| BackgroundDiabetic cardiomyopathy(diabetic cardiomyopathy,DCM)is a disease caused by diabetes that changes the structure and function of myocardium independent of diabetic macrovascular complications,which is related to the metabolic abnormality of diabetes.DCM is the main cause of morbidity and mortality in developed countries,and the incidence of this disease increases with the incidence of obesity and type 2 diabetes.The course of DCM is long and complex,from a subclinical stage characterized by subtle structural and functional abnormalities,to severe diastolic heart failure with normal ejection fraction,followed by systolic dysfunction,resulting in heart failure with decreased ejection fraction.In view of its serious clinical harm,basic and clinical studies in this field follow.However,so far,there are no special guidelines for diagnosing patients or constructing treatment system in clinical practice.Therefore,at present,the effective methods for the prevention and treatment of the disease are still limited,and the incidence of DCM remains high,prompting us to constantly explore its occurrence and development mechanism,and explore potential prevention and treatment strategies.The pathogenesis of DCM is extremely complex.At present,it is believed that insulin resistance and high glucose and high insulin stimulation are the source and initiating factors of DCM.Structural remodeling such as myocardial fibrosis caused by metabolic disorders is an important pathological manifestation of the occurrence and development of DCM.Insulin resistance or hyperglycemia and hyperinsulinemia were observed in DCM,which were characterized by impaired myocardial insulin signal pathway,dysfunction of mitochondria and endoplasmic reticulum and so on.These pathophysiological changes lead to fibrosis,hypertrophy,cardiac diastolic dysfunction,and eventually lead to systolic heart failure.Therefore,around metabolic abnormalities,myocardial fibrosis as a starting point,actively looking for intervention targets in this area,from the source to block the unique metabolic abnormalities of type 2 diabetes,it is possible to achieve the containment of DCM.Adenosine monophosphate dependent kinase(AMP-actived protein kinase α,AMPK))is a key signal molecule in the regulation of energy metabolism and myocardial interstitial fibrosis in type 2 diabetes mellitus.AMPK plays a variety of functions in cellular energy metabolism,which can promote the absorption of blood glucose,reduce the levels of blood glucose and blood lipids,and increase insulin sensitivity,which is closely related to the regulation of energy balance.Recently,it has been found that AMPK signal pathway plays an important role in the process of myocardial interstitial fibrosis:AMPK can be used as a target of ERK to inhibit the proliferation of cardiac fibroblasts induced by angiotensin II;AMPK can inhibit the activation of transcriptional downstream induced by TGF-β,thus achieving the effect of anti-fibrosis.To sum up,AMPK is a key signal molecule of diabetic myocardial fibrosis and may play an important role in the occurrence and development of DCM.Therefore,can the effective regulation of myocardial fibrosis be achieved through the regulation of AMPK pathway,so as to curb the occurrence of DCM?However,little is known about the precise regulation mechanism of AMPK pathway.Cell death-induced DFF45-like effector(Cell death-inducing DNA fragmentation factor 45-like effector protein,CIDE)family is a group of new genes which can induce cell apoptosis,which was discovered at the end of the 20th century.In addition to promoting apoptosis,CIDEC,as an important regulator of glucose and lipid metabolism,is closely related to the occurrence of insulin resistance and is a key molecule in the regulation of metabolic abnormalities peculiar to type 2 diabetes,which provides an idea for us to intervene in DCM.Studies have shown that there is a homozygous nonsense mutation(E186X)of CIDEC in patients with adipose metabolism disorder