The Cardioprotective Mechanisms Of SGLT2 Inhibitors

Background:Cardiovascular disease（CVD）is one of the most serious diseases,which contributes to the poor health outcomes.Although intracoronary thrombolysis and cardiac interventional therapies have been widely used in the treatment of myocardial infarction（MI）,the morbidity and mortality rate of MI are still rising,suggesting the defects in MI treatment.Recently,sodium-glucose co-transporter-2（SGLT2）inhibitor,a new generation of hypoglycemic drugs,has been reported to reduce cardiovascular mortality in patients with diabetes.Nevertheless,the protective mechanism remains elusive.Given that cardiomyocytes do not express SGLT2 and autophagy plays a key role in ischemic heart disease,whether the drug acts directly on the heart to produce cardioprotection and the regulatory mechanism of MI-induced autosis of cardiomyocytes need to be elucidated.Purpose:1.To reveal the direct target（s）and main regulatory pathways of SGLT2inhibitors in cardiomyocytes,through which the drugs fulfil cardioprotection against ischemia injury;2.To clarify the cardioprotective mechanism of SGLT2 inhibitors against MI through weakening of autosis.Methods and Results:1.db/db mice（mimic type 2 diabetes,T2DM）pretreated with SGLT2 inhibitor Empagliflozin（EMPA）,were compared with mice pretreated with DMSO（Vehicle control）,or metformin,and post-treated with EMPA,respectively.Administration of EMPA before MI significantly improved the 1-week survival rate after MI in db/db mice contrast to DMSO group（87.5%vs.23.8%,P=0.0002）,with increased cardiac function,reduced fibrotic scars,and alleviated degree of hypertrophy of cardiomyocytes in the MI border zone.Despite the equivalent hypoglycemic effect,EMPA ameliorated the 1-week survival rate after MI compared with Metformin group（87.5%vs.50%,P=0.0334）.Nevertheless,there was no statistically significant difference in the survival rate between Metformin and DMSO group（50%vs.23.8%）.In streptozotocin-induced diabetic mice（mimic type 1 diabetes,T1DM）,pretreatment of EMPA significantly raised the 1-week survival rate after MI compared with DMSO group（91.7%vs.55.0%,P=0.0325）,with increased cardiac function,reduced fibrotic scars,and alleviated degree of hypertrophy of cardiomyocytes in the MI border zone.These findings further demonstrate the cardioprotection of EMPA in multiple diabetes models,which isn’t benefiting from hypoglycemia.2.Although administration of EMPA before MI didn’t result in hypoglycemia,the increased survival rate and cardiac function,and suppressed fibrotic scars were also observed in wild-type mice,compared to DMSO group.3.To mimic myocardial ischemia and/or reperfusion stress,we established glucose deprivation（GD）,an in vitro cell culture model,in the rat cardiomyocyte cell line（H9c2）,primary rat neonatal cardiomyocytes（Rat-CMs）,and human-induced pluripotent stem cell–derived cardiomyocytes（hi PSC-CMs）.We demonstrated that pretreatment with EMPA significantly rescued the GD-induced cell death.Moreover,EMPA improved myocyte contractility in Rat-CMs.In addition,we indicated that EMPA itself rather than its metabolites,conferred the protective effects on cardiomyocytes against starvation.4.To explore whether EMPA directly targets cardiomyocytes,we innovatively combined cellular subfraction analysis and high-performance liquid chromatography（HPLC）to reveal the distribution of EMPA in cultured cardiomyocytes,through which we detected the enrichment of EMPA on the cell membrane.After screening 357membrane transporter/ion channel compound library,we determined potential candidate targets of EMPA on cardiomyocytes,including sodium channel,proton pump,NHE1 and P2X7 receptor.Utilizing molecular docking prediction,we confirmed that NHE1 might be the direct target of SGLT2 in cardiomyocytes.5.Overexpression of NHE1 raised cardiomyocytes’sensitivity to glucose deprivation,leading to the significant reduction of cell survival compared with the negative control Lenti-NC,while EMPA reversed the detrimental effects of overexpression of NHE1 on cardiomyocytes.In contrast,in NHE1 knockout cell lines,which were obtained through CRISPR/Cas9 system and sh RNA,GD-induced cardiomyocytes death was attenuated.Meanwhile,EMPA had no impact on the NHE1knockout cell lines.6.RNA-seq analysis discovered that knockout of NHE1 leaded to the variations in the pathway related to ion transport and autophagy.Using an adenovirus expressing m RFP-GFP tandem fluorescent-tagged LC3,we indicated that EMPA could block autophagic flux resulted from GD.Overexpression of NHE1 aggravated GD-induced cardiomyocyte autophagic death,which was reversed by EMPA.In contrast,NHE1knockout significantly decelerated the cell death induced by tat-beclin1 and GD.7.Compared to WT mice,Nhe1^-/-mice exhibited reduced myocardial infarction size in response to the ischemia-reperfusion（I/R）,comparable to the protective effect of EMPA administration in WT mice.We further disclosed the significantly increased survival rate and reduced cardiac infarct size after MI in NHE1 cardiomyocyte-specific knockout mice than WT mice.8.We demonstrated that the autophagy level of the border heart was significantly attenuated at 1-week post MI in WT mice pretreatment with EMPA,wth the down-regulation of Beclin1,which plays a central role in autosis.Moreover,in beclin1^+/-mice,the increased survival,reduced fibrotic scars and further decreased autophagic flux were ascertained.Conclusions:Taken together,our findings have demonstrated for the first time that cardiomyocyte NHE1 might be the potential target of EMPA,where EMPA produce the cardioprotection effects in MI through the reduction of autophagic death.Our work proposed the spatial-temporal window effect of membrane receptors and the concept that interventions of cardiomyocyte autosis could prevent and treat MI.These findings provide new insights in drug development for ventricular remodeling and heart failure after MI,especailly targeting cardiomyocyte NHE1 and autosis.