Protective Effect And Mechanism Of DHA Against H9C2 Cardiomyocyte Injury Caused By Lipid Overload

Objective: In recent years,the number of patients with type 2 diabetes mellitus(T2DM)has been increasing.Diabetes-related cardiac complications is the main cause of death in T2 DM patients.Clinical and animal experiments found that supplementation of n-3 polyunsaturated fatty acids(n-3 PUFAs)can counteract cardiac hypertrophy and improve cardiac function.In our preliminarily experiment we found that the levels of n-3 PUFAs docosahexaenoic acid(DHA)in the myocardium of T2 DM rats induced by low-dose STZ(streptozotocin)in the high-fat diet were significantly decreased.And accompanied with left ventricular hypertrophy,left ventricular systolic function decreased significantly.But supply with DHA all the levels were improved.Apoptosis is an orderly death controlled by genes which refers to maintain homeostasis environment.It involves a series of gene expression and regulation.Our previous studies shown that apoptosis increased in heart of T2 DM rats has improved by DHA supplemention.However,the protective effects and mechanism of DHA and other n-3 PUFAs aganist cardiomyocyte apoptosis needs to be proved at the cellular level.Cytoskeleton refers to the structure of protein fibers in eukaryotic cells.The cytoskeleton plays an important role not only in maintaining cell morphology,subjecting to external forces,maintaining orderly internal structure,but also in many important life activities.In cardiomyocytes,the cytoskeleton and its binding proteins form a dynamic system.Reported in the literature,cytoskeleton remodeling is seen in the animal and cell models of cardiac hypertrophy and heart failure.Our previous experiments shown that myofibril fiber texture in heart of T2 DM rats blurred,irregular arrangement and dissolved,and can be improved by DHA supplemention.The effect DHA and other n-3PUFAs on the cardiomyocyte skeleton also need to be confirmed at the cellular level.Based on above purpose,we treated rat cardiomyocyte H9C2 with high concentrations of palmitic acid(C16:0)to mimic the high lipid environment,resulting in cell damage,and then investigate the protective effects and mechanism of n-3 PUFA DHA aganst the palmitic acid-induced injure.Methods:1 MTS assay to detect the cell viability,screening the appropriate C16: 0 and DHA concentration.2 Western Blot was used to detect the expression of Cleaved Caspase3,Cleaved PARP,PARP and FADD protein.3 Real time RT-PCR was used to detect the expression of Caspase3,9,12,TNF-α,HO-1 mRNA.4 TUNEL staining was used to detect different concentrations of C16: 0,n-3 PUFAs and AA treatment of apoptosis(green).5 The effect of C16: 0 on the cytoskeleton of H9C2 cardiomyocytes was observed by the method of phalloidin staining.The effect of C16:0,n-3PUFAs and AA treatment group on cardiomyocyte skeleton(red)were observed.6 Western Blot was used to detect the expression of TNNT2,Calpain1,Calpain2 and CalpainS1 protein,and the changes of TNNT2,Calpain1,Calpain2 and CalpainS1 protein after DHA supplementation.7 Real time-RT PCR was used to detect the expression of Calpain1 mRNA and the analysis of GraphPad Prism5.8 SPSS17.0 statistical software for processing analysis.The data were expressed as x ± SD,and compare with paired t-test.The difference was statistically significant with P <0.05.Results:1 Lipid overload caused H9C2 viability decrease and DHA can improve the decreased cell viabilityAfter treated with C16:0 from 50 to 400 μmol / L for 24 hours the viability of H9C2 cells was significantly lower than that of BSA control group(P <0.05),and the higher the concentration of C16:0 was,the lower the viability of H9C2 cardiomyocytes was.DHA at 10,100 μmol/L can revere the decreased viability.2 C16:0-induced cardiomyocyte viability decrease was associated with the oxidative stress increase and inflammatory responseThe higher the concentration of C16:0 was,the higher the mRNA expression of HO-1 and TNF-α was.3 Lipid overload-caused H9C2 viability decrease is related to endoplasmic reticulum apoptotic pathwayAfter treated with C16:0 for 24 h,as the concentration of C16:0 gradually increased,the number of TUNEL positive cells(green),the expression of Caspase3 mRNA,the protein expression of Cleaved Caspase3,PARP and Cleaved PARP also increased.But the protein level of FADD was not found.These results indicated that C16:0 induced-cardiomyocyte viability decrease is related to apoptosis rather than necrosis.After treated with C16:0 for 24 h,as the concentration of C16:0 gradually increased,the mRNA expression of Caspase12 but not Caspase9 also increased.So the apoptosis caused by lipid overload is related to endoplasmic reticulum apoptotic pathway.4 DHA and other n-3 PUFAs can improve the lipid overload-apoptosis injuryThe C16:0-induced increase in number of TUNEL positive cells(green),mRNA expression of Caspase3,protein expression of Cleaved Caspase3,PARP and Cleaved PARP was reversed by n-3 PUFAs DHA,EPA and LNA,but not n-6 PUFAs AA.5 Lipid overload-caused viability decrease of H9C2 is related to the change of cytoskeletonAfter treated with different concentrations of C16:0 for 24 h,as the concentration of C16:0 gradually increased,the number of irregular and pleomorphism cells(red)and the protein expression of Calpain1,2,S1 also increased.But the protein expression of TNNT2 decreased.These results indicated that decrease in cell viability caused by lipid overload is associated with the increasing expression of Calpain 1,2,S1,which causes the degradation of TNNT2,leading to changes in cytoskeleton morphology.6 DHA and other n-3 PUFAs can improve the cytoskeleton morphology changes caused by lipid overloadC16:0-induced increase in number of irregular and pleomorphism cells(red),the protein expression of Calpain 1,2,S1 and the mRNA expression of Calpain1 and C16:0-induced decrease in protein expression of TNNT2 were reversed by n-3 PUFAs DHA,EPA and LNA but n-6 PUFAs AA.The results showed that n-3 PUFAs DHA,EPA and LNA could inhibit the change of cytoskeleton and improve the cell viability caused by lipid overload.Conclusion:1 DHA can improve the lipid overload-induced loose of H9C2 cardiomyocyte viability.2 Possible protective Mechanisms of DHA against viability loose:(1)To inhibit the apoptosis though endoplasmic reticulum apoptotic pathway;(2)To improve the cytoskeleton damage though downregulating the expression of Calpain to reduce the degradation of TNNT2.