and insulin resistance diabetes,and there is a significant negative correlation between the expression of human CIDEC and insulin resistance index.FSP27-/-mice showed a decrease in the level of triglyceride,an increase in the rate of lipid metabolism and an increase in insulin sensitivity.Therefore,CIDEC can regulate the occurrence and development of abnormal glucose and lipid metabolism and insulin resistance at the same time,and play an important role in the regulation of abnormal metabolism in type 2 diabetes.In addition,CIDEC/FSP27siRNA transfection could restore the activity of AMPK,further increase the expression of UCP3 and reduce the synthesis of SERBP and perilipin protein.However,CIDEC/AMPK signal pathway has not been reported in DCM.In summary,AMPK plays a significant role in the regulation of myocardial interstitial fibrosis and energy metabolism in type 2 diabetes.CIDEC is closely related to glucose and lipid metabolism and can regulate AMPK signal pathway,so we speculate that CIDEC is overexpressed in diabetic state,and may affect the process of DCM through the regulation of AMPK,but it has not been reported yet.In view of the above hypotheses,we will take DCM rats as a model to study the role of CIDEC in the occurrence and development of DCM,specifically inhibit the expression of CIDEC by CIDEC-shRNA interference technique,and explore the regulation mechanism of CIDEC/AMPK signal pathway on collagen synthesis and the feasibility of reversing DCM at the integral level.Objectives1.To establish a DCM rat model to detect myocardial fibrosis,myocardial hypertrophy and cardiac function changes in type 2 DCM rats,and to explore the role of CIDEC/AMPK signal pathway in the occurrence and development of DCM.2.CIDEC-shRNA adenovirus was used to transfect DCM rats to observe the changes of myocardial fibrosis,myocardial hypertrophy and cardiac function after CIDEC gene silencing,and to study the mechanism of CIDEC gene silencing improving DCM at the level of whole animal.Methods1.Induction of DCM rat model and animal groupsForty male Sprague-Dawley(SD)rats(4 weeks of age,120-140g)were purchased from Beijing Huafukang Animal Experimental Center,China.The animals were housed under standard laboratory conditions(25℃ temperature,60-70%relative humidity,and 12h light-dark cycles).After 1 week of acclimatization,intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed,and fasting blood glucose(FBG)and fasting insulin(FINS)were measured.The rats were then randomized into control group(n=10)and diabetic group(n=30).The type 2 diabetic group was fed a high-fat diet(34.5%fat,17.5%protein,48%carbohydrate;Beijing HFK BioTechnology Co.Ltd,China),and the control group received normal chow.After 4 weeks,IPGTT and IPITT were performed again,and blood was sampled through the jugular vein.FBG and FINS were measured again,and the insulin sensitivity index[ISI=ln(FBG×FINS)1]was calculated.After a 4-week high-fat diet,streptozotocin(STZ,Sigma,St.Louis,MO;27.5 mg/kg i.p.in 0.1 mol/L citrate buffer,pH 4.5)was administrated at once in rats with insulin resistance.One week after STZ administration,rats with FBG>11.1 mmol/L in two consecutive analyses were considered diabetes.After 12 weeks of diabetes,rats showed an increase in E/e’ were deemed as DCM rats,then the DCM rats were randomly divided into three groups:DCM group(n=15),DCM+vehicle group(n=15),DCM+CIDEC shRNA group(n=15).2.Blood analysesAfter rats fasted overnight,we collected jugular blood.Fasting blood glucose(FBG)was analyzed by glucose oxidase method.Total cholesterol(TC),low density lipoprotein(LDL),high density lipoprotein(HDL),triglycercide levels(TG)and Free fatty acids(FFA)were analyzed with use of the Bayer 1 650 blood chemistry analyzer(Bayer,Tarrytown,NY USA).Fasting insulin level was measured by ELISA.ISI was calculated.3.IPGTT and IPITTGlucose tolerance was assessed by IPGTT after rats fasted for 12 h.A bolus of glucose(1 g/kg i.p.)was injected,and blood samples were collected sequentially