A Study Of The Impact Of ADAM17 And ACE2 Gene Intervention On Diabetic Cardiac Remodeling And Underlying Molecular Mechanisms

BackgroundIn recent years,the prevalence of diabetes has increased rapidly worldwide.The leading cause of death in diabetic patients is diabetic cardiovascular complications.Diabetes could cause metabolic disorders of cardiomyocytes,intracellular calcium overload,micro vascular lesions in myocardial wall,etc.,which lead to cardiomyocyte hypertrophy and apoptosis,myocardial fibrosis,and thus ventricular remodeling.Diabetic cardiomyopathy(DCM)is a type of cardiac dysfunction unique to patients with diabetes.Patients with DCM often have impaired left ventricular diastolic function,with or without left ventricular systolic dysfunction.The common pathological changes were left ventricular enlargement,myocardial interstitial fibrosis,cardiomyocyte hypertrophy and cardiomyocyte apoptosis.The molecular mechanism of diabetic ventricular remodeling is complex and the key regulatory mechanisms are unknown,which hinders the development of effective therapeutic targets and drugs.The renin-angiotensin system(RAS)plays a key role in the progression of heart failure,angiotensin-converting enzyme inhibitors(ACEI),angiotensin receptor blockers(ARB)and aldosterone receptor antagonists have been shown to improve cardiac remodeling and dysfunction which have become cornerstone drugs in the treatment of heart failure.However,these classical RAS members as therapeutic targets have certain limitations.A disintegrin and metalloprotease protein-17(ADAM 17),a member of the Zinc ion dependent metalloprotease superfamily,is a transmembrane protein composed of 824 amino acids,its function is to lyse a variety of cytokines,cell adhesion factors,growth factors,receptors,ligands and proteases anchored to the cell membrane through proteolysis.Recent studies have shown that ADAM 17 could convert angiotensin-converting enzyme 2(ACE2)on cell surfaces,and the converted ACE2 exodomain remains ACE2 active.The metalloproteinase region of ADAM17 plays a major role in ACE2 shedding.The metalloproteinase region of AD AM17 plays a major role in ACE2 shedding.Studies have shown that the serum levels and activity of soluble ACE2 increased in patients with heart failure,which is associated with cardiac insufficiency.This mechanism may involve the increase of ADAM 17 activity caused by RAS activation,which promotes the exfoliation of ACE2 as a substrate for AD AM17 into the peripheral blood.However,the causal relationship between AD AM 17 and cardiac remodeling and its molecular mechanisms have been poorly studied.So far,no studies have reported the effect of ADAM 17 gene modification on DCM and its molecular mechanism.Our previous studies showed that cardiomyocyte specific knockout of AD AM17 improved myocardial fibrosis,cardiomyocytes apoptosis,and left ventricular function in DCM mice.This preliminary study revealed the beneficial effect of AD AM17 in DCM mice,but the detailed mechanism has not been fully elucidated,and the following key scientific questions remain unanswered:(1)The expression and function of AD AM17 in different types of cells in the myocardium are unknown,and the location of cell apoptosis when ADAM 17 knockout is unknown;(2)In the study of animal phenotypes,an important defect of previous studies was the lack of ADAM17α-MHCKO non-diabetic group(ADAM17α-MHCKO Control group).The effect of ADAM 17 gene knockout alone on left ventricular morphology and function of mice was unclear.(3)The effect of ADAM 17 on substrate ACE2 and its downstream products Ang Ⅱ and Ang-(1-7)is unknown,so it is not possible to determine whether ADAM 17 knockdown improves DCM by increasing ACE2.(4)In the previous study,undifferentiated H9c2 cell lines were used,which did not have the phenotypic characteristics of cardiomyocytes and could not represent the primary cardiomyocytes.The difference between differentiated H9c2 cells and primary cardiomyocytes remains unclear.(5)The relationship between ADAM 17 and glucose metabolism is unclear,and the pathway through which ADAM17 is affected in diabetes is unclear.AMPK is a key regulatory molecule in glucose metabolism,but the relationship between ADAM 17 and AMPK is not clear.