| Type2diabetes mellitus (T2DM) is associated with increasedcardiovascular risk, which include a2-3fold increase in cardiac death and anincreased risk of CHF (congestive heart failure). During the past3decades,numerous epidemiologic and observational studies have shown that dietaryn-3polyunsaturated fatty acids (n-3PUFAs), especially eicosapentaenoic acid(EPA;20:5n-3) and docosahexaenoic acid (DHA;22:6n-3) which enriched infish oil, protect against several types of cardiovascular diseases (CVDs) suchas myocardial infarction, arrhythmia, atherosclerosis, or hypertension. For thisreason, the American Heart Association (AHA) currently recommends1g/dEPA+DHA for individuals with established disease including T2DM.However, molecular mechanism of the cardioprotective effect of DHA inT2DM remains unclear.In the present study, we used a high-fat diet (HFD) plus a low-dose ofSTZ to induce the type2diabetic rat, which is known to develop manyfeatures reported in patients with uncontrolled type2diabetes mellitus,including hyperglycemia, polyuria, and loss of body weight. Firstly, thecomposition of free fatty acids including long chain n-3PUFA and theperoxisomal fatty acid β-oxidation activity were assayed in the diabetic heart.Secondly, the cardioprotective effects of dietary DHA supplementation wereinvestigated by determination of left-ventricular function, serum and cardiacDHA content, myocardium morphology change and genomic mRNAexpression in type2diabetic rats. Finally, in order to explore the molecularmechanism of the cardioprotective effect of DHA, the cardiac troponincomplex including troponin T, C and I expression, calpain activity andexpression were studied in the T2DM heart and in C16:0treated H9C2myocardial cell line. Part Ⅰ Reduction of n-3PUFAs, specifically DHA and EPA, andenhancement of peroxisomal beta-oxidation in type2diabetic rat heart.Objective: The PUFA composition and peroxisomal beta-oxidation werestudied in the T2DM rat heart.Methods:1Serum and cardiac fatty acids composition was determinedby capillary gas chromatography.2The total activity of fatty acidbeta-oxidation was determined by spectrophotometry.3The peroxisomalbeta-oxidation activity was determined by luminometric assay.4Correlationbetween cardiac PUFA content and peroxisomal beta-oxidation activity wasdetermined with the spearman correlation test.Results:1Changes of PUFAs composition in T2DM serum and heartIn serum, PUFAs, including C22:4n-6(~614%), were significantlyincreased in the DM group compared with the control group. Only the LNA(C18:3n-3) and EPA (C20:5n-3) were62%and19%lower, respectively, inDM serum than in CON serum. Additionally, there was no significant changein n-6/n-3ratio between the two groups.In the heart, PUFAs, including C18:2n-6(~17%) and C22:4n-6(~157%),were significantly increased in the DM group compared with the control group.C24:0was29%lower in the DM heart than in the CON heart. Mostimportantly, we found that all the n-3series PUFAs, including LNA (~45%),EPA (~100%), DPA (~42%) and DHA (~48%), were dramatically decreased inthe DM hearts compared to the CON hearts. Moreover, accompanied with aslight increase in the sum of n-6PUFAs (~11%), the dramatic decrease in sumof n-3PUFAs made the n–6/n-3ratio~115%higher in the DM hearts than inthe CON hearts.2The activity of peroxisomal beta-oxidation were increased in T2DMheartThe total fatty acid β-oxidation activities of DM hearts (P<0.01)increased by76%compared with CON hearts.The peroxisomal beta-oxidation activity in the DM hearts (P<0.01) was increased by30%compared to the CON hearts.3EPA, DPA and DHA content were negative correlated with peroxisomalbeta-oxidation in T2DM heartA correlation analysis showed that there were negative correlationsbetween the abundance of EPA (r=0.67, P<0.001), DPA (r=0.63, P<0.001)and DHA (r=0.83, P<0.001) and the activity of peroxisomal beta-oxidationin rat hearts.Part ⅡCardioprotective effect of DHA in type2diabetic rat heartObjective: In this part, we studied the left-ventricular