from the tail vein at 0,15,30,60,and 120 min and tested for glucose.Plasma glucose was measured with a One-Touch Glucometer(LifeScan,Milpitas,CA).The mean area under the receiver operating characteristic curve(AUC)was calculated for glucose.To evaluate insulin tolerance,IPITT was performed after rats fasted for 4 h.A bolus of insulin(1 unit/kg i.p.)was administered,and blood samples were taken for glucose measurement as described above.4.EchocardiographyEchocardiography involved use of the Vevo2000 imaging system(VisualSonics,Toronto,Canada).During echocardiography,rats were anesthetized by intraperitoneal injection with 0.04mg/g body weight of pentobarbitol sodium.Images were obtained from two-dimensional,M-mode,pulsed-wave Doppler and tissue Doppler imaging.All measurements were performed by the same observer and were the average of six consecutive cardiac cycles.Wall thickness and LV dimensions were obtained from a longaxis view at the level of chordae tendineae.Diameters of left ventricular end diastole(LVEDd),left ventricular end-diastolic posterior wall(LVPW),and septum thickness,as well as left ventricular ejection fraction(LVEF)and fractional shortening(FS),were measured according to the American Society of Echocardiography guidelines.The mitralvalve pulsed Doppler and mitral annulus tissue Doppler recordings were obtained from the apical four-chamber view.After pulsed Doppler and tissue Doppler,we evaluated transmitral early diastolic flow velocity(E)and mitral annulus early diastolic velocity(e’)and calculated E/e’.5.Hemodynamic measurementsRats under deep anesthesia as described above.Millar catheter(SPR-869,Millar Instruments Inc.,Houston,TX)was advanced from the right carotid artery into the left ventricle.After a 5-min period of stabilization,the parameters were acquired and recorded.The myocardial contractility was assessed according to the left ventricular systolic peak pressure(LVSP)and the maximum rate of ascending pressure change in the left ventricle(+dp/dtmax).Myocardial relaxation was evaluated according to the left ventricular enddiastolic pressure(LVEDP)and the maximum descending rate of left ventricular pressure(dp/dtmax).6.Cardiac collagen morphometric analysesInterstitial and perivascular fibrosis were also evaluated by Picrosirius red staining.Sections were stained with 0.5%sirius red(Sigma)in saturated picric acid for 25 min.Collagen was stained red.Dark green-stained collagen fibers were quantified as a measure of fibrosis in Masson trichrome-stained sections.The collagen volume fraction(CVF)and perivascular collagen area/luminal area(PVCA/LA)were analyzed by quantitative morphometry with automated image analysis(Image-Pro Plus,Version 5.0)as reported previously.The collagen content of myocardial tissue was determined by hydroxyproline assay.Tissue hydrolysate was detected by use of an ELISA kit(F15649;Westang Bio-Tech,Shanghai,China).Paraffin sections underwent immunohistochemistry by a microwave-based antigen retrieval method.The sections were incubated with primary rabbit polyclonal anti-laminin,anti-collagen Ⅰ and anti-collagen Ⅲ antibodies overnight and then with a matching biotinylated secondary antibody for 30 min at 37℃.The results were observed under ×200 magnification with an Olympus microscope.Western blot analyses were used to collagen Ⅰ and Ⅲ semiquantitative determination,and collagen Ⅰ/Ⅲ ratio was calculated.7.Histology and morphometric analysesParaformaldehyde(4%)-fixed hearts were bisected transversely at the midventricular level,embedded in paraffin,and cut into 4-μm sections.Hematoxylin-eosin staining and laminin immunohistochemistry staining were performed on myocardial tissue to facilitate fiber cross-sectional measurements.The myocyte cross-sectional area was assessed under×400 magnification by an Olympus microscope(Olympus,Japan)within the left ventricle,and a mean was obtained by quantitative morphometry with automated image analysis(Image-Pro Plus,Version 5.0).Myocardial frozen