Effects Of Drp1-regulated Fatty Acid Oxidation On Oxidative Stress And Apoptosis In Diabetic Cardiomyocytes

BackgroundDiabetic cardiomyopathy（DCM）is a diabetes-induced cardiac dysfunctional disease characterized by early apoptosis,fibrosis,and myocardial remodeling,followed by diastolic and systolic dysfunction,and eventual progression to heartfailure.The pathogenesis of DCM has not been fully elucidated and may be related to mitochondrial dysfunction,oxidative stress,inflammation,lipotoxicity,calciumhomeostasis imbalance,insulin resistance,and endoplasmic reticulum stress.Indiabetic cardiomyocytes,elevated expression of dynamin-related protein 1（Drp1）and increased mitochondrial fission lead to structural disruption and dysfunction ofmitochondria,which in turn leads to impaired oxidative phosphorylation,increased production of reactive oxygen species（ROS）,and impaired energy metabolism.Furthermore,in diabetic cardiomyocytes,fatty acid（FA）uptake is increased due to insulin resistance and impaired glucose utilization.However,when FA uptake by cardiomyocytes exceeds mitochondrial fatty acid oxidative（FAO）,lipotoxicsubstances accumulate in cardiomyocytes,which in turn induce oxidative stress and increased apoptosis.It has not been reported whether the regulation of mitochondrial division by Drp1 can improve FAO in diabetic cardiomyocytes,thereby reducingoxidative stress,inhibiting apoptosis,and ultimately improving diabetic myocardial function.ObjectiveTo investigate the effect and mechanism of Drp1 on oxidative stress andapoptosis in H9c2 cardiomyocytes cultured with high glucose.The intention is to provide some theoretical basis for exploring protective strategies for diabeticcardiomyocytes.Methods1.Rat embryonic H9c2 cardiomyocytes were divided into normal glucose（NG）group（5.5 mmol/L glucose+19.5 mmol/L mannitol）and high glucose（HG）group（33 mmol/L glucose）and cultured continuously.H9c2 cardiomyocytes from the HG group were selected for group intervention,which were divided into HG+Dr group（33 mmol/L glucose+Drp1 si RNA）,HG+NC group（33 mmol/L glucose+Control si RNA）,HG+Dr+Et group（33 mmol/L glucose+Drp1 si RNA+26.3?mol/LEtomoxir）,and HG+NC+Et group（33 mmol/L glucose+Control si RNA+26.3?mol/L Etomoxir）.The groups were intervened separately for 48 hours under different conditions.Etomoxir,a specific inhibitor of CPT1,also inhibits fatty acid oxidation by inhibiting CPT1 activity.2.The IC₅₀ of Etomoxir in high glucose cultured H9c2 cardiomyocytes was detected by the CCK-8 assay to determine the optimal intervention concentration of Etomoxir.Drp1 si RNA was transfected with Lipofectamine RNAi MAX reagent and the effect of transfection was verified by Western blot.3.Mitochondrial proteins were extracted from each group of H9c2cardiomyocytes using the Mitochondrial Extraction Kit,and the expression of mitochondrial Drp1 and CPT1B proteins was detected by Western blot.The mitochondrial morphology of each group of H9c2 cardiomyocytes was observed by transmission electron microscopy.The cardiomyocyte viability was measured by CCK-8 in each group.Oil Red O staining was used to detect intracellular lipids in each group of cardiomyocytes.The DCFH-DA probe was used to detect the intracellular ROS content in each group of cardiomyocytes.Cardiomyocyte apoptosis was detected by TUNEL in each group.The NADPH/NADP⁺values,reduced glutathione（GSH）,and free fatty acid（FFA）content in cardiomyocytes of each group were measured by absorbance.Firefly luciferase was used to detect the intracellular ATP content of each group of cardiomyocytes.The expression levels of Drp1,CPT1B,Bcl-2,c-Caspase-3,and c-Caspase-9 proteins were measured by Western blot in each group of cardiomyocytes.Results1.The IC₅₀ of Etomoxir in H9c2 cardiomyocytes cultured with high glucose was determined to be 26.23?mol/L,and the optimal intervention concentration of Etomoxir was 26.3?mol/L.Validation analysis demonstrated that Drp1 si RNA transfection of high glucose cultured H9c2 cardiomyocytes was successful and could be used for subsequent experiments.Drp1 and CPT1B protein expression levels in mitochondria of H9c2 cardiomyocytes followed the same trend as total intracellular Drp1 and CPT1B protein expression levels.Drp1 protein expression was elevated,the mitochondrial division increased and mitochondrial structure was destroyed,and CPT1B protein expression level decreased.When Drp1 protein expression was inhibited,the mitochondrial division was reduced,the mitochondrial structure was intact,and the CPT1B protein expression level increased.2.Compared with the NG group,H9c2 cardiomyocytes in the HG group showed increased Drp1 expression（P<0.05）,decreased mitochondrial volume and increased structure disruption（P<0.0001）,decreased CPT1B expression（P<0.0001）,decreased cell viability（P<0.001）,increased intracellular lipid accumulation（P<0.0001）,increased FFA content（P<0.01）,decreased NADPH/NADP⁺values（P<0.0001）,decreased GSH content（P<0.0001）,increased ROS production（P<0.0001）,decreased ATP production（P<0.0001）,increased TUNEL positive（P<0.0001）,increased expression of proapoptotic proteins（P<0.05）and decreased expression of antiapoptotic proteins（P<0.05）.3.Compared with the HG+NG group,H9c2 cardiomyocytes in the HG+Dr group showed reduced Drp1 expression（P<0.0001）,increased mitochondrial volume and structure integrity（P<0.01）,increased CPT1B expression（P<0.01）,increased cell viability（P<0.05）,reduced intracellular lipid accumulation（P<0.001）,reduced FFA content（P<0.01）,increased NADPH/NADP⁺values（P<0.01）,increased GSH content（P<0.001）,decreased ROS production（P<0.001）,increased ATP production（P<0.001）,decreased TUNEL positive（P<0.01）,decreased expression of pro-apoptotic proteins（P<0.05）and increased expression of anti-apoptotic proteins（P<0.05）.4.After the application of Etomoxir to inhibit CPT1 activity,the effects of inhibition of Drp1 protein expression that promotes fatty acid oxidation,attenuates oxidative stress,and reduces apoptosis were reversed,and the damage to H9c2cardiomyocytes cultured in high glucose was aggravated.Conclusion1.H9c2 cardiomyocytes cultured in high glucose had elevated Drp1 protein expression,increased mitochondrial division and structure disruption,reduced fatty acid oxidation,enhanced oxidative stress,and increased apoptosis.2.Inhibition of Drp1 protein expression in H9c2 cardiomyocytes cultured in high glucose resulted in reduced mitochondrial division,increased structure integrity,increased fatty acid oxidation,reduced oxidative stress,and decreased apoptosis.3.The protective effect of inhibiting the Drp1 protein expression in diabetic cardiomyocytes may be related to the Drp1/CPT1 signaling pathway.