function, DHAcontent, microstructure and morphology of myocardial cell and cardiacgenomic mRNA expression to evaluate the cardioprotective effect of dietaryDHA supplementation in T2DM.Methods:1T2DM rats were induced by the combination of a HFD and alow-dose STZ, and some of the T2DM rats were fed with a HFD plus DHAsupplementation.2The cardiac DHA content was determined by gas capillarychromatography.3The LV function was determined by M-typeechocardiogram.4Microstructure and morphology of myocardial cell wasdetermined by transmission electron microscope (TEM).5The filamentstructure of myocardial cell was determined by laser scanning confocalmicroscope (LSM) after phalloidin staining.6The changes of genomic mRNAexpression were determined by gene chip technique.7The differentiallyexpressed genes were further analyzed by GO and Pathway analysis.Results:1The T2DM rats weight gain after dietary supplementation of DHA.There was no significant change in fasting blood glucose(FBG) betweenthe DM and DM+DHA group.1.1Body weightAt the end of the experiment (32wks), the body weight was significantlylower in the DM group than in the CON group (P<0.01). And the body weightwas significantly higher in the DM+DHA group than in the CON group(P<0.01), but not than in the CON group (P>0.05). 1.2FBGAt the end of the experiment (32wks), the FBG level was significantlylower in the DM and DM+DHA group than in the CON group (P<0.01). Andthere was no significant change in FBG level between the DM and DM+DHAgroup (P>0.05).2Diets supplemented with DHA increased the DHA content in type2diabetic rat heartsThe DHA content of rat hearts in DM+DHA group was not only farhigher than that in DM group (about4times, P<0.01), but also higher thanthat in control group (about2times, P<0.01).3Diets supplemented with DHA improved the cardiac function of type2diabetic rats3.1Parameters of left ventricular hypertrophyThe left ventricular mass (LV mass) in DM group was higher than that inCON group (P<0.01), but there was no differences in DM+DHA groupcompared to the other two groups (P>0.05). LV mass/body weight index inDM group was significantly higher than that in the CON group and DM+DHA group.3.2Parameters of LV pump function and systolic functionCompared with the CON group, systolic LV volume (LV Vol; s) in theDM group were higher, while LV ejection fraction (%LVEF) and LV fractionalshortening fraction (%LVFS) were lower (P<0.01). However, all the aboveparameters were significantly improved in DM+DHA group (P<0.01), andno significant difference was seen compared with CON group (P>0.05). Therewas no significant difference in diastolic LV volume (LV Vol; d) among thethree groups (P>0.05).4Diets supplemented with DHA improves LV myocardial microstructureof T2DM ratsIn CON group, the mitochondria were typically intact and neatlyarranged. Cardiac muscle fibres were abundant with regular arrays ofmyofibrils closely arranged within sarcomere. And the M line and Z line were distinct and regular arrays.In DM group, different sizes of mitochondria were disarranged and with ahigher matrix density compared with CON group. Myofibrils were irregulararrays, fracture and dissolved within increased sarcomere length. And the Mline and Z line were disarranged and indistinct.In DM+DHA group, mitochondria were also disarranged and with ahigher matrix density. But the structure of myofibrils were almost normalcompared with CON group, excepted the increased sarcomere length.5Diets supplemented with DHA improves myofibrils structure of T2DMratsIn CON group, myofibrils were distinct and regular arrays. In DM group,myofibrils were indistinct and irregular arrays, and a lot of myofibrils werefracture and dissolved. In DM+DHA group, the structure of myofibrils werealmost normal compared with CON group.6The genomic mRNA expression was improved in T2DM heart afterdietary supplementation of DHA.6.1Differential expressed genes between two groups: There were109up-and224down-regulated genes in the DM group than in the CON