The Effect And Mechanism Of Dapagliflozin And MST1 In Diabetic Cardiomyopathy

Dissertation 1 The Protective Effect and Mechanism of Dapagliflozin against Cardiac Fibrosis in Diabetic Cardiomyopathy1.BackgroundDiabetes mellitus is defined as a group of metabolic diseases with hyperglycemia as the main feature resulted from relative or absolute deficiency of insulin secretion,insulin resistance,or both.Moreover,the increasing prevalence rate of diabetes has made it an increasingly serious public health problem worldwide,among which type 2 diabetes accounts for more than 90%.At present,the main cause of death in patients with diabetes can be attributed to cardiovascular complications.Diabetic cardiomyopathy(DCM),one of the most common complications to increase the risk of heart failure in diabetic patients,refers to the abnormality of myocardial structure and function which can't be explained with hypertension,valvular disease or coronary artery disease.The pathogenesis of DCM is complex and interrelated.Hyperglycemia and metabolic disorders can lead to cell apoptosis,extracellular matrix(ECM)deposition,oxidative stress and inflammation,etc.Cardiac fibrosis,caused by excessive deposition and cross-linking of ECM,is closely related to ventricular remodeling and the impairment of cardiac systolic and diastolic function during the process of DCM.ECM is mainly generated by transdifferentiated fibroblasts,the origin of which has not been fully clarified,including resident fibroblasts,endothelial cells,epicardial cells,bone marrow-derived cells and other sources.In the course of cardiac fibrosis,it has been reported that approximately 27-35%of fibroblasts originate from endothelial cells.This process of cell transdifferentiation is called endothelial-to-mesenchymal transition(EndMT),in which endothelial cells gradually lose part of their endothelial properties and acquire a fibroblast-like phenotype under the pro-fibrotic stimulation.In the aspect of molecular mechanism,the TGF-?/Smad signaling pathway plays a vital part in myocardial fibrosis and EndMT,and can mediate the up-regulation of EndMT-related transcription factors(Snail 1,Snail2,Twist1l,Twist2).Sodium-glucose cotransporters 2(SGLT2)inhibitors are a new class of hypoglycemic agents approved by FDA in 2013,which can directly act on SGLT2 receptors in proximal tubules of the kidney to inhibit glucose reabsorption and thus reduce blood glucose.Currently,SGLT2 inhibitors approved by FDA and widely used in clinical practice include dapagliflozin,canagliflozin and empagliflozin.Based on extensive clinical trials and the 2019 ESC guidelines,SGLT2 inhibitors have been prioritized for use in diabetic patients with CVD due to potential cardiovascular protection.Importantly,previous studies have found that the protective effect of SGLT2 inhibitors against heart failure may be independent of its glycemic control effect,and the potential mechanism still requires further elucidation.Adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)is a widelyexpressed cellular energy sensor which can be activated by phosphorylation of Thur172 residues.Studies have shown that activation of AMPK could inhibit TGF-?/Smad-mediated fibroblasts transdifferentiation and improve cardiac fibrosis in diabetic rats.However,it is not clear whether dapagliflozin,an SGLT2 inhibitor,can improve diabetic cardiac fibrosis through activation of AMPK.2.Objective(1)To establish type 2 diabetic rat model and explore the effect and mechanism of dapagliflozin on myocardial interstitial and perivascular fibrosis(2)To investigate the effect of dapagliflozin on high glucose-induced EndMT and its molecular mechanisms in human umbilical vein endothelial cells(HUVECs)(3)To investigate the effect of dapagliflozin on the proliferation,transdifferentiation,and collagen production of high glucose(HG)-stimulated cardiac fibroblasts(CFs)(4)To explore the role of AMPK?/TGF-?/Smad signaling in the anti-EndMT effects of dapagliflozin3.Methods(1)Type 2 diabetic rat model was constructed through high-fat feeding for insulin resistance combined with intraperitoneal injection of STZ.Dapagliflozin or metformin were given to rats through drinking water.Rats were randomly placed in 4 groups:CON group,DCM group,DCM+dapagliflozin(DAPA)group and DCM+metformin(MET)group.In study design,we chose metformin for positive control.(2)Left ventricular ejection fraction(LVEF),fractional shortening(FS),E/A and E '/A'were analyzed by echocardiography to evaluate cardiac systolic and diastolic function.(3)After euthanasia of the rats,the heart weight and body weight were measured.Hematoxylin and Eosin(HE)staining was performed to show cardiac structure.Masson's Trichrome,Picrosirius Red and immunochemical staining were used for the detection of collagen deposition in cardiac interstitial and perivascular area and oxidative stress.Cardiac collagen content and the expression of fibrosis indicators were measured via western blot and qRT-PCR.(4)Immunohistochemical and immunofluorescence staining were used for the detection of localization and expression of EndMT markers in the endothelium of peripheral vessels in rat hearts.The expression of EndMT markers and downstream signaling protein were measured via western blot and qRT-PCR.