The Role Of Mitochondrial Oxidative Stress And Homeostasis In Diabetes-induced Atrial Remodeling And The Effects Of Pioglitazone Use

Objective:The mechanisms underlying in diabetes-related atrial fibrillation（AF）development have not been fully elucidated.Our previous studies suggested that oxidative stress and inflammation play important roles in diabetic atrial remodeling,and PPAR-γagonist pioglitazone attenuates diabetes-induced atrial structural and electrical remodeling via antioxidant and anti-inflammatory actions.Indeed,mitochondria are the most important cellular source of reactive oxygen species（ROS）in most of the mammals.Thus,in this study,we aimed to further evaluate the roles of mitochondrial oxidative stress and homeostasis in the atrial remodeling in diabetes and the potential effects of pioglitazone use.Methods:Healthy adult Japanese white rabbits were randomly divided into control（C）,diabetic（DM）,and pioglitazone-treated DM（Pio,4 mg/kg/day）groups.Type 1diabetic rabbit model was induced by intravenous injection of alloxan,rabbits were sacrificed for relevant experiments after 8 weeks of modeling.Cardiac structure and function were detected by echocardiography.The cross-sectional area of left atrial myocytes was determined by HE staining,the degree of left atrial fibrosis was determined by Masson staining,and the distribution and expression of left atrial sympathetic nerve remodeling related proteins were analyzed by immunohistochemistry and Western blot.The electrocardiogram and hemodynamic parameters of the rabbits were measured.The cardiac conduction function was evaluated by electrophysiological studies of isolated hearts by Langendofff perfusion system and the inducibility of AF was detected.The intracellular calcium transient（CaT）of the isolated rabbit atrial myocytes was detected by confocal microscopy under 1 Hz field stimulation.Mitochondrial morphology was observed by electron microscopy,and mitochondrial ROS production rate,respiratory control rate（RCR）and membrane potential（MMP）levels were measured.Serum PPAR-γand PPAR-α,and oxidative stress and inflammation-related markers levels were measured by ELISA.Mitochondrial biogenesis-related proteins such as PGC-1α,mitochondrial fusion and fission-related proteins were detected by Western blot,as well as the oxidative stress,inflammation and fibrosis related proteins.In the cell experiments,the mouse atrial cardiomyocyte cell line HL-1 cells were transfected with PGC-1αsmall interfering RNA（siRNA）,and hydrogen peroxide（H₂O₂）was administrated to form an oxidative stress state for analyzing whether pioglitazone specifically regulated mitochondrial oxidative stress and homeostasis via the PPAR-γ/PGC-1αsignaling pathway.Results:（1）Compared with the C group,the expression of TGF-β1 and sympathetic remodeling-related proteins TH and NGF were significantly increased in the left atrium of the DM group.Also,the left atrial diameter,cardiomyocyte cross-sectional area and interstitial fibrosis were significantly increased in the DM group compared with the C group.Interatrial conduction time and atrial refractory effective period dispersion were also significantly increased in the DM group than the C group,along with a higher incidence of inducible AF.However,pioglitazone prevented these abnormalities.There were no significant differences in hemodynamics and surface ECG parameters among the 3 groups;（2）Pioglitazone increased serum SOD activity,decreased mitochondrial ROS production,inhibited the levels of oxidative stress and inflammation markers in serum（8-OHdG and hs-CRP）and left atrium（NF-κB）of diabetic rabbits;（3）Pioglitazone suppressed atrial mitochondrial structural damage induced by diabetes,and increases mitochondrial RCR and MMP levels in diabetic rabbits;（4）Serum PPAR-γlevels in the DM group were significantly lower than that in the C group,while Pio group showed a significantly higher PPAR-γlevels than DM group;Western blot results showed that expression levels of PGC-1αand other mitochondrial biogenesis（NRF1 and TFAM）,fusion（Opa1 and Mfn1）and fission（Drp1）related proteins were significantly decreased in the left atrium in the DM group.However,these changes were reversed by pioglitazone treatment;（5）The mean CaT amplitude in the left atrial myocytes of the DM group was significantly increased under 1 Hz field stimulation,and pioglitazone significantly inhibited this change;（6）In HL-1 cell line,transfected with PGC-1αsiRNA blunted the effect of pioglitazone on improving mitochondrial oxidative stress and MMP collapse in H₂O₂-treated cells.Conclusion:Pioglitazone could reduce mitochondrial ROS production,inhibit oxidative stress and inflammation,improve mitochondrial homeostasis remodeling(structure,function,biogenesis,fusion and fission,and Ca²⁺handling),and subsequently reverse diabetes-induced atrial remodeling,reduce the risk of AF through the PPAR-γ/PGC-1αsignaling pathway.