sections(5-μm)were stained with Oil Red O(Sigma)for 10 min,washed,and then counterstained with hematoxylin for 30 s.A Nikon microscope(Nikon,Melville,NY)was used to capture the Oil Red O-stained tissue sections with ×600 magnification.Paraffin sections underwent immunohistochemistry by a microwave-based antigen retrieval method.The sections were incubated with primary rabbit polyclonal anti-advanced glycosylation end-products(AGEs)and anti-interleukin(IL)-6 antibodies overnight and then with a matching biotinylated secondary antibody for 30 min at 37℃.The results were observed under ×200 magnification with an Olympus microscope.8.The expression of CIDEC/AMPKa signaling pathwayReal time PCR and Western blot were used to determine the level of CIDEC mRNA and protein expression in cardiac tissue.Phospho-AMPKα and AMPKα were determined by use of western blot,then the ratio of phospho-AMPKα/AMPKa was analyzed.9.CIDEC gene silencing in vivoAfter 12 weeks of diabetes,the DCM+CIDEC shRNA group were injected 2.5×1010 plaque-forming units of an adenovirus harboring shRNA against a CIDEC gene(CIDEC shRNA,Hanbio Biotechnology Co.,Ltd.,Shanghai,China)and the DCM+vehicle group was injected an empty virus(vehicle)as control.Adenovirus transfer was repeated in 2 weeks.Results1.Development of metabolic disorders in DCM ratsData of metabolic tests revealed that lipid metabolic disturbance,glucose tolerance impairment and insulin resistance was generated after a 4-week high-fat diet,and was further aggravated after STZ injection in diabetic group.In brief,a high-fat diet and lowdose STZ induced obesity,insulin resistance,moderate hyperglycemia and hyperlipidemia in T2DCM rats,resembling the state of human type 2 diabetes.2.Deterioration of left ventricular(LV)function in DCM ratsLVEDd,LVEF,FS,and E/e’ were evaluated in order to investigate alterations in LV expansion,systolic and diastolic function.Similar to the pattern observed in humans,diastolic dysfunction preceded systolic dysfunction,beginning from 2 to 3 months after induction of DM.The results unraveled that E/e’ in diabetic rats begun to appear a slight increase after 1 week of STZ injection,showed a moderate increase after 6 weeks of diabetes and existed a striking increase after 12 weeks of diabetes compared with control.Our model was in the relative early stages of DCM,LVEF and FS were mildly impaired couple with an increase of LVEDd after 12 weeks of diabetes.3.Myocardial morphometric changesThe DCM group showed cardiac hypertrophy and fibrosis with a scattered,small,inordinate and nonuniform pattern,as well as damaged and irregular collagen network structure in the interstitial and perivascular areas.Body weight and heart weight to body weight ratio were higher in DCM group than control group with LV myocyte size enlarged.Collagen Ⅰ and Ⅲ were elevated in DCM rats compared with the control.The ratio of collagen Ⅰ/Ⅲ were elevated more significantly.Myocardial lipid analysis showed myocardial accumulation of triglycerides in DCM group.DCM rats had higher Oil Red O-staining areas than control.Moreover,protein levels of IL-6 and AGEs were significantly elevated in DCM group versus control by use of immunohistochemistry.Thus,cardiac ectopic lipid deposition and aberrant inflammation developed in DCM rats.4.Detection of cardiac CIDEC expression in DCM ratsOur data showed that relative mRNA and protein levels of CIDEC were elevated in heart in diabetic rats compared with control.With CIDEC shRNA transfection,CIDEC relative mRNA was diminished 62.5%and protein expression was reduced by 41%,implicating the transfection was effective.5.The underlying mechanism of CIDEC gene silencing alleviating DCM5.1 CIDEC gene silencing ameliorated metabolism in DCM ratsAt the end of the experiment,serum TC,TG,FFA,LDL,FBG and FINS levels were significantly higher in DCM group than control,conversely HDL and ISI were lower.By CIDEC gene silencing,levels of the above metabolic indices were restored.By IPGTT and IPITT,the levels of