(5)HUVECs were exposed to HG for inducing EndMT,with treatment of dapagliflozin in different concentrations.CCK-8 assay was conducted for cell viability detection.The expression of EndMT markers and downstream signaling protein were measured via immunofluorescence double-staining,western blot and qRT-PCR.DCFH-DA probe was used for detection of intracellular reactive oxygen species(ROS)level.(6)To explore the role of AMPK,Compound C and AMPKa siRNA were used for inhibition of AMPK,for activation,AICAR and A-769662 were selected.(7)CCK-8 assay,immunofluorescence staining and western blot were performed for determining the effect of dapagliflozin on the proliferation,transdifferentiation,and collagen production of CFs treated with HG.4.Results(1)At the end of the study,significantly reduced LVEF,FS,E/A ratio,and E'/A' were shown in untreated diabetic rats,indicating the systolic and diastolic dysfunction,with the elevated cardiomyocyte diameter and heart weight to body weight ratio,which could be improved by dapagliflozin or metformin treatment.(2)Compared with the control group,an elevated level of collagen deposition was detected in the interstitial and perivascular region of diabetic hearts,with the upregulation of fibrosis indicators.DAPA treatment could attenuate these fibrotic changes,which was comparable to metformin.(3)In contrast to the control group,the expression of endothelial markers was downregulated,with the up-regulated expression of mesenchymal markers and EndMT-related transcription factor in hearts of diabetic rats,which was inhibited after DAPA treatment.(4)HG induced EndMT in HUVECs,reflected in decreased levels of endothelial markers,and elevated levels of mesenchymal markers and EndMT-related transcription factors.However,the impaired cell viability and EndMT transition were reversed with DAPA treatment in a dose-dependent way.(5)Reduced phosphorylation level of AMPKa and up-regulated expression of TGF-? and Smad4 were observed in both diabetic hearts and HG-treated HUVECs.DAPA treatment effectively restored phosphorylation of AMPK?,which was comparable to metformin,and inhibited expression of TGF-? and Smad4.(6)After inhibition of AMPK,the antagonistic effect of DAPA on HG-induced EndMT and TGF-?/Smad signaling was blocked.Moreover,activation of AMPK could directly suppress the TGF-?/Smad signaling pathway and EndMT transition induced by HG.(7)DAPA could inhibit the proliferation,transdifferentiation and collagen synthesis of CFs in a dose-dependent way.(8)After DAPA treatment,the up-regulated NOX4 expression in diabetic hearts and HGstimulated HUVECs was inhibited.The increased ROS production induced by HG in HUVECs was also suppressed by DAPA treatment.However,the antioxidant effect of DAPA was eliminated by AMPKa siRNA,suggesting that the antagonistic effect of DAPA on the oxidative stress of HG-induced HUVEC was dependent on AMPK activation.5.Conclusion(1)DAPA treatment attenuated cardiac remodeling and cardiac dysfunction in the rat model of type 2 diabetes.(2)DAPA treatment improved diabetic cardiac fibrosis through inhibiting cardiac EndMT and reducing cardiac interstitial and perivascular collagen deposition.(3)DAPA treatment inhibited EndMT and the transdifferentiation of CFs induced by HG,decreasing collagen synthesis and secretion.(4)DAPA treatment antagonized EndMT through AMPK-mediated inhibition of TGF?/Smad signaling pathway.Dissertation 2 The Role and Mechanism of MST1 in Type 2 Diabetic Cardiomyopathy1.BackgroundDiabetes mellitus(DM)refers to a group of metabolic disorders featured by persistently high blood glucose and has become one of the leading causes of death worldwide.Cardiovascular complication is currently the primary cause of death among patients with diabetes.Diabetic cardiomyopathy(DCM)is known as the abnormality of myocardial structure and cardiac dysfunction independent of coronary artery disease,hypertension and major valve disease.The early clinical characteristics of DCM include cardiac hypertrophy and diastolic dysfunction,which could progressively develop to systolic dysfunction and eventual decompensated heart failure.The unique clinical course of DCM involves complex pathophysiological mechanisms,including hyperglycemia,insulin resistance,lipid metabolic disorder,RAAS activation,mitochondrial dysfunction,oxidative stress,extracellular matrix(ECM)deposition,apoptosis,and so forth.Cardiomyocyte apoptosis runs through the whole process of DCM,which can lead to the loss of cardiac contractile unit and ultimate heart failure.In the context of diabetes,long-term metabolic disorders can result in excessive production of reactive oxygen species(ROS)and impaired antioxidant capacity.Studies have supported mitochondria as the primary source of ROS load in multiple organs of diabetic patients and animals.Mitochondrial ROS can directly contribute to mitochondrial dysfunction,which will in turn aggravate the production of ROS,forming a vicious cycle and impairing cell function and survival.In addition,oxidative stress has been reported to promote apoptosis through a mitochondrialdependent or independent pathway.Mammalian Sterile 20-like kinase 1(MST1),a serine/threonine kinase,serves as a key component of the Hippo signaling pathway,and participates