Mechanisms And Preventions Of Striated Muscle Injury Induced By Statin And Lipids

1.BackgroundStatins(3-hydroxy-3-methylglutaryl monoacyl coenzyme A reductase inhibitors)are widely used in the clinic,particularly in the field of cardiovascular disease,and have been the cornerstone of primary prevention and secondary prevention of cardiovascular disease.Statins have well-established lipid-lowering effects,not only strongly reducing total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C)and weakly lowering triglycerides(TG),but also elevating high-density lipoprotein cholesterol(HDL-C).In addition,statins have anti-inflammatory,endothelial protection,inhibition of thrombosis,anti-atherosclerotic,and plaque stabilizing effects,which markedly reduce morbidity and mortality of coronary heart disease.Even studies have argued that statins can shrink atherosclerotic plaques,breaking the traditional perception that'coronary heart disease is irreversible'.Despite the great clinical benefits,statins also have significant side effects,such as myalgia,myositis,rhabdomyolysis,liver injury,diabetes,central nervous system disorders.These side effects are dose related and marked individual variability,and although the overall incidence is not high,this limits the drug-use in some patients,particularly in patients with multiple risk factors for coronary heart disease and those who require secondary prevention,where statins cannot deliver the value.So,it is of great theoretical value and clinical significance to study the molecular mechanisms and interventions of statin-induced side effects.As a major side effect of statins,statin-induced muscle damage(including myalgia, myositis and rhabdomyolysis)not only makes some patients intolerant to statins,but also affects exercise rehabilitation in some patients with established cardiovascular disease.Multiple mechanisms have been demonstrated to be involved in statin myotoxicity,such as decreased sarcolemma or T-tubule cholesterol,decreased CoQ10,activation of the phosphoinositide 3-kinase(PI3K)/Akt pathway,and impaired mitochondrial function.But its detailed molecular mechanisms are still not fully elaborated.Since it has been reported that muscle apoptosis may play a critical role in statin-induced muscle injury,we focused on ameliorating statin myotoxicity by preventing statin-induced skeletal muscle apoptosis.MicroRNAs(miRs)are single stranded RNA molecules of about 20 nucleotides that can repress the translation of mRNA or degrade mRNA by targeting the 3'-untranslated region(3'-UTR)of specific mRNAs with partial complementarity for the purpose of post transcriptional regulation.Our research group has reported that ectopic expression of miR-133a(which is specifically and highly expressed in skeletal and cardiac muscle)in endothelial cells under pathophysiological conditions,and statins inhibit ectopic expression of miR-133a to ameliorate endothelial dysfunction under pathological conditions,suggesting that statins are able to regulate Moy miRNA gene expression.It has been reported that miR-208 and 133a/b are biomarkers of skeletal muscle toxicity,miR-21 is involved in cardiomyocyte apoptosis,and miR-103/107 regulate myocardial ischemia-reperfusion injury.Thus,miRs play an important role in regulating muscle cell function and determining muscle cell fate.Muscle specific miRs(miR-1,miR-133,and miR-206)regulate skeletal muscle development,but whether they are involved in statin induced skeletal injury remains unclear.The function of miR-1 has been confirmed by several studies that overexpression of miR-1 leads to apoptosis of some types of cells,so we hypothesized that statins could cause apoptosis of muscle cells and then produce skeletal muscle toxicity by affecting the expression of miR-1.Based on the above hypothesis,we explored whether statins have an effect on the expression of miR-1 a,which in turn produces skeletal muscle toxicity.Further,we will select the potential target genes of miR-1a through the database,and then validate the target genes of miR-1a through dual luciferase reporter assay,the expression level of target gene mRNA and the expression level of target gene protein.The whole experiment was performed by both cellular and animal levels.2.Research purpose(1)Whether statins have an effect on the expression of miR-1a;(2)Whether interfering with the expression of miR-1a is able to aggravate or alleviate statin-induced skeletal muscle toxicity;(3)The target genes of miR-1a were screened and validated;(4)After determining the target gene of miR-1a,interfering with the expression of the target gene,to see whether there is an effect on statin induced muscle toxicity,and then to judge whether miR-1a produced muscle damage