blood glucose in DCM group were significantly higher than control at all of the time points tested.By CIDEC gene silencing,levels of blood glucose declined dramatically at all of the time point tested.Similarly,the AUC across the time for glucose level was higher in DCM group than control,which was decreased by CIDEC gene silencing.These results demonstrated that DCM rats showed severe insulin resistance,glucose and lipid metabolic disturbance,which could be attenuated by CIDEC gene silencing.5.2 CIDEC gene silencing reverses myocardial hypertrophy,lipids deposition and inflammation in DCMWith CIDEC gene silencing,the body weight,heart weight to body weight ratio,myocyte size and protein level of brain natriuretic protein(BNP)were significantly decreased,coincide with amelioration of cardiac hypertrophy in DCM+CIDEC shRNA group compared with DCM+vehicle group.With CIDEC gene silencing,Oil Red O-staining areas,IL-6 and AGEs were reduced,suggesting CIDEC gene therapy reduced inflammation and ectopic lipid deposition.5.3 CIDEC gene silencing attenuated myocardial interstitial fibrosisThe scattered and inordinate pattern became dense rules with a decrease of CVF,PVCA/LA and collagen content by CIDEC gene silencing,suggesting CIDEC gene silencing reversed cardiac hypertrophy and fibrosis.5.4 Mechanism of CIDEC mediated cardiac fibrosis in DCM ratsCardiac fibrosis is an important pathogenic feature in DCM.Then we focused the role of CIDEC on cardiac fibrosis and the involved mechanism in vivo.The results showed that CIDEC was increased,the phosphorylation of AMPKαwas downregulated,and syntheses of collagen Ⅰ and Ⅲ were elevated in DCM rats.However,these changes could be restored by CIDEC gene silencing,indicating CIDEC-AMPKαsignaling pathway may participate in myocardial interstitial fibrosis in DCM.Thus,we suggested that DCM rats expressed excessive CIDEC,which resulted in suppression of AMPKαphosphorylation and further induced promotion of collagen syntheses.5.5 CIDEC gene silencing recovered cardiac function in DCM ratsAt the end of the experiment,by use of echocardiography,LVEF and FS were deteriorated,besides E/e’ and LVEDd elevated in DCM group versus control group.Deterioration of cardiac function was recovered by CIDEC gene silencing,suggesting CIDEC gene silencing improved LV dysfunction.By cardiac catheterization,LVEDP was increased and the absolute values of ± dp/dt max were decreased in DCM group versus control group.The LVESP among the four groups showed no statistical difference.Increased LVEDP and decreased absolute values of±dp/dt max in DCM rats were reversed by CIDEC gene silencing,which reconfirmed the beneficial effect of CIDEC shRNA on improving LV diastolic dysfunction.Conclusions1.DCM rats have significant disorder of glucose and lipid metabolism,which is characterized by insulin resistance,hyperglycemia and hyperlipidemia,which is basically consistent with the clinical characteristics of type 2 diabetes.2.Left ventricular diastolic dysfunction is the main manifestation of DCM rats,followed by systolic dysfunction.3.DCM rats showed myocardial fibrosis,myocardial hypertrophy,ectopic lipid deposition and inflammation,and increased expression of CIDEC in myocardial tissue,suggesting that CIDEC is involved in the pathological process of DCM.4.Up-regulation of CIDEC expression and inhibition of AMPKα signal transduction pathway significantly increase the synthesis of type Ⅰ and Ⅲ collagen and the proportion of collagen Ⅰ/Ⅲ,resulting in myocardial interstitial fibrosis,which is an important molecular mechanism for the occurrence and development of type 2 DCM.5.CIDEC gene silencing can improve left ventricular systolic and diastolic function in DCM rats by upregulating AMPKα-Thy172 phosphorylation,reducing collagen Ⅰ and Ⅲsynthesis,reducing collagen Ⅰ/Ⅲ ratio,and alleviating myocardial interstitial fibrosis.6.CIDEC gene silencing can reduce the disorder of glucose and lipid metabolism,myocardial hypertrophy,ectopic lipid deposition