group. Therewere90up-and135down-regulated genes in the DM group than in theDM+DHA group. There were73up-and84down-regulated genes in theDM+DHA group than in the CON group.Same differential expressed genes: There were exactly the same23up-and41down-regulated genes in the DM and DM+DHA group than in theCON group. And there were exactly the same34up-and79down-regulatedgenes in the DM group than in the DM+DHA and CON group.We further investigated the differential expressed genes by GO andPathway analysis online. The results were followed:6.2The GO analysis including Molecular Function, Biological Process andCellular Component analysis.6.2.1The differential expressed genes were involved in19CellularComponents, and the typical Cellular Components were ribosome, extracellular region, ATP synthase complex, mitochondrial matrix, nucleusand nucleosome.6.2.2The differential expressed genes were involved in29Molecular Function,and the typical Cellular Components were structural constituent of ribosome,translation initiation factor activity, electron carrier activity, hydrogen iontransporter activity, ATP and ATPase binding, GTP binding and GTPaseactivity, short-branched-chain-acyl-CoA dehydrogenase activity, fructose-bisphosphatase activity, NAD binding, pyruvate dehydrogenase kinase activity,oxidoreductase activity, phosphoinositide3-kinase binding and SH2domainbinding.6.2.3The differential expressed genes were involved in28Biological Process,and the typical Cellular Components were protein biosynthesis, translationalelongation, translational initiation, ATP synthesis coupled proton transport,positive regulation of chronic inflammatory response, cellular response toreactive oxygen species, transformed cell apoptosis and caspase activation viacytochrome c, endosome to lysosome transport, glucose metabolism,mitochondrial electron transport and negative regulation of lipoprotein lipaseactivity.6.3The Pathway analysisThe differential expressed genes were involved in14Pathway, and thetypical Pathway were Type I diabetes mellitus, p53signaling pathway,Apoptosis, Oxidative phosphorylation, Ribosome, Glycolysis/Gluconeo-genesis, Tight junction, Proteasome and Regulation of actin cytoskeleton.Part ⅢThe mechanism of cardioprotective effect of DHA on myofibrilsdissolved and fracture in T2DM rat heartObjective: To explore the reason of myofibrils dissolved and fracture intype2diabetic rat cardiac muscle tissues and the molecular mechanism of theprevention effect of DHA on all the above pathological changes.Methods:1The expression of myocardial TNNC1, TNNT2and TNNI3was observed at protein level by Western Blot.2The expression of myocardialTNNC1, TNNT2and TNNI3was observed at mRNA level by Real time PCR. 3The myocardial Calpain activity was detected by AMC method.4Theexpression of myocardial calpain1,2, and4was observed at protein level byWestern Blot.5The expression of myocardial calpain1,2, and4wasobserved at mRNA level by Real time PCR.6MTT assay was used to screenthe appropriate concentration of C16:0treatment in H9C2cells.7WesternBlot and Real time PCR methods were used to detected that whether all theabove phenomena can be inhibited by DHA intervention.8Effects of DHA onC16:0-induced apoptosis in H9C2cells wer detected by flow cytometryanalysis.9Cell apoptosis was determined by Hoechst33258staining in H9C2cells.10Effects of DHA on Caspase3and Caspase12were detected both atprotein and mRNA level by Western Blot and Real time-PCR respectively.Results:The results of in vivo experiments1The protein level of related molecules involved in cardiac troponincomplex were decreased in T2DM rats, while the protein level of thatwere normal in T2DM rats supplemented with DHA1.1The protein level of related molecules involved in cardiac troponincomplex were significantly decreased in T2DM ratsWestern results showed that the protein levels of myocardial TNNC1,TNNT2and TNNI3in DM group were significantly decreased compared withthe control group. Immunohistochemical staining results similarly