through this target gene.3.Research methods3.1 Extraction and differentiation of skeletal muscle satellite cellsExtraction:Gastrocnemius muscles of 10-day C57BL/6 mouse were dissected,minced,digested with collagenase ? and pancreatin followed by cell sieve filtration to remove impurities,and satellite cells were isolated by taking advantage of the difference in the attachment rate between satellite cells and fibroblasts.Differentiation:Satellite cells were differentiated with DMEM medium containing 2%horse serum,after 3 days the cells started to differentiate,after 5 days the satellite cells had almost fully differentiated into mature skeletal muscle cells and some skeletal muscle cells would fuse into myotubes.3.2 Transfection of miR-1a mimics and inhibitor in vitroAfter muscle satellite cells were well differentiated,miR-1a mimics and inhibitor were mixed with serum-free medium and Lipofectamine 3000 according to the recommended protocol by Lipofectamine 3000,and the mixture was added to 6-well cell culture plates keeping 4-6h,after which the medium was changed to normal medium.3.3 Construction of animal model and its intervention protocolApoE knockout(ApoE-/-)mice(background:C57/BL6),6-8 weeks old,were bred in the animal house of cardiovascular laboratory,Qilu Hospital of Shandong University.The experimental animals were divided into five groups:Normal control(CON)group,PBS gavage(vehicle)group,simvastatin gavage(statin)group,lentivirus negative control(vector)group,and lentivirus mediated miR-1a inhibition(miR-1a inhibitor)group.There were 16 mice in each group,half male and half female.Simvastatin was suspended in PBS and mice in the drug treated group were intragastrically administrated with simvastatin at a dose of 20 mg/kg/day for 8 weeks.Mice in the miR-1a inhibitor group were injected with lentivirus via tail vein at a dose of 1.5×107 TU/mouse at 0 week and repeated once at 4 weeks.3.4 Total RNA extraction and real-time quantitative polymerase chain reaction(PCR)Total RNA was extracted from skeletal muscle cells and analyzed for quality control and concentration according to the kit's instructions,reverse transcribed to cDNA according to the reverse transcription kit's instructions.Real-time quantitative PCR was performed according to the kit's instructions to determine the Ct value of target RNA and U6.3.5 Western blotThe total protein of the cells or tissues was extracted,and the gray values of the target protein and internal reference were determined by the following steps:Electrophoresis,transmembrane,incubating antibodies,photographing,and quantitative analysis.3.6 TUNEL assayCells(green fluorescence):Following the instructions of the kit,the cells were punched,incubated,nucleus stained,etc.,then the cells were fixed on glass slides,photographed using a fluorescence scanning microscope and analyzed quantitatively by a software.Tissues(chromogenic method):Paraffin sections were deparaffinized,hydrated,incubated,colored,and photographed using a light microscope.3.7 Flow cytometryThe treated cells were digested with trypsin without EDTA,and treated according to the requirements of the kit.The cells were added into the designated test tube and detected on the computer.3.8 Cell Counting Kit-8(CCK8)Cells were seeded in 96 well plates and treated according to the pre-designed scheme.After the cells were washed with PBS,CCK8 reagent was added into the wells,and the plates were incubated in incubators.Then the absorbance of each well was measured by enzyme reader,and the absorbance data were analyzed.3.9 Double luciferase report experimentHEK293 cells were seeded in 12 well plates.Two plasmids(WT-MAP3K1-UTR or MT-MAP3K1-UTR),miR-1a mimics and NC were co-transfected into HEK293 cells.The fluorescence intensity was measured twice according to the instructions of the kit.Finally,the fluorescence intensity was analyzed.3.10 MAP3K1 cDNA and siRNA transfectionAccording to the instructions of lip3000,MAP3K1 cDNA and siRNA were mixed with serum-free medium and lip3000,and then added into the cell culture medium.After 4-6 hours,the normal medium was changed to new culture medium.After 48 hours,the next experiment was carried out.3.11 Detection of serum muscle toxicity index in miceCreatine kinase(CK)and lactate dehydrogenase(LDH)in serum of mice were detected according to the instructions of the individual kits.3.12 Detection of exercise capacity in miceReferring to the previous methods,forelimb grip strength test,hanging grid test,running tolerance test were performed to detect the exercise capacity of mice.3.13 Hematoxylin eosin(HE)stainingParaffin sections were dewaxed and hydrated,placed in hematoxylin and eosin for a specific time,dehydrated and transparent.Finally,these