and inflammation in DCM rats.BackgroundDiabetic cardiomyopathy(DCM)is described as structural and functional changes in myocardium caused by specific metabolic abnormalities of diabetes,which is independent of diabetic macrovascular complications.Clinical studies have shown that DCM is characterized by diastolic heart failure as the early manifestation.Myocardial interstitial fibrosis is the main mechanism of diastolic heart failure and also lead to the impairment of systolic function with the increase of collagen concentration,suggesting that myocardial interstitial fibrosis plays an important role in the occurrence and development of DCM.Although myocardial fibrosis caused by diabetes increased morbidity and mortality of DCM,there has been no effective treatment to improve heart failure yet.Therefore,it’s important to find a common way that improves glucose metabolism and reduces myocardial fibrosis and to study the underlying mechanism of myocardial fibrosis for seeking the preventive and therapeutic strategy of DCM.Cardiac fibroblasts(CFs),the ultimate effector cells in the process of fibrosis,is a potential target for anti-fibrosis treatment of myocardial fibrosis in DCM.However,it is a continuous research area for screening the intrinsic intervention targets of CFs.We have found that CIDEC is closely associated with myocardial fibrosis in DCM at the integral level.DCM rats showed obvious myocardial fibrosis and the increased ratio of type Ⅰ/Ⅲcollagen,accompanied with the increased expression of CIDEC and the decreased activity of AMPKα/ACC signaling.Furthermore,the silence of CIDEC gene can alleviate myocardial fibrosis by activating AMPKα/ACC signaling.Therefore,CIDEC/AMPKαsignaling pathway is involved in myocardial fibrosis of DCM.However,at the cellular level,it needs to be further explored whether CIDEC is a potential intervention target for collagen synthesis and how CIDEC and AMPKα localize and interact with each other in CFS.Initially,CIDEC was identified as an apoptotic protein based on its homology with DNA cleavage factor.However,recent studies have redefined it as a metabolic phenotype that contributes to human health and regulates lipid droplet dynamics and lipid metabolism,which is closely related to metabolic balance.Localization in mitochondria is necessary for CIDE protein to induce apoptosis and DNA fragmentation.Puri et al.discovered that CIDEC was highly expressed in adipocytes and localized on lipid droplets,which played a key role in lipid metabolism.Therefore,these proteins are considered as lipid droplet related proteins.Other studies suggested that CIDEC may also exist in endoplasmic reticulum.However,it is not well understood that the subcellular localization and function of CIDEC in CFS under insulin-resistant state.At the integral animal level,we found that CIDEC could regulate AMPKα in the preliminary study.However,how does CIDEC regulate AMPKα to play a role in the process of insulin resistance-induced collagen synthesis in CFs,and which subtype it acts on?AMPK exists in the form of heterotrimer complex,including α,β and γ subunits.Among them,α subunit is the catalytic subunit,including two subtypes considered as α1 and α2.Salt Ian et al.found that most of the AMPK complex located in the nucleus contained a2 subtype rather than al subtype,suggesting that AMPKα2 may be involved in the direct regulation of gene expression.Studies have also shown that AMPKα2 can regulate Ang-Ⅱ-induced collagen synthesis in abdominal aortic aneurysm,and it is a target of metformin to alleviate myocardial fibrosis.However,it is unclear whether AMPKα2 play a vital role in insulin resistance-induced collagen synthesis in CFs,let alone the subcellular localization and mechanism of different subtypes.Taken all together,CIDEC/AMPKα signaling pathway may play an important role in insulin resistance-induced collagen synthesis in CFs.However,it’s unclear that the subcellular localization and interaction between CIDEC and AMPKα exist in CFs