(6)Previous studies have shown that ADAM 17 could affect autophagy and apoptosis of cardiomyocytes in diabetes,but the specific regulatory mechanism is unclear,and the causal relationship between autophagy and apoptosis of cardiomyocytes is unclear.To solve the above scientific problems,we designed and carried out a series of in vivo and in vitro experiments.In addition,what role does ACE2 play in improving the efficacy of DCM by cardiomyocyte specific ADAM 17 knockout?Does the effect of ADAM 17 on cardiomyocyte apoptosis and autophagy disappear when ACE2 is disturbed?In order to clarify these important scientific questions,we constructed animal and cell models of ADAM 17 and ACE2 gene dual interference in vivo and in vitro for further investigation.Part Ⅰ The study of the impact of ADAM17 gene intervention on diabetic cardiac remodeling and underlying molecular mechanismObjectives1.To investigate the effects of cardiomyocyte specific ADAM 17 knockout on left ventricular remodeling and cardiac function in diabetic mice.2.To investigate the causes of ADAM 17 expression and enzyme activity increase in diabetic mice.3.To investigate how cardiomyocyte specific knockout AD AM 17 regulates AMPK signaling pathway.4.To investigate the effect of cardiomyocyte specific knockout of AD AM17 on autophagic flux and the concrete links affecting autophagy.5.To investigate the molecular mechanism of ADAM 17 interference in primary cardiomyocytes in vitro.Methods1.Construction of knockout miceAD AM17 gene specific knockout mice were constructed by CRISPR-Cas9 and obtained by crossing ADAM17flox/flox mice with α-MHC-Cre mice.2.Groups of animal experiments and construction of DCM modelAnimal experiments are divided into four groups:the ADAM7fl/fl non-diabetic group(ADAM17fl/fl Control group),the AD AM17α-MHCKO non-diabetic group(ADAM17α-MHCKO Control group),and the ADAM17fl/fl diabetic group(ADAM17fl/fl DM group)and AD AM17α-MHCKO diabetes group(ADAM17α-MHCKO DM group).3.EchocardiographyThe left ventricular systolic and diastolic functions of type Ⅱ diabetic mice were evaluated by ultrasonic measurements after successful DCM modeling.4.Collection of mouse tissues and pathological examinationAfter euthanasia,heart specimens were taken from mice and put into 4%paraformaldehyde or-80℃ refrigerator for subsequent pathology and molecular biology experiments.HE,Masson staining,immunofluorescence staining,TUNEL and co-localization cardiomyocyte fluorescence staining were performed on the heart tissue sections.5.Extraction of neonatal rat primary cardiomyocytes(NRCMs)and differentiated H9c2 cellsPrimary cardiomyocytes were extracted from newborn Wistar rats(1d-3d)by differential adhesion method.H9c2 cells were cultured with low serum and stimulated by RA to induce phenotypic differentiation into related adult cardiomyocytes.6.Groups of cell experimentsAccording to the experimental design,the grouping of cell experiments is as follows:(1)Vehicle group;(2)High glucose/palmitate group(GP group);(3)High glucose/palmitate+scrambled negative control siRNA group(GP+NC-siRNA group);(4)High glucose/palmitate+ADAM17 siRNA group(GP+ADAM17-siRNA group).In addition,in order to explore the concrete link of ADAM 17 affecting autophagic flux,chloroquine(CQ)and its blank solvent were added to the above four groups,which were divided into 8 groups,respectively:Vehicle group,Vehicle+CQ group,GP group,GP+CQ group,GP+NC-siRNA group,GP+NC-siRNA+CQ group,GP+ADAM17-siRNA group,GP+ADAM17-siRNA+CQ group.7.RFP-GFP-LC3B autophagic flux detectionPremo autophagic flux assay reagent RFP-GFP-LC3B was used to detect the dynamic processes of autophagy at different stages.8.Transcriptome RNA sequencingCardiac tissues of ADAM17α-MHCKO DM group and AD AM17fl/fl DM group were frozen with liquid nitrogen for sequencing.9.Western BlotProtein was extracted from animal heart tissues and cells and then subjected to SDS-gel electrophoresis.After transmembrane,closure,incubation of primary and secondary antibodies,luminescence development was performed.10.Statistical analysisAll data were tested for normal distribution before analysis.For data conforming to normal distribution,unpaired T-test was used to compare the differences between the two groups.Continuous variables were expressed by means of mean ± standard error(SEM).In all statistical analyses,p<0.05 was considered statistically significant.Results1.ADAM17 expression was significantly increased in DCM miceCompared with the control group,the mRNA and protein expression levels of ADAM17 and the enzyme activity of ADAM17 in DCM group were significantly increased.2.Distribution of ADAM17 expression in the