The Interventive Effects Of TSA On Myocardial Ischemia-reperfusion Injury In Rat Through Endoplasmic Reticulum Stress Regulation And Its Mechanism

The endoplasmic reticulum (ER) is an important membranous organelle in theeukaryotic cells and its main functions include nascent polypeptide folding, assembly,modification and secretion; lipids synthesis and calcium storage. In fact, ER isextremely sensitive to stress stimuli like ischemia, hypoxia and etc. Those stimuli cancause unfolded or misfolded proteins in the ER continuously accumulated and breakthe ER homeostasis, which is termed as endoplasmic reticulum stress (ERS).Moderate ERS can initiate the unfolded protein response (UPR) to recover the ERhomeostasis. However, when the stress persists or strength is too strong, UPR isunable to alleviate the pressure brought by unfolded or misfolded proteins, ERS willinitiate the apoptosis, resulting in tissue damage.The resupply the blood of ischemic myocardium will cause further irreversibledamage in heart, this phenomenon is known as myocardial ischemia and reperfusion(I/R) injury. The mechanism of I/R injury is complicated, but ERS-induced apoptosishas been shown to be one of the most important pathophysiological mechanismsleading to myocardial I/R injury. Therefore, I/R injury may be alleviated throughregulation of ERS.Trichostatin A (TSA) is known to be one of the most effective histone deacetylase(HDAC) inhibitors by now. TSA can inhibit the myocardial autophagy, apoptosis andinflammatory cytokines production, which has an interventive effect on a variety ofheart disease. In myocardial I/R injury model, TSA can reduce the myocardial infarctsize and improve cardiac function as well as inhibit the cardiomyocyte apoptosis, butthe mechanism remains unclear. Moreover, studies have shown that HDAC canparticipate in the transcriptional regulation of ERS proteins GRP78the CHOP,therefore, we speculate that TSA may regulate ERS and prevent the myocardial I/Rinjury through ERS regulation. To verify this hypothesis, we studied the interventiveeffects of TSA on myocardial ischemia-reperfusion injury in rat through endoplasmic reticulum stress regulation and explored its mechanism from the following fouraspects:1. Screening of ERS-related genes affected by TSA by bioinformatics methodSo far, there is no report found on the effect of TSA on ERS gene expression.Therefore, we first used bioinformatics method to analyze the cDNA microarray byTSA treatment in order to screen ERS-related genes affected by TSA. The resultsshow that there ERS genes, including caspase12, were significant changed after TSAtreatment.2. Effect of TSA on ERS induced by tunicamycin (Tm) in H9c2cellsThe gene chip screening showed that TSA can affect ERS genes expression inmice, but it is still unknown whether cardiomyocytes ERS in rat can be influenced byTSA, so we conducted the following studies:(1) Tm is the commonly used ERS inducer in vitro. In order to clarify the optimalworking concentration of Tm in H9c2cells, we detected the H9c2cell viability afterTm by CCK-8and determined1μM as its working concentration. Real-timequantitative PCR showed that mRNA expressions of ERS genes GRP78CHOP andCaspase12were changed after different time with Tm, which indicated that Tm caninduce ERS in H9c2.(2) In order to investigate the impact of TSA on ERS induced by Tm, we first usedimmunofluorescence staining and western blot methods to observe the expressionchanges of ERS marker protein GRP78. The results show that, compared with thecontrol group, GRP78expression in Tm group was significantly raised (P<0.01); TSAfurther increased GRP78expression in comparion to Tm group (P<0.05). Thedetection of Caspase12expression by Western blot showed that Tm can increaseprocaspase-12and active caspase-12expression (Tm vs control, all P<0.05), however,procaspase-12(Tm+TSA vs Tm, P<0.05) and active caspase12in the Tm+TSAgroup (Tm+TSA vs Tm, P<0.01) were significantly reduced, suggesting that TSA canaffect the ERS induced by Tm.