paraffin sections were sealed,photographed and analyzed.3.14 ImmunohistochemistryThe paraffin sections were dewaxed,hydrated,blocked,incubated with antibody,color developed,dehydrated,transparent and sealed.The pictures were taken by an electron microscope.Finally,the quantitative analysis of the target protein was carried out by a software.4.Results4.1 Effects of statins on miR-1a expression in skeletal muscle cellsSkeletal myocyte miR-1a expression increased gradually with increasing statin stimulation time;similarly,miR-1a expression has a dose-dependent manner under the stimulation of statin.4.2 Effects of interfering of miR-1a expression on statin-induced cytotoxicity in skeletal muscle cellsStatin caused an increase in the ratio of cleaved-caspase3,7,9/pro-caspase3,7,9 and a decrease in the ratio of bcl-2/bax in skeletal muscle cells,and after inhibiting the expression of miR-1a,the ratio of cleaved-caspase3,7,9/pro-caspase3,7,9 and the ratio of bcl-2/bax increased.However,elevating miR-1a expression at the base of statin stimulation,the ratio of cleaved-caspase3/pro-caspase3 was further increased,and the ratio of bcl-2/bax was further decreased.Similar to WB results,TUNEL and flow cytometry results showed that skeletal muscle cell apoptosis was increased by statin,while the level of apoptosis was decreased after the miR-1a inhibition.CCK8 results showed that inhibition of miR-1a expression could alleviate statin-induced cytotoxicity in skeletal muscle cells.4.3 Mitogen activated protein kinase kinase kinase 1(MAP3K1)is a target gene of miR-1aAccording to the screening results of the database,MAP3K1 is a potential target gene of miR-1a.After overexpression of miR-1a in HEK293 cells,the expression of MAP3K1 mRNA and protein were obviously decreased.After miR-1a mimics was co-transfected with WT-MAP3K1-UTR,the fluorescence intensity decreased significantly,but the fluorescence intensity did not change after co-transfection with MT-MAP3K1-UTR.4.4 Overexpression of MAP3K1 ameliorates statin-induced apoptosis in skeletal muscle cellsStatin reduced intracellular MAP3K1 expression in skeletal muscle cells in a time-dependent and concentration-dependent manner.Statin increased the ratio of cleaved-caspase-3,7,9/pro-caspase-3,7,9 and decreased the ratio of bcl-2/bax in skeletal muscle cells,and based on the statin stimulation,overexpression of MAP3K1 decreased the ratio of cleaved-caspase-3,7,9/pro-caspase-3,7,9 and increased the ratio of bcl-2/bax.Flow cytometry results were consistent with WB,after overexpression of MAP3K1,the level of statin induced apoptosis in skeletal muscle cells decreased.CCK8 results showed that statin-induced muscle cytotoxicity was alleviated after overexpression of MAP3K1.4.5 Statins cause skeletal muscle cell injury via the miR-1a-MAP3K1 pathwayInhibition of miR-1a expression alleviated statin induced elevation of cleaved-caspase3,7,9/pro-caspase3,7,9 ratio and decrease of bcl-2/bax ratio,whereas this protective effect of miR-1a inhibitor was almost abolished after inhibition of MAP3K1.Flow cytometry and TUNEL results also showed that inhibiting the expression of MAP3K1 blunted the anti-apoptotic effect of miR-1a inhibitor.CCK8 results indicated that the cytoprotective effect of miR-1a inhibitor disappeared after inhibition of MAP3K1.4.6 Inhibition of miR-1a expression significantly ameliorates statin-induced muscle histomorphological disarrayLongitudinal sections of muscle(HE staining)showed that statin clearly caused loose muscle fibers and disordered walking,while after inhibiting the expression of miR-1a,the muscle fibers became compacted and the walking became smooth and regular.Cross-sections of muscle(HE staining)showed that statins markedly disordered the arrangement of myofibers with variable sizes in diameter,while these abnormal changes were significantly alleviated after inhibiting the expression of miR-1a.Quantitative analysis revealed that statin significantly decreased myofiber cross-sectional area(CSA),which was upregulated by miR-1a inhibitor.4.7 Inhibition of miR-1a expression improves statin-elevated indicators of muscle toxicity and enhances exercise capacity in miceThe content of CK and LDH in serum of mice was increased by statin,and after inhibiting the expression of miR-1a,the content of CK and LDH in serum was significantly decreased.In addition,statin decreased forelimb grip strength,hang time,exhausting running time,exhausting running distance in mice,and these indices were significantly improved by miR-1a inhibitor.4.8 Inhibition of miR-1a expression ameliorates statin-induced apoptosis in gastrocnemius muscle of miceIn gastrocnemius muscle of mice,the expression level of miR-1a was increased by statin,the