heartADAM 17 was abundantly expressed in cardiomyocytes,but sparsely expressed in fibroblasts and endothelial cells.In addition,immunofluorescence analysis revealed more intensive staining of ADAM 17 in the myocardium of diabetic mice than in control mice.3.Body weight,blood glucose and serum lipid profileCardiomyocyte specific knockout of ADAM17 did not affect BW,FBG,TG and TC levels,glucose tolerance and insulin sensitivity in mice.4.Cardiomyocyte specific knockout of ADAM17 significantly ameliorated left ventricular remodeling and cardiac function in diabetic miceCardiomyocyte specific knockout ADAM 17 significantly improved left ventricular remodeling and systolic and diastolic functions in diabetic mice,but ADAM 17 gene knockout alone did not affect left ventricular remodeling and function in physiological mice.5.Cardiomyocyte specific knockout of ADAM17 significantly improved myocardial fibrosis and cardiomyocyte apoptosis in diabetic miceThe results of Masson showed that cardiomyocyte specific knockout of ADAM 17 could significantly reduce myocardial fibrosis in diabetic mice,while ADAM 17 gene knockout alone did not affect myocardial fibrosis in mice under physiological conditions.TUNEL staining and WB results showed that cardiomyocyte specific knockout of ADAM 17 could significantly reduce the apoptosis of myocardial cells in diabetic mice,but did not affect the apoptosis of myocardial cells in physiological mice.The results of in vitro experiments are consistent with those of in vivo experiments.6.ADAM17 was essential for cleaving ACE2 in vivo and in vitroThis suggests that ADAM 17 may only affect the posttranscriptional expression level of ACE2 during DCM.Serum ACE2,AngⅡ and Ang-(1-7)levels in mice showed that ADAM 17 plays a key role in regulating ACE2-Ang Ⅱ/Ang-(1-7)axis in both physiological and pathological states.The results of in vitro experiments are consistent with those of in vivo experiments.7.ADAM17 plays an important role in regulating AMPK/mTOR signaling pathway and TFEB expression in cardiomyocytesThe results of WB reveal that AD AM17 knockout reversed AMPK pathway inactivation and inhibition of TFEB protein expression in diabetes mellitus.in DCM mice.The results of in vitro experiments are consistent with those of in vivo experiments.In addition,GP stimulation inhibited nuclear translocation of TFEB in cardiomyocytes,while AD AM 17 inhibition significantly enhanced nuclear translocation of TFEB in cardiomyocytes.ADAM 17 affects the AMPK/mTOR signaling pathway by regulating phosphorylation of 4EBP1 and p70S6K proteins.8.Cardiomyocyte specific knockout of ADAM 17 significantly improved autophagic flux of cardiomyocytesAutophagy flux was measured using autophagy inhibitor CQ and RFP-GFP-LC3B.It was found that GP stimulation inhibited autophagic flux in cardiomyocytes,and ADAM 17 deficiency in cardiomyocytes improved autophagic flux.ADAM 17 may inhibit autophagic flux by attenuating the formation of autophagosome at an early stage of autophagic flux,and ADAM 17 deficiency in cardiomyocytes could improve autophagic flux.9.The effect of ADAM 17 deficiency on autophagosome formation-related proteinsBy detecting the protein expression levels of Atg3,Atg5,Atg7,Atg12 and Beclin-1,it was found that AD AM 17 plays an important role in the formation of autophagosomes,and the deficiency of ADAM 17 in cardiomyocytes can promote the formation of autophagosomes in the autophagic flux.10.ADAM17 deficiency reduces apoptosis by improving autophagic flux in cardiomyocytesIn NRCMs,the results showed that ADAM 17 deficiency reduced apoptosis by improving autophagic flux in cardiomyocytes.11.Effect of ADAM17 on ADRA1A/AMPK-mediated cardiac apoptosis and autophagyAutophagy and apoptosis were measured after selective AMPK inhibitor Dorsomorphin was applied.The results showed that ADAM 17 deficiency improved autophagy and apoptosis of cardiomyocytes through AMPK,and the improvement effect of AD AM17 deficiency on autophagy and apoptosis of cardiomyocytes disappeared when AMPK was inhibited.12.HIF-1α may be the upstream mediator of AD AM17 in DCM miceHIF-1α may be the upstream mediator of AD AM17 up-regulation in DCM mice,which also explains the increased expression and activity of ADAM 17 in the heart of diabetic mice.Conclusion1.Cardiomyocyte specific knockout of AD AM17 significantly alleviates cardiomyocyte apoptosis and myocardial fibrosis in diabetic mice,and improves left ventricular remodeling