(3) To further observe the impact of TSA on apoptosis in H9c2cell induced by ERS, we detected H9c2cell apoptosis rates by AnnexinV-FITC/PI double staining. Theresults showed that the rate of apoptosis was significantly higher in Tm group (Tm vscontrol, P<0.01) while lower in Tm+TSA group (Tm+TSA vs Tm, P<0.05),suggesting that TSA can reduce the ERS-induced apoptosis in H9c2cells.(4) HDAC2has been reported to be closely related to heart disease. In order toobserve whether HDAC2is involved in ERS, and whether TSA can inhibit theactivity of HDAC2, we detected the HDAC2activity by colorimetric method. It isfound that compared with the control group, given Tm significantly increasedHDAC2activity (Tm vs control, P<0.05); TSA significantly reduced HDAC2activity(Tm+TSA vs Tm, P<0.05), suggesting that HDAC2may be involved in the ERSregulation.3. Effect of TSA on ERS induced by myocardial I/R injury in rats.Male Wistar rats were used and pretreated with saline or TSA (0.05,0.1and0.2mg·kg–1) once daily intraperitoneally for5days. Whether myocardial I/R injury caninduce ERS, we first established the myocardial I/R model by occlusion/release ofthe left anterior descending coronary artery. Immunohistochemistry was used to detectthe changes of ERS key proteins GRP78、XBP1and CHOP after I/R injury. It wasfound that GRP78, XBP1and CHOP were elevated greatly in I/R group, comparedwith Sham group. The results were further confirmed by western blot analysis on ERSkey proteins GRP78and CHOP expression. Next, whether pre-administration of TSAcan influence myocardial I/R-induced ERS? To solve this problem, Western blotmethod was used to quantitative detect the expression changes of ERS key proteinsGRP78, CHOP, Caspase12and ATF4. Results showed that, compared with the Shamgroup, GRP78, of CHOP, Caspase12and ATF4proteins expression were increasedafter myocardial I/R, while GRP78expression was increased and expression of CHOP,Caspase12and ATF4proteins were reduced in the I/R+TSA group. Furthermore, itwas found that TSA significantly reduced the myocardial infarct size, plasmaactivities of lactate dehydrogenase and creatine kinase in a dose-dependent manner inrats. Accompanied by the reduced injury, TSA also markedly reduced I/R-induced myocardial apoptosis (30min/24h) by the TUNEL assay.4. Regulation mechanism of TSA in ERS genes expressionTo further explore the regulation mechanism of TSA in the ERS critical genesexpression, we detected the acetylation levels of histone H3and H4at the promoter ofgenes grp78and chop using chromatin immunoprecipitation (ChIP) analysis. It wasfound that acetylation level of histone H4at grp78gene promoter region was elevatedby TSA. However, the histone H3acetylation level of chop gene promoter waslowered. In addition, we combined MAPPER database and gene chip to predict thepossible transcriptional factors, which can be regulated by TSA and also be involvedin the transcription of ERS-related genes. Results showed that AhR protein expressioncan be influenced by TSA treatment and AhR participated in the transcriptionregulation of ERS-related genes like chop at the promoter region.In summary, we can conclude as follows:1. Three ERS genes are screened by DNA microarray analysis of TSA treatment withbioinformatics method, suggesting that TSA is likely to be involved in the ERSregulation.2. TSA can upregulate the expression of the ERS marker protein GRP78, butdown-regulate the expression of critical ERS apoptosis protein Caspase12in vitro.TSA reduced the ERS-induced H9c2apoptosis rate, suggesting that TSA can regulatethe ERS and inhibit cardiomyocyte apoptosis. Moreover, HDAC2may be involved inthe ERS regulation.3. TSA can increase GRP78experession while reduce CHOP, Caspase12and ATF4expression in the ERS induced by I/R injury in rat. Further studies showed that TSAcan inhibite I/R-induced myocardial apoptosis and alleviate the myocardial I/R injury.It means that TSA can prevent I/R injury by regulating the ERS.4. TSA can regulate the transcription of grp78and chop genes by changing theacetylation levels of histone H3and H4at gene promoter region. Furthermore, wealso predicted that TSA can regulate transcription factor AhR to indirectly affect chopgene expression.