in the regulation of multiple cellular processes,such as proliferation,differentiation,apoptosis and autophagy.A number of studies have suggested that MST1 can mediate oxidative stress-induced apoptosis,indicating that MST1 is closely related to oxidative stress.However,it hasn't been clarified that whether MST1 can directly promote oxidative stress as an upstream regulatory molecule.Activation of MST1 has been observed in diabetic heart tissue and high glucose-stimulated cardiomyocytes,whereas the effect and mechanism of MST1 on oxidative stress in type 2 DCM,as well as on myocardial apoptosis related to lipid metabolism disorder have not been fully elucidated.2.Objective(1)To establish type 2 diabetic rat model and explore the role and mechanism of MST1 in the development of DCM with MST1-shRNA lentivirus intervention(2)To investigate the role and mechanism of MST1 in palmitic acid(PA)-induced oxidative stress and apoptosis of cardiomyocytes with MST1-siRNA intervention(3)To explore the effect of MST1 on oxidative stress and apoptosis and its mechanism in cardiomyocytes using adenovirus for overexpression3.Methods(1)Type 2 diabetic rat model was constructed through high-fat feeding combined with STZ injection,and the rats were then injected with lentivirus.Rats were randomly placed in 4 groups:CON+shNC group,CON+shMST1 group,DCM+shNC group,DCM+shMSTl group.(2)Echocardiography was performed to measure left ventricular ejection fraction(LVEF),fractional shortening(FS),E/A and E '/A',for assessing cardiac systolic and diastolic function.(3)Masson's Trichrome,Picrosirius Red and immunochemical staining were conducted for the detection of collagen deposition in rat hearts.(4)The level of apoptosis in rat hearts was measured through TUNEL staining and western blot detection of apoptosis-related proteins.DHE staining was used for the detection of oxidative stress in rat hearts.(5)MST1 expression was inhibited by small interfering RNA to explore its role and mechanism in PA-induced apoptosis and oxidative stress of cardiomyocytes.In addition,verification through overexpression of MST1 by adenovirus transfection was conducted.(6)The cell viability was detected through CCK-8 assay.The level of apoptosis in cardiomyocytes was assessed through TUNEL staining and western blot detection of apoptosis-related proteins.DHE and DCFH-DA probe were used for measurement of intracellular ROS level.(7)MitoSOX staining was used for detection of the ROS produced by mitochondria.Mitochondrial DNA oxidative damage in cardiomyocytes was detected with co-localization analysis by immunofluorescence of 8-hydroxydeoxyguanosine(8-OHdG)and MitoTracker.(8)ATP content,mitochondrial membrane potential(TMRM staining)and transcription level of genes related to mitochondrial biosynthesis in cardiomyocytes were detected to evaluate the mitochondrial function.4.Results(1)Compared with the control group,a marked upregulation of MST1 expression wasobserved in diabetic hearts.Compared with DCM+shNC group,rats in DCM+shMST1 group exhibited increased LVEF,FS,E/A and E '/A',suggesting that cardiac systolic function and diastolic dysfunction were significantly improved in diabetic rats after knockdown of MST1.(2)The increase of extracellular collagen deposition in hearts induced by diabetes was markedly improved after MST1 knockdown,suggesting that inhibition of MST1 expression can alleviate cardiac fibrosis in diabetic rats.(3)Compared with the control group,the proportion of TUNEL-positive cells and the fluorescence intensity of DHE in diabetic hearts were much higher,while knockdown of MST1 markedly decreased the level of myocardial apoptosis and oxidative stress in hearts of diabetic rats.(4)MST1 was up-regulated in PA-stimulated primary cardiomyocytes.After PA treatment,reduced survival rate and increased apoptosis level and intracellular ROS level were detected in cardiomyocytes,which was markedly reversed after MST1 knockdown.(5)PA induced the increment of mitochondrial ROS production and mitochondrial DNA oxidative damage.In contrast,MST1 inhibition alleviated the mitochondrial ROS-related injury in cardiomyocytes.(6)Compared with the BSA control group,PA treatment significantly reduced the mitochondrial membrane potential and ATP level in cardiomyocytes.On the other hand,the mRNA levels of TFAM and PGC-1? were down-regulated after PA treatment.However,mitochondrial dysfunction induced by PA was significantly improved after MST1 knockdown.(7)Compared with the NC group,the ratio of TUNEL-positive cardiomyocytes and intracellular ROS level were significantly increased after MST1 overexpression,with the increment of mitochondrial ROS and mitochondrial DNA oxidative damage in cardiomyocytes.5.Conclusion(1)Up-regulation of MST1 was observed in the heart tissue of type 2 diabetic rats,and MST1 knockdown ameliorated the ventricular remodeling and cardiac dysfunction of diabetic rats.(2)MST1 gene silencing reduced oxidative stress,cell apoptosis and cardiac fibrosis in myocardial tissue of diabetic rats.(3)PA treatment significantly elevated the expression of MST1 in primary cardiomyocytes.(4)MST1 mediated PA-induced oxidative stress and apoptosis of cardiomyocytes by promoting mitochondrial ROS production and mitochondrial dysfunction.