV vector is an ideal vector for gene therapy of heart disease. ④ CBA promoter recombinant AAV9 vectors have high liver tropism, it can mediate exogenous gene to be expressed in the liver stably and for a long-term, it is an ideal vector system for gene therapy of liver disease. ⑤Targeted activation of cardiac ERK1/2 signaling pathway can stimulate endogenous myocardial damage resistance, so it is key targets for prevention and treatment of the aging myocardial ischemia-reperfusion injury. The Ca MEK gene can be used as small molecule drugs of gene therapy, it can targeted activate ERK1/2 signaling pathway, reduce myocardial infarction, inhibit myocardial apoptosis, prevent myocardial ischemia- reperfusion injury in aging mice to effectively protect the ageing myocardial ischemia.