The Role Of ALDH2in Protective Effect Against Myocardial Ischemia Reperfusion Injury During Isoflurane Preconditioning:a Study Of Its Mechanism

Objective:Confirm the critical role of ALDH2in isoflurane-induced cardioprotection and detect the functional significance of manipulating ALDH2expression by construction of ALDH2knockdown adenovirus and constitutively active ALDH2mutant adenovirus.Methods (1) according to the sequence of ALDH2and the principle of SiRNA,we designed a siRNA refer to Cortinez, G’s paper, target the ALDH2gene we synthesis small hairpin siRNA, after denature and refolding,formed double strand ALDH2-siRNA.Using the DNA recombination technology,we linked ALDH2-siRNA and lined RetroQ vector. After Identification by restriction endonuclease digestion and replication, we cut the Rat-ALDH2-SiRNA from RetroQ-Rat-ALDH2and link it to pShuttle-Basic-ERFP shuttle vector, get the pShuttle-RFP-Rat-ALDH2-siRNA vector. Finally we transfer pShuttle-RFP-Rat-ALDH2-siRNA to pAdeno vector to get pAdeno-RFP-Rat-ALDH2-siRNA vector.(2) Previous studies demonstrated that the constitutively active ALDH2amino acid487must be Glu not Lys, and Thr185, Thr412and Ser279must be constitutively phosphorylated. Accordingly, we obtained constitutively active mutant ALDH2(CA-ALDH2) by nucleotide substitutions leading to the mutations Lys487Glu, Thr185Asp, Thr412Asp, and Ser279Asp introduced into the wild-type rat ALDH2cDNA.Using the DNA recombination technology, We linked CA-ALDH to pShuttle-CMV-RFP shuttle vector,the pShuttlet-CA-ALDH2-RFP vector. We transfer pShuttle-CA-ALDH2-RFP to pAdeno vector to get pAdeno-CA-ALDH2-RFP vector.Results:(1)Successfully constructed pAdeno-RFP-Rat-ALDH2-siRNA vector, after Identification by restriction endonuclease digestion, there is no mutation in the sequence, which is in our expectation.(2) Using the site-mutation technology we constructed pAdeno-CA-ALDH2vector after Identification by restriction endonuclease digestion, and got the right sequence, which Laid the foundation for our next experiments. Conclusions:Successfully constructed pAdeno-RFP-Rat-ALDH2-siRNA vector,Western blot analysis showed siRNA can inhibite ALDH2gene expression in rat Myocardial cells;Successfully constructed pAdeno-CA-ALDH2vector,Western blot analysis showed the CA-ALDH2can expressed in Myocardial cells quite well. Objective:Elucidate the role of ALDH2and in anti-apoptosis effect induced by isoflurane pretreatment by implementing specific activation or suppression on ALDH2gene, where neonatal rat cardiomyocytes were infected by pre-prepared ALDH2knockdown and constitutively active mutant adenovirus vectors.Methods:primary cultured one-day old neonatal SD rat cardiomyocytes were randomly and homogeneously divided into different experimental groups as follows: vector-infected with and without isoflurane, Adeasy-Si-ALDH2-treated with and without isoflurane; vector-infected with and without isoflurane, Adeasy-CA-ALDH2-treated with and without isoflurane. Exposure to isoflurane was performed by incubating the cells for5minutes in0.5mM isoflurane (approximately1.0minimum alveolar concentration) in0.5%FBS DMEM. The isoflurane-containing medium was removed immediately before the onset of hypoxic conditions and the cells were washed with phosphate-buffered saline(PBS). Simulated I/R was achieved by culturing the Isolated neonatal cardiomyocytes in0.5%FBS DMEM in a hypoxia chamber, saturated with5%CO2/95%N2at37℃for24h and following reoxygenation for12h using0.5%FBS DMEM in the normal incubating condition. At the end of H/R, the apoptosis pathway proteins(Cleaved caspase-3and full-length caspase-3) in different cell groups were determined by Western Blot analysis; the caspase-3enzyme activity of cell lysate in different groups was detected by Caspase-3Colorimetric assay kit; apoptosis index was performed using terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) with an in situ cell death detection kit, according to the manufacturer’s protocol for cultured cells and the lactate dehydrogenase(LDH)activity released from the cytosol of damaged cells into the culture medium was measured by Cytotoxicity Detection Kit (LDH, Roche). The role of ALDH2downregulation or activation in isoflurane-induced myocardial anti-apoptotic effect was evaluated by the values above.Results:Isoflurane preconditioning attenuated cardiomyocytes apoptosis induced by H/R injury in vitro:After24h of hypoxia followed by12h of reoxygenation we observed significant cardiomyocyte apoptosis demonstrated by increased DNA fragmentation using TUNEL staining, by laser scanning cytometry and caspase3activity in the vector control(Ad-RPF) group (Figure1B-E). Pretreatment with isoflurane significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase3activity and leakage of LDH (P<0.05, Figure1B-E). However, when ALDH2was downregulated (Figure1A) in cardiomyocytes by Ad-Si-ALDH2, increased TUNEL positive staining level, more intense cleaved caspase-3staining, caspase3activity and LDH release (P>0.05, FigurelB-E) were observed, which supports the hypothesis that ALDH2might play a critical role in isoflurane-induced cardioprotection. Immunoblotting analysis showed a substantial increase in ALDH2level in Ad-CA-ALDH2-RFP-infected cells compared to Ad-RFP (Figure2A). Constitutively active ALDH2induced by Ad-CA-ALDH2significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase3activity and leakage of LDH (P<0.05, Figure2B-E), which were not further increased by isoflurane treatment, i.e. no significant difference existed between control and isoflurane-pretreatment group. This suggests that activated (or phosphorylated) ALDH2involves isoflurane-induced cardioprotection.Conclusions:Isoflurane preconditioning alleviated in vitro H/R injury by activation of ALDH2. Downregulated ALDH2gene leads to a great increase in apoptotic cardiomyocytes. Constitutively activated ALDH2(upregulated ALDH2gene) results in a significant inhibition of H/R induced cardiomyocytes apoptosis, which indicates that activated ALDH2involves isoflurane-induced cardioprotection. This result supports our hypothesis that phosphorylated ALDH2may play a critical role in isoflurane-induced cardioprotection. The anti-apoptotic mechanism of ALDH2may involves inhibition of caspase-3and hence inhibition of apoptotic pathway. Objective:Reveal the role and mechanism of endogenic ALDH2and its phosphorylation at specific site in anti-apoptosis effect induced by isoflurane pretreatment by means of pharmacologic activation or inhibition of ALDH2in vivo.Methods:The acute myocardial I/R injury model was performed by LAD ligation. To confirm isoflurane-induced APC, a minimal alveolar concentration of isoflurane of1.0(2.1%) was started at the end of the stabilization period and administered for30minutes, followed by30minutes of washout before coronary occlusion. Rats were randomly assigned to one of the following groups subjected to different protocols:Sham groups, non-ischemic control group of sham-operated rats without and with isoflurane:rats were opened chest without ligating LAD for160minutes (n=5,respectively); I/R group andIsoP+I/R group:40minutes of myocardial ischemia and120minutes of reperfusion without and with isoflurane (n=5,respectively). To evaluate the role of ALDH2in isoflurane-induced cardioprotection, the direct activator of ALDH2, Alda-44(40μM), was given5minutes prior to ischemia in the groups without and with isoflurane (n=8, respectively), and the ALDH2inhibitor, cyanamide (5mM), was given without and with isoflurane (5minutes prior to isoflurane)(n=8, respectively). At the end of the reperfusion, the heart was removed and infarct size(IS) and area at risk(AAR) were assayed by Evans Blue and TTC staining. Before removing the heart,5ml blood samples were taken. Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were assayed using commercial kits by an automatic analyzer7600; signal pathway proteins (Phos-ALDH2and Total-ALDH2in different groups) were assayed by Western Blot analysis.Results:(1) Cardioprotective effect induced by Isoflurane preconditioning: Regional myocardial ischemia for40minutes by LAD ligation followed by120minutes of reperfusion markedly increased the leakage of LDH, CK-MB and myocardial infarctsize compared to sham controls(P<0.01). Isoflurane-induced APC significantly reduced the I/R-induced increase in LDH, CK-MB release in rat heart and substantially decreased I/R-induced myocardial infarct size (P<0.05). As shown in Figure1.(2) Phosphorylation of ALDH2participated in cardioprotection induced by IsoP:Pretreatment with isoflurane prior to ischemia increased the phosphorylation of ALDH2compared to I/R groups (P<0.01). The ALDH2inhibitor, cyanamide, significantly inhibited isoflurane-induced activation of ALDH2(P<0.05). The direct activator of ALDH2, Alda-44, substantially increased the phosphorylation of ALDH2P<0.01), but did not enhance the phosphorylation of ALDH2by isoflurane (图2A) Consistent with ALDH2phosphorylation,we observed that isoflurane and Alda-44markedly attenuated I/R-induced leakage of LDH and CK-MB in plasma, as well as myocardial infarct size (P<0.01,图2B-D) However, the isoflurane-induced decrease in LDH and CK-MB release, and reduction of infarct size was significantly blocked by the ALDH2inhibitor cyanamide (P<0.01, Figure2B-D). These findings