ratio of cleaved-caspase3,7,9/pro-caspase3,7,9 was increased,the ratio of bcl-2/bax was decreased,and after inhibiting the expression of miR-1a,these indicators of apoptosis were significantly improved.Immunohistochemistry showed a similar trend in cleaved-caspase 3 expression in gastrocnemius muscle.TUNEL results showed that the level of apoptosis in the gastrocnemius was increased by statin,and statin-induced apoptosis was significantly improved by miR-1a inhibitor.5.Conclusion(1)Statin increased the expression of miR-1a in skeletal muscle cells;(2)MAP3K1 is a target gene of miR-1a;(3)Statin induce apoptosis of skeletal muscle cells through miR-1a-MAP3K1 pathway,resulting in skeletal muscle toxicity.1.BackgroundObesity is a chronic metabolic disease caused by many factors.With the development of social economy and the improvement of living standards,the incidence of obesity is increasing year by year,which has become a global public health problem.According to statistics,the number of obese patients in more than 70 countries has at least doubled since 1980.Therefore,the global obesity problem is in urgent need of attention.Obesity has been shown to be an independent risk factor for cardiovascular disease.Obesity can lead to a variety of cardiovascular diseases,such as cardiomyopathy,coronary heart disease,hypertension,arrhythmia,heart failure and so on.Long term obesity can lead to extensive infiltration of fat into the myocardium,and can lead to myocardial steatosis seriously,namely fatty heart.Obese cardiomyopathy can be diagnosed if there are structural and functional abnormalities in the heart of obese patients,which cannot be explained by hypertension,diabetes,coronary heart disease,valvular heart disease and other diseases.Obese cardiomyopathy can be manifested as diastolic heart failure and(or)systolic heart failure,with no obvious symptoms at the initial stage,and NYHA grade ? cardiac function at the later stage.It can develop from thickening of ventricular wall and narrowing of cardiac cavity to thinning of ventricular wall and enlargement of cardiac cavity,and finally to dilated cardiomyopathy.Obese cardiomyopathy is caused by a variety of mechanisms,such as insulin resistance,lipid damage,inflammation,mitochondrial dysfunction,endothelial dysfunction,changes in cardiac microcirculation,changes in cardiac conduction system,production of endogenous toxins,production of reactive oxygen species,etc.So far,the exact mechanism of obese cardiomyopathy is still unclear.Clinically,there is no other effective intervention except to let patients lose weight.Therefore,it is of great practical need and clinical value to study and screen effective measures for the treatment of obese cardiomyopathy.G protein coupled receptor 40(GPR40),also known as free fatty acid receptor 1(FFAR1),belongs to the G protein coupled receptor family and has seven transmembrane structures.It has been confirmed that it is expressed in heart,liver,kidney,nerve tissue,airway and other organs.It can be combined and activated by free fatty acids in the body and participate in a variety of physiological functions.GPR40 can participate in glucose and lipid metabolism,and later found that it can also participate in other important physiological functions,and can improve the pathophysiological process of many organs.Overexpression of GPR40 can improve glucose tolerance and increase insulin secretion in obese mice.Activation of GPR40 can promote neural development and alleviate diabetes related cognitive impairment in mice.GPR40 can reduce the neuroinflammatory reaction,reduce brain edema and promote the recovery of neurological function after brain injury.GPR40 agonists can reduce the level of oxidative stress and inflammation,reduce lipid deposition and fibrosis in nonalcoholic fatty liver disease.In addition,GPR40 can improve depression and anxiety,relieve pain,alleviate renal fibrosis,and promote the repair of bronchial epithelial cells.In the cardiovascular field,studies have found that fat emulsion can reduce ischemia-reperfusion injury through GPR40.There are few studies on the role of GPR40 in other cardiovascular diseases.Since GPR40 plays a protective role in a variety of disease models,and it is closely related to glucose and lipid metabolism,we speculate whether GPR40 has a certain protective effect on obese cardiomyopathy?Therefore,we stimulated H9c2 cells with palmitic acid(PA)to simulate high-fat environment,and fed rats with high-fat diet to establish obesity model,to see whether GPR40 is protective against lipotoxicity at both in vitro and in vivo levels and determine the molecular mechanisms.2.Research purpose(1)The change of GPR40 expression in cardiomyocytes in high-fat