The Effects And Mechanisms Of Empagliflozin On Cardiac Function Improvement In Heart Failure Rat

Background and purposeHeart failure(HF)is the final outcome of various cardiovascular diseases.A variety of independent risk factors for cardiovascular diseases can induce or aggravate the occurrence of HF,such as diabetes,hypertension,and myocardial hypertrophy.Diabetes is an important independent risk factor in the development of HF,and it is also an important cause of death in HF patients.Diabetes is often accompanied by obesity,hypertension and insulin resistance,which can lead to cardiac hypertrophy and further aggravate HF.Studies have shown that in patients with heart failure,diabetic patients have a two-fold increase in the risk of death compared with non-diabetic patients.Therefore,how to improve cardiac function while treating diabetes is an urgent problem to be solved.SGLT2 inhibitors(SGLT2is)are a class of hypoglycemic drugs with the effect of improving cardiac function.The improvement of cardiac function caused by SGLT2 is is observed in both diabetic and non-diabetic people,indicating that the improvement may be independent of glucose lowering.However current research on the mechanism of SGLT2 is improving cardiac function is still incomplete.Among the existing SGLT2 inhibitors,the cardiac function improvement effect of empagliflozin(EMPA)is the most significant.Therefore,we take EMPA as the research object in this study.We established a rat model of HF by ligating the anterior descending coronary artery,and administered EMPA to the therapy groups.Then we estimated the cardiac function of rats in different groups to figure out whether EMPA improved cardiac function in HF rats.We went through transcriptomics and proteomics to compare the differentially expressed genes and proteins between the control and the therapy group and to find out the key factor of how EMPA improves cardiac function.According to the sequencing results,we speculated that EMPA improved cardiac function by suppressing cardiac hypertrophy.Angiotensin ?(Ang ?)was used to induce an in vitro cardiomyocyte hypertrophy model,and the western blot method was used to detect the expression of key molecules on ERK1/2 pathway in hypertrophic cardiomyocytes treated with EMPA.We hypothesized that EMPA could inhibit cardiomyocyte hypertrophy and improve cardiac function by inhibiting ERK1/2 pathway and its downstream molecules.MethodThe anterior descending coronary artery of Wistar rats was ligated to construct a myocardial infarction model,and intragastric administration was started 4 weeks after myocardial infarction.The rats were divided into control group(sham operation group),model group(HF group),low-dose and the high-dose EMPA treatment group.The first two groups were given normal saline,and the latter two groups were given4 mg/kg/d and 10 mg/kg/d of EMPA for 3 weeks.After the administration,the heart function was evaluated by echocardiography and then sacrificed.Take the hearts to observe their general morphology,then separate the left ventricles for HE and Masson staining;separate the non-infarcted area of left ventricular for transcriptomics and proteomics detection.According to the sequencing results,it is speculated that EMPA may function through improving cardiac hypertrophy,which was verified by in vitro experiments.An in vitro model of H9C2 cardiomyocyte hypertrophy was constructed using Ang ?.We measured the cell viability and cell area under different Ang ? concentration and time,so as to determine the appropriate conditions for Ang ? to induce cardiomyocyte hypertrophy.After the model was successfully constructed,the cells were divided into the control group(blank group),the Ang ? group and the Ang ?+EMPA group.The expression of key proteins in the classical hypertrophy pathway(ERK1/2 pathway)was detected by Western blot,and the results were analyzed to illustrate the mechanism of how EMPA regulated cardiac hypertrophy.Results1.EMPA improves cardiac function in HF ratsCompared with the HF group,in the high-dose EMPA treatment group,the left ventricular ejection fraction and the left ventricular fractional shortening were significantly increased,and the myocardial fibrosis and left ventricular hypertrophy index were significantly reduced.In the low-dose treatment group,these indexes showed an improvement trend compared with the HF group,but there was no significant difference.2.Transcriptomics and Proteomics SequencingCompared with the HF group,the high-dose treatment group showed significant differences in the omics expression.Among them,75 differentially expressed genes were detected in the transcriptomics(46 genes were up-regulated and 29 were downregulated),and the proteomics detected 80 differentially expressed proteins(53proteins were up-regulated and 27 were down-regulated).The enrichment analysis of GO and KEGG pathways suggested that this process may be related to myocardial hypertrophy,and multiple differentially expressed genes and proteins are also closely related to cardiomyocyte hypertrophy: down-regulation of Nppa,G0s2,Fn14,and up-regulation of Prakaa2 in the transcriptomics,and down-regulation of Myh7,Pin1,Fhl1 and up-regulation of Nmnat3 in the proteomics.Among these molecules,Nppa(ANP)has been used as a biomarker for HF and myocardial hypertrophy,so we took ANP as the research subject during in vitro experimental.3.EMPA can alleviate cardiomyocyte hypertrophy induced by Ang ?Ang ?(1?M,24h)successfully established cardiomyocyte hypertrophy model.The results of q PCR detection of cardiac hypertrophy marker genes(ANP,BNP)and WGA staining showed that compared with the Ang ? group,the degree of cardiomyocyte hypertrophy in the Ang ?+EMPA group was significantly improved.4.EMPA inhibits the expression of key molecules of the ERK1/2 pathwayAng ? induced an up-regulation of the expression levels of p-ERK1/2 and its downstream proteins in H9C2 cells.After EMPA treatment,the phosphorylation level of ERK1/2 was reduced,and then the expression levels of p-GATA4 and ANP were thereby decreased,which inhibited cardiomyocyte hypertrophy.ConclusionEMPA improves cardiac function by inhibiting cardiac hypertrophy through the ERK1/2 pathway.