and cardiac dysfunction.2.In type Ⅱ diabetic mice,the expression and activity of ADAM 17 protein were significantly increased by activating HIF-1α,and the level of ACE2 protein was significantly decreased,and the content of soluble ACE2 in serum was significantly increased.3.The AMPK signaling pathway is activated by ADRA1A after ADAM 17 knockout in cardiomyocytes.4.AD AM17 knockout of cardiomyocytes promotes the formation of autophagosome in autophagic flux,improves autophagic flux,and further reduced apoptosis of cardiomyocytes.Part Ⅱ The study of the impact of ADAM17 and ACE2 gene alone and combination intervention on diabetic cardiac remodeling and underlying molecular mechanismsObjectives1.To investigate whether ADAM 17 deficiency can improve diabetic ventricular remodeling and cardiac function in mice through ACE2.2.To investigate whether ADAM 17 knockout still continue to improve myocardial fibrosis and apoptosis in diabetic mice when cardiac ACE2 is inhibited.3.To investigate the autophagy and apoptosis of cardiomyocytes in vitro when ADAM 17 and ACE2 were inhibited simultaneously.Methods1.Groups of experimental animals and construction of DCM modelADAM17α-MHCKO mice were obtained after breeding.The cre-negative mice born in the same litter(ADAM17fl/fl mice)were used as the control group in animal experiments.ADAM17fl/fl mice and AD AM17α-MHCKO mice were given AAV9-cTnT-shACE2 via tail vein.Animal experiments were divided into four groups:ADAM17fl/fl DM+Vector group,ADAM17α-MHCKO DM+Vector group,ADAM17fl/fl DM+AAV9-shACE2 group,ADAM17α-MHCKO DM+AAV9-shACE2 group.2.EchocardiographyThe same as Part Ⅰ.3.Collection of mouse tissues and pathological examinationThe same as Part Ⅰ.4.Extraction of neonatal rat primary cardiomyocytes(NRCMs)Primary cardiomyocytes were extracted from newborn Wistar rats(1d-3d)by differential adherence method.5.Groups of cell experimentsAccording to the experimental design,the grouping of cell experiments is as follows:(1)Vehicle group;(2)GP group;(3)GP+NC-siRNA group;(4)GP+ADAM17-siRNA group;(5)GP+ACE2-siRNA group;(6)GP+ADAM17-siRNA+ACE2-siRNA group.6.Western BlotProtein was extracted from animal heart tissues and cells and then subjected to SDS-gel electrophoresis.After transmembrane,closure,incubation of primary and secondary antibodies,luminescence development was performed.7.Statistical analysisThe same as Part Ⅰ.Results1.Weight,fasting blood glucose and blood lipid of mice in each groupCardiomyocyte specific knockout of ADAM17 gene and ACE2 knockout of cardiac tissue did not affect BW,FBG,TG and TC levels in mice.2.ACE2 plays a key role in improving cardiac remodeling and cardiac function in mice after AD AM17 knockoutThe results showed that ADAM17 knockout could not continue to improve left ventricular remodeling and cardiac systolic and diastolic functions in diabetic mice when ACE2 was inhibited in the heart,suggesting that ADAM17 deficiency can improve cardiac remodeling and cardiac function in DCM through ACE2.3.ACE2 plays a key role in improving myocardial fibrosis in DCM mice by ADAM17 knockoutThe results of Masson showed that ADAM17 gene knockout could not continue to improve myocardial fibrosis in diabetic mice when ACE2 was inhibited in the heart,suggesting that ADAM17 deficiency plays a key role in improving myocardial fibrosis in DCM mice through ACE2.4.ACE2 plays a key role in improving cardiomyocyte apoptosis in DCM mice by ADAM17 knockoutThe results of TUNEL and WB experiment showed that ADAM17 gene knockout could not continue to improve cardiomyocytes apoptosis in diabetic mice when ACE2 was inhibited in the heart,suggesting that AD AM17 deficiency plays a key role in improving cardiomyocytes apoptosis in DCM mice through ACE2.5.The role of ACE2 in improving autophagy of cardiomyocytes in DCM mice after ADAM17 knockoutACE2 plays a key role in the improvement of myocardial autophagy by ADAM17 through AMPK pathway,while ADAM17 deficiency plays a key role in the improvement of myocardial autophagy through ACE2.Conclusions1.When ACE2 was inhibited in the heart,AD AM17 gene knockout could not continue to improve ventricular remodeling and cardiac function in DCM,but ADAM17 deficiency could improve cardiac remodeling and cardiac function in DCM through ACE2.2.ADAM17 deficiency is induced by ACE2 to improve myocardial fibrosis and cell apoptosis in DCM mice.3.ADAM17 deficiency is induced by ACE2 to improve cardiomyocytes autophagy in DCM mice.