and how CIDEC/AMPKα signaling pathway mediates collagen synthesis.We established insulinresistant CF model to study the interaction and subcellular localization of CIDEC and AMPKα,and the role of CIDEC/AMPKα signaling pathway in collagen synthesis.Further we explored that the regulatory mechanism of CIDEC knockdown on insulin resistanceinduced collagen synthesis through the specific inhibition of CIDEC expression by CIDEC shRNA adenovirus transfection at the cellular level.Objectives1.To establish insulin-resistant cardiac fibroblast model,and to study the role of CIDEC in insulin resistance-induced collagen synthesis at the cellular level.2.To explore the subcellular localization of different AMPKα subtypes in CFs and their interaction with CIDEC.3.Constructing the recombinant adenovirus vector of rat CIDEC and transfecting CFs to study the important role of CIDEC/AMPKα signaling pathway in insulin resistanceinduced collagen synthesis.MethodsNeonatal rat CFs were cultured,and real-time PCR(RT-PCR),western blot,single and double immunofluorescent staining,duolink? in situ PLA,co-immunoprecipitation(Co-IP)and adenovirus transfection technology were performed.1.To simulate the insulin resistance of type 2 diabetes in vitro,insulin-resistant cardiac fibroblast model were induced by high glucose and high insulin.The cells were divided into normal glucose group(5.5 mmol/L)and insulin-resistant group(15mmol/L glucose and 104μU/ml insulin).The expression of CIDEC mRNA and protein,the phosphorylation of AMPKα,the contents of type Ⅰ and type Ⅲ collagen and the ratio of collagen Ⅰ/Ⅲ were analyzed.2.The CFs were divided into normal glucose(NG)group and insulin-resistant(IR)group.The intracellular localization of CIDEC and AMPKα subtypes,the colocalization and interaction between CIDEC and AMPKα1+α2,and the interaction between CIDEC and AMPKα2 were observed.3.CIDEC overexpression adenovirus(Flag+GFP labeled)was constructed to transfect the primary cultured neonatal rat CFs.The biological interaction between CIDEC and AMPKαl+α2 and between CIDEC and AMPKα2 proteins was detected by Co-IP technique.4.CIDEC shRNA adenovirus(GFP labeled)was constructed to transfect normal and insulin-resistant CFs.After inhibiting the expression of CIDEC mRNA and protein,the effect of insulin resistance on the phosphorylation of AMPKα,the expression of collagen type Ⅰ and Ⅲ,and the ratio of collagen Ⅰ/Ⅲ were observed.5.In NG group,IR group and IR+CIDEC-shRNA group,AMPK inhibitor compound C was added to inhibit the phosphorylation of AMPKα.After inhibiting AMPKα activation,the effects of CIDEC shRNA adenovirus transfection on insulin resistance-induced collagen synthesis and collagen Ⅰ/Ⅲ ratio were observed in CFs.Results1.Induction of insulin-resistant cardiac fibroblast modelInsulin-resistant cardiac fibroblast model was successfully induced by incubating with high glucose and high insulin(15mmol/L glucose and 104μU/ml insulin,insulin-resistant stimulation).We observed that relative mRNA of CIDEC peaked at 24h after insulin-resistant stimulation(P<0.001),while the protein level of CIDEC peaked at 48h(P<0.01).The results also demonstrated that effect of insulin resistance reached the top at 48h poststimulation,so the following insulin-resistant cardiac fibroblasts were incubated with insulin-resistant stimulation for 48h.2.Insulin resistance promoted collagen synthesis and disproportion in CFsCollagen Ⅰ and Ⅲ syntheses and collagen Ⅰ/Ⅲ ratio were elevated in insulin-resistant CFs compared with normal CFs(P<0.05～P<0.001).3.Insulin resistance inhibited phosphorylation of AMPKα and ACCThe phosphorylation of AMPKα was downregulated in insulin-resistant CFs compared with normal CFs(P<0.001).4.Insulin resistance promoted CIDEC nuclear translocationWith insulin-resistant stimulation,the fluorescent IOD of CIDEC increased significantly in nucleus(P<0.001),while the fluorescent IOD in cytoplasm decreased(P<0.05),accompanied by a more than 2 times increase in the ratio of nucleus to