suggest that isoflurane-mediated cardioprotection is mainly mediated by activation of ALDH2.Conclusions:LDH and CK-MB leakage from rat heart I/R injury is significantly reduced by APC effect by isoflurane preconditioning, with a decrease in infarct size caused by upregulation of ALDH2phosphorylation. We suggest isoflurane-induced cardioprotective effect may essentially result from ALDH2activation. Objective:Measure the activity of two PKC subtypes (PKCε and PKCδ) in isoflurane pretreatment against myocardial I/R injury by detection of mitochondrial translocation of both subtypes with Western blotting. Identify the role of both subtypes in ALDH2-mediated isoflurane pretreatment by application of inhibitors of both subtypes. Explore endogenic PKC and ALDH2signaling pathway and its molecular regulation mechanism.Methods:(1) To verify that PKCε participates in the phosphorylation of ALDH2, the PKCε inhibitor, PKCε v1-2(1μM), was given5minites prior to ischemia without and with isoflurane (n=8, respectively,).Analyze the concentration of LDH and CK-MB in the rat Serum. Evans Blue-TTC stainning to analyze infarct area in different groups (I/R, I/R+IsoP, I/R+εvl-2and I/R+IsoP+εvl-2group). Signaling pathway proteins(Phos-ALDH2, Total-ALDH2, Mito-PKCε and Total-PKCε in Four different groups) were assayed by Western Blot analysis.(2) To identify the effect of isoflurane preconditioning on PKCδ activation and translocation, we measured mito-PKCδ and total PKCδ expression in all groups (Sham, I/R, Sham+IsoP and I/R+IsoP group).(3) To identify the role PKCδ plays in phosphorylation of ALDH2, the PKCδ inhibitor, Rottlerin(1μM), was given5minites prior to ischemia without and with isoflurane (n=8).Analyze the concentration of LDH and CK-MB in the Serum. Signaling pathway proteins(Phos-ALDH2, Total-ALDH2in Four different groups) were assayed by Western Blot analysis.Results:(1) PKCε is involved in isoflurane-induced phosphorylation of ALDH2and cardioprotection:PKCε translocation to mitochondria and then phosphorylation of ALDH2is required to protect the heart from I/R injury. Compared with control group without isoflurane preconditioning, isoflurane pretreatment significantly enhanced mitochondrial PKCε concentration in cardiomyocytes (P<0.01), accompanied with significant increase in phosphorylated ALDH2in cardiomyocytes (P<0.01), whileεV1-2, a PKCε inhibitor, could significantly inhibit this effect (P<0.01). Here we demonstrate that pretreatment with isoflurane resulted in elevated mitochondrial levels of PKCε accompanied by phosphorylation of ALDH2. Isoflurane-induced phosphorylation of ALDH2was inhibited by the PKCε inhibitor, PKCεV1-2. Because mitochondrial translocation of PKCε occurs rapidly, with a corresponding decline in cytosolic PKCε levels, and because the total cellular PKCε levels do not change (FigurelA), our data suggest that pretreatment with isoflurane enables dynamic mitochondrial translocation of PKCε in response to I/R. Consistent with PKCε translocation to mitochondria, PKCεV1-2had a detrimental effect on isoflurane-induced attenuation of LDH and CK-MB leakage and the decrease in myocardial infarct size (Figure1B-D);(2) Pretreatment with isoflurane resulted in decreased PKCδ activity and mitochondrial levels in cardiomyocytes. Our data shows that compared to sham groups, I/R led to a significant increase in PKCδ levels in mitochondria of cardiomyocytes (P<0.01), Isoflurane preconditioning greatly inhibits mitochondria translocation of PKCδ in cardiomyocytes, and significantly decreases mitochondria PKCδ concentration in cardiomyocytes (P<0.01)(Figure2A-B).(3) Decreased level of PKCδ translocation to mitochondria involves isoflurane preconditioning-induced cardioprotection:Isoflurane-afforded inhibition of ALDH2phosphorylation level decrease (Figure3A-B) and LDH and CK-MB release (Figure3C-D) by I/R was mimicked by Rottlerin. Western blot showed that inhibition of PKCδ significantly elevated phosphorylation of ALDH2regardless of isoflurane preconditioning. Similarly, isoflurane preconditioning inhibits PKCδ activity, thus inhibiting its mitochondria translocation to act against I/R-induced myocardial injury.Conclusions:Pretreatment with isoflurane resulted in significantly elevated mitochondrial levels of PKCε accompanied by phosphorylation of ALDH2, in the same time attenuated translocation of PKCδ. The bi-directional regulation of PKC two subtypes activation level during I/R would further activate ALDH2, and guarantee resistance to ischemia-reperfusion injury. Isoflurane preconditioning may activate PKC (PKCεand PKCδ)-ALDH2signaling pathway to induce protective effect against myocardial I/R injury.