environment;(2)The effect of GPR40 interference on the function of myocardium in high-fat environment;(3)If GPR40 can affect the function of myocardium in high-fat environment,the detailed molecular mechanism of GPR40 will be explored.3.Research methods3.1 Construction of animal model and its interventionSixty Wistar rats of about 150g were purchased from animal center of Shandong University and fed into animal room of Qilu Hospital of Shandong University.They were divided into four groups with 15 rats in each group:Control group,obesity group,vector group and GPR40 overexpression group(GPR40 group).All rats were adaptively fed for one week.One week later,the control group continued to be fed with normal diet,while the obesity group,vector group and GPR40 overexpression group were fed with high-fat diet.After 12 weeks of feeding,if the weight of rats fed with high-fat diet was more than 20%higher than that of rats fed with normal diet,the obesity model was successfully constructed.At the 13th week,the obese group,vector group and GPR40 overexpression group were injected with normal saline,empty vector virus and GPR40 overexpression virus via tail vein respectively.At the end of the experimental period(4 weeks later),echocardiography was performed,and then euthanasia was performed.3.2 Echocardiography The rats were put into the anesthesia box and given continuous isoflurane inhalation.After the anesthesia took effect,the rats were placed on the operating platform,fixed their limbs,and continued to be given inhalation anesthesia with trachea.The chest hair of rats was removed with depilatory cream,and the heart was imaged with VEVO 2100 imaging system.The heart rate,left ventricular ejection fraction(LVEF),left ventricular shortening rate(LVFS),left ventricular posterior wall diastolic thickness(LVPWd)and interventricular septum thickness diastolic(IVSTd)were measured and analyzed.After that,four chamber cardiac imaging was performed to measure and analyze the peak value of E/A.3.3 Treatment of H9c2 cellsH9c2 rat cardiomyocytes were purchased from the American Type Culture Collection(ATCC)and cultured in high glucose medium containing 10%fetal bovine serum.The cells were divided into four groups:Control group,high fat group(PA group),negative control group(vector group)and GPR40 overexpression group(GPR40 group).The cells were transfected with GPR40 overexpression vector or empty vector for 48 hours,and then stimulated with PA for 24 hours.After that,the total protein or RNA was extracted.3.4 Determination of blood pressure in ratsThe rats were fixed on a flat plate.After a period of calm,the systolic and diastolic blood pressure were measured with a rat non-invasive sphygmomanometer,and the mean blood pressure was calculated.Each rat was measured 3 times and the average value was taken.3.5 Determination of serum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)and serum glucoseAfter anesthesia,cut off the tail tip of rats,squeeze out blood drops on the blood test strip,and insert it into the blood glucose detector to determine the blood glucose of rats.After opening the thoracic cavity of rats,the left ventricle was punctured with a syringe needle for blood collection,and then the blood was slowly injected into the coagulation promoting tube.After 30 minutes,the blood was centrifuged at the speed of 3000 rpm at room temperature for 15 minutes,and the supernatant was removed for the determination of blood lipid.TG,TC and LDL-C test kits were used to determine the content of serum lipids.3.6 Determination of superoxide dismutase(SOD),malondialdehyde(MDA)and NADPH oxidase(NADPH oxidase)in animal serumThe frozen serum was removed from the-80? freezer,dissolved and returned to room temperature.In strict accordance with the requirements of the instructions of SOD,MDA and NADPH oxidase assay kits,We formulate the detection solution and implement the detection,use the absorbance obtained for the calculation of indicators in each group,and analyze the difference between each group.3.7 Total RNA extraction and real-time quantitative polymerase chain reaction(PCR)Total RNA was extracted from skeletal muscle cells and analyzed for quality control and concentration according to the kit's instructions,reverse transcribed to cDNA according to the reverse transcription kit's instructions.Real-time quantitative PCR was performed according to the kit's instructions to determine the Ct value of target RNA and U6.3.8 Western blotThe total protein of the cells or tissues was extracted,and the gray values of the target protein and internal reference were determined by the following steps:Electrophoresis,transmembrane,incubating antibodies,photographing,and quantitative analysis.3.9 Hematoxylin