cytoplasm of CIDEC fluorescent IOD.The observation was also confirmed by western blot analyses of nuclear and cytoplasmic components respectively.5.Changes of subcellular localization and expression of different AMPKα subtypes in insulin-resistant CFsThen subcellular localizations of AMPKα1+α2,AMPKα1 and AMPKα2 were simultaneously detected.The results demonstrated that AMPKα l+α2 was distributed both in cytoplasm and nucleus in normal CFs.In insulin-resistant CFs,the fluorescent IOD of AMPKα1+α2 decreased in cytoplasm(P<0.05)but increased in nucleus(P<0.05).The results further demonstrated AMPKαl was distributed almost completely inside cytoplasm in normal CFs.Moreover,the fluorescent IOD of AMPKαl decreased significantly after insulin-resistant stimulation(P<0.05),suggesting that insulin-resistant stimulation decreased the distribution of AMPKαl in the cytoplasm.Conversely,AMPKα2 was observed to distribute almost completely inside nucleus in normal CFs.In insulin-resistant CFs,the nuclear fluorescent IOD of AMPKα2 increased markedly compared the normal(P<0.001).Based on the results,we speculate that the excessive CIDEC in nucleus maybe interact with AMPKα2 under insulin-resistant state.6.Interaction and co-localization between CIDEC and AMPKαl+α2 in CFsThe results of Co-IP showed that CIDEC could directly interacted with AMPKαl+α2.The results of duo-link? in situ PLA further proved that the fluorescent IOD of the positive region(CIDEC and AMPKαl+α2 interaction)was decreased in cytoplasm(P<0.05),but it was significantly increased in nucleus(P<0.001),and the nucleocytoplasmic ratio was also strikingly increased(P<0.001)in IR group compared with NG group.The changes of CIDEC and AMPKαl+α2 co-localization distribution and area in insulin-resistant CFs detected by double immunofluorescent staining was consistent with the results of duolink?in situ PLA.7.Interaction and localization between CIDEC and AMPKα2 in CFsThe results of duo-link? in situ PLA demonstrated that CIDEC could biochemically interact with AMPKα2 in CFs,and the positive areas(almost all in nucleus)were markedly increased under the insulin-resistant state(P<0.001).The results of Co-IP further confirmed that CIDEC could directly interact with AMPKα2.Therefore,we elucidated that in nucleus of CF,excessive CIDEC induced by insulin resistance biochemically interacted with AMPKα2.8.Mechanism of CIDEC facilitating collagen syntheses and disproportion in insulinresistant CFsIn order to explore the mechanism of CIDEC facilitated collagen syntheses,we transfected insulin-resistant CFs with CIDEC shRNA.The results unraveled that mRNA and protein levels of CIDEC were significantly declined by CIDEC shRNA transfecting(P<0.001),contributing to reduction of collagen Ⅰ and Ⅲ syntheses and collagen Ⅰ/Ⅲ ratio(P<0.05～P<0.001),which was accompanied by upregulation of AMPKα phosphorylation(P<0.001).In order to further confirm CIDEC facilitated collagen syntheses via AMPKα,we added AMPK inhibitor compound C into the culture medium 2h before CIDEC shRNA transfecting.We found that AMPKα phosphorylation were dramatically decreased(P<0.01～P<0.001),accompanied with increases of collagen Ⅰ and Ⅲ syntheses and collagen Ⅰ/Ⅲ ratio in IR+CIDEC-shRNA+cC group versus IR+CIDEC-shRNA group(P<0.05～P<0.001),suggesting compound C could offset the effect of CIDEC gene silencing on reducing collagen syntheses in insulin-resistant CFs.These findings clarified that excessive CIDEC downregulated phosphorylation of AMPKα,further resulting in elevation of collagen syntheses in insulin-resistant CFs.Conclusions1.Insulin resistance induces the excessive expression of CIDEC and promotes CIDEC nuclear translocation in myocardial fibroblasts.2.CIDEC promotes collagen syntheses and collagen Ⅰ/Ⅲ ratio via inhibiting the activation of AMPKα.3.CIDEC knockdown reduces insulin resistance-induced collagen synthesis and the ratio of collagen Ⅰ/Ⅲ through upregulating AMPKα phosphorylation. |
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