indicatedthat the protein level of myocardial TNNT2in DM group was decreasedcompared with the control group. Real time PCR results showed that there wasno significant differences in the expression of myocardial TNNC1, TNNT2and TNNI3at mRNA level between DM group and CON group (P>0.05).All the above results suggested that the down-regulation of TNNC1,TNNT2and TNNI3protein levels were associated with myofibrils dissolvedand fracture in type2diabetic rat cardiac muscle tissues. And the decrease ofTNNC1, TNNT2and TNNI3proteins were not due to the reduced expression.1.2The protein level of related molecules involved in cardiac troponincomplex were normal in T2DM rats supplemented with DHA Western results showed that there were no significant differences in theprotein levels of myocardial TNNC1, TNNT2and TNNI3between DM+DHAgroup and CON group. However, the protein levels of myocardial TNNC1,TNNT2and TNNI3in DM+DHA group were significantly increasedcompared with DM group. These results were further confirmed byImmunohistochemical staining assay. Real time PCR results showed that theexpression of myocardial TNNC1, TNNT2and TNNI3at mRNA levels werenormal in DM+DHA group (P>0.05).All the above results suggested that diets supplemented with DNA couldprevent the down-regulation of TNNC1, TNNT2and TNNI3proteins whichwere associated with myofibrils dissolved and fracture in type2diabetic ratcardiac muscle tissues.2The total calpain activity was increased in DM heart, but not inDM+DHA2.1Increased total calpain activity in T2DM heartThe cardiac normal (natural) and maximum (CaCl2) calpain activity wassignificantly higher in DM group than in CON group (P<0.01). After addedcalcium, the total activity of calpain (maximum/natural) was increased91%and135%in DM and CON group respectively, indicating that the calcium wasslightly increased in T2DM heart.2.2The cardiac total calpain activity was normal in T2DM rats supplementedwith DHAThe cardiac normal (natural) and maximum (CaCl2) calpain activity wassignificantly lower in DM+DHA group than in DM group (P<0.01). Afteradded calcium, the total activity of calpain (maximum/natural) was increased122%in DM+DHA group, indicating that the calcium concentration wasnormal in T2DM rats supplemented with DHA.3The expression of Calpain were increased in T2DM rats, while theexpression of Calpain was normal in T2DM rats supplemented with DHA3.1The expression of Calpain were increased in T2DM ratsWestern results showed that the protein levels of myocardial Calpain1,2 and4in DM group were significantly increased compared with the controlgroup. Real time PCR results showed that the expression of myocardialCalpain1,2and4mRNA in DM group were significantly increased comparedwith the control group (P<0.01).All the above results suggested that the reasons of Calpain activityaugmented were the up-regulation of μ-Calpain and m-Calpain both at proteinand mRNA level.3.2The expression of Calpain was normal in T2DM rats supplemented withDHAWestern results showed that there were no significant differences in theexpression of myocardial Calpain1,2and4proteins between DM+DHAgroup and control group. However, the expression of myocardial Calpain1,2and4proteins in DM+DHA group were significantly decreased comparedwith DM group. Real time PCR results showed that the expression ofmyocardial Calpain1,2and4at mRNA level were significantly decreasedcompared with DM group (P<0.01). Compated with control group, theexpression of Calpain1and2at mRNA level in DM+DHA groups have nodifferences, while the expression of Calpain4at mRNA level was decreased(P<0.01).All the above results suggested that diets supplemented with DNA couldprevent the up-regulation of μ-Calpain and m-Calpain which were associatedwith Calpain activity.The results of in vitro experiments4The concentrations of C16:0treatments in H9C2cells were screened bythe MTT assayAfter different concentrations of C16:0treatments for12h and24h, thecell survival rates in C16:0treated groups displayed a significant