eosin(HE)stainingParaffin sections were dewaxed and hydrated,placed in hematoxylin and eosin for a specific time,dehydrated and transparent.Finally,these paraffin sections were sealed,photographed and analyzed.3.10 Immunohistochemistry(IHC)The paraffin sections were dewaxed,hydrated,blocked,incubated with antibody,color developed,dehydrated,transparent and sealed.The pictures were taken by an electron microscope.Finally,the quantitative analysis of the target protein was carried out by a software.3.11 Masson stainingParaffin sections were deparaffinized,hydrated,and processed strictly according to the procedures recommended by the Masson staining kit.Pictures were taken with an electron microscope.3.12 Enzyme linked immunosorbent assay(ELISA)Strictly follow the instructions of ELISA kit to determine the concentration of target protein.3.13 DHE detectionThe cells on the climbing pieces were incubated with DHE probe.Cell climbing slides were buckled onto glass slides,fixed with nail polish,and photographed under a fluorescence microscope.The present procedure was performed under light protected conditions.3.14 TUNEL detectionFollowing the instructions of the kit,the cells were punched,incubated,nucleus stained,etc.,then the cells were fixed on glass slides,photographed using a fluorescence scanning microscope and analyzed quantitatively by a software.3.15 Co-Immunoprecipitation(co-IP)The binding ability of SIRT1 and LKB1 was determined by co-IP.4.Results4.1 The expression of GPR40 in cardiomyocytes increased in high-fat environment Compared with the normal diet fed rats,the expression of GPR40 in high-fat diet fed rats was higher;compared with the control group,the expression of GPR40 in H9c2 cells stimulated by PA was higher.4.2 Overexpression of GPR40 can improve myocardial fibrosis in high-fat environmentFrom the results of western blot and immunohistochemistry,overexpression of GPR40 in obese rats can reduce the expression of collagen ?,collagen ?,connective tissue growth factor(CTGF)and transforming growth factor-?(TGF-?).Masson results showed that overexpression of GPR40 reduced myocardial fibrosis in obese rats.4.3 GPR40 attenuated myocardial inflammation by inhibiting the NF-?B pathway in high-fat environmentOverexpression of GPR40 can reduce the mRNA and protein expression of IL-1?,IL-6 and TNF-? in the heart of obese rats.In mechanism,overexpression of GPR40 reduced the activation of NF-? B pathway and inhibited p65 translocation to the nucleus.4.4 GPR40 may improve the level of oxidative stress in myocardium under high lipid environment through intracellular NRFWestern blot and immunohistochemistry showed that overexpression of GPR40 could reduce the expression levels of NOX2,NOX4,3-NT and 4-HNE in the heart of obese rats.The mechanism of anti-oxidative stress may be related to the increase of Nrf2 expression in nucleus.4.5 Overexpression of GPR40 can improve the level of myocardial apoptosis in high-fat environmentIt can be seen from both in vitro and in vivo that overexpression of GPR40 can reduce the level of cleaved-caspase-3 and increase the ratio of bcl-2/bax in myocardium of obese rats,that is,it can improve the level of cardiac apoptosis4.6 Overexpression of GPR40 can improve cardiac hypertrophy and worsen cardiac function in obese ratsCompared with the normal rats,the heart of obese rats was significantly hypertrophied and diastolic function was significantly decreased.After the overexpression of GPR40,the thickness of ventricular wall and interventricular septum was decreased,and diastolic function was significantly improved.4.7 Overexpression of GPR40 can activate SIRT1/LKB1/AMPK pathway,which is inhibited by obesityFrom both in vitro and in vivo levels,it has been shown that the SIRT1/LKB1/AMPK pathway was inhibited in the myocardium under high-fat environment and was activated after overexpression of GPR40.Mechanistically,overexpression of GPR40 not only elevated SIRT1 expression but also increased SIRT1/LKB1 binding.4.8 Overexpression of GPR40 can protect myocardium from obesity by SERT1 in ratsOn the basis of overexpression of GPR40,siRNA was used to inhibit the expression of SIRT1.The myocardial protective effect of overexpression of GPR40 in high-fat environment disappeared.This showed that GPR40 works through SIRT1.5.Conclusion(1)The expression of GPR40 in myocardium increased in high-fat environment;(2)Overexpression of GPR40 improves cardiac remodeling and cardiac dysfunction in obese rats.The mechanism may be that overexpression of GPR40 has beneficial effects on anti-oxidative stress,anti-apoptosis and anti-inflammation;(3)The beneficial effect of GPR40 overexpression on myocardium may be achieved by activating SIRT1/LKB1/AMPK pathway.