decrease in adose-dependent manner ranging from100to500μmol/L. Therefore,100,150,200and250μmol/L of C16:0were selected in further assays to explore theircytotoxic effects on H9C2cells by24h. 5C16:0could simulate the phenomena of Calpain degradating troponinfound in vivoWestern results showed that100,150,200and250μmol/L C16:0treatment could up-regulate the expression of Calpain1,2and4in aconcentration-dependent manner in H9C2cells, while down-regulate theexpression of TNNC1, TNNT2and TNNI3in a dose-dependent manner.Results from spearman correlation test studies indicated that the protein levelof Calpain and troponin were negatively correlated.All the above results suggested that under the present experimentalconditions, we use C16:0to successfully simulate the phenomena of troponindegradation by Calpain found in vivo. Therefore, we choose the most relevant"200μmol/L C16:0treatment for24h in H9C2cells" as a follow-upexperiment cell model.6The inhibition effect of DHA on troponin degradation by calpain wasverified in vitroWestern results showed that12.5,50,100μmol/L DHA treatment couldreversed the increasing of Calpain1,2and4protein level, and decreasing ofTNNC1TNNT2and TNNI3protein level induced by200μmol/L C16:0treatment for24h in H9C2cells. Real time PCR results also show that12.5,25,50,100μmol/L DHA treatment could reversed the increasing of Calpain1,2and4mRNA expression induced by200μmol/L C16:0treatment for24h inH9C2cells (P<0.01).7The calpain inhibition effect of DHA was proven by reversing ofcalpain-induced apoptosis7.1DHA reversed the C16:0induced apoptosis in H9C2cellAfter100,150,200and250μmol/L C16:0treatment for24h, the earlyand late apoptosis rate was significantly increased in H9C2cell (P<0.01).FCM and MTT results showed that12.5,25,50,100μmol/L DHA treatmentcould reversed the increased apoptosis and decreased survival rate by200μmol/L C16:0treatment for24h in H9C2cells (P<0.01).Hochest33258staining results also showed that50μmol/L DHAtreatment could significantly deceased the apoptosis morphology (multiple nuclear fragments) by200μmol/L C16:0treatment for24h in H9C2cells.7.2C16:0induced apoptosis was reversed by DHA through inhibition of“Calpain-Caspase12-Caspase3” pathwayWestern results showed that Caspase12and activated Caspase3(but nottotal Caspase3) was significantly increased by100,150,200and250μmol/LC16:0treatment for24h in H9C2cells. And12.5,50,100μmol/L DHAtreatment could reversed the increased Caspase12and activated Caspase3protein level by200μmol/L C16:0treatment for24h in H9C2cells. Real timePCR results show that the mRNA level of Caspase12but not Caspase3wasincreased by200μmol/L C16:0treatment for24h in H9C2cells. Real timePCR results also show that12.5,25,50,100μmol/L DHA treatment couldreversed the increasing of Caspase12mRNA expression induced by200μmol/L C16:0treatment for24h in H9C2cells (P<0.01).From the results above, the calpain inhibition effect of DHA was provedagain by its inhibition of “Calpain-Caspase12-Caspase3” pathway indirectly.Conclusions:1The increased peroxisomal beta-oxidation activity resulting in significantlydecreased n-3PUFAs (especially DHA and EPA) and doubled n-6/n-3ratioin T2DM heart.2The cardiac function was decreased, accompanied with plenty fracturedand dissolved myofibrils in T2DM rat heart. Dietary supplementation withDHA reversed the decline of DHA in T2DM heart. And DHA alsoimproved the cardiac function, protected myofibrils structure and improvedgenomic mRNA expression of the T2DM heart.3The increased expression and activity of μ-Calpain and m-Calpain resultingin increased degradation of troponin complex proteins, therefore causedfractured and dissolved myofibrils and disarranged sarcomere in T2DMheart.4DHA can effectively inhibit the transcriptional increasing of μ-Calpain andm-Calpain, therefore inhibit the cascade reaction of degradation of troponincomplex proteins, fractured and dissolved myofibrils and disarranged sarcomere in T2DM heart. |
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