Effects And Mechanisms Of IP3R1/GRP75/VDAC1 Complex In Diabetic Atrial Remodeling

Objective:There are several risk factors for atrial fibrillation（AF）,including coronary heart disease,hypertension and diabetes.In the setting of diabetes,atrial electrical remodeling and structural remodeling caused by mitochondrial oxidative stress play a key role in the occurrence and maintenance of atrial fibrillation.Endoplasmic reticulum stress is currently considered to be the key link from homeostasis to dysfunction,and is a common cellular and molecular mechanism of metabolic diseases such as insulin resistance,obesity and diabetes.This study mainly explores the role of endoplasmic reticulum stress and mitochondrial oxidative stress in diabetic atrial remodeling and the research the role of IP3R1/GRP75/VDAC1complex.Methods:We extract and culture primary neonatal rat atrial myocytes and divide them into 4 groups:normal control group（NG）,high-glucose group（HG）,high-glucose+sodium phenylbutyrate（4-PBA）group and 4-PBA group.In the high-glucose treatment group,primary atrial myocytes were treated with 25m M glucose for 48 hours.In addition,HL-1 cells were cultured with Claycomb medium and divided into the following 4 groups:Negative Control（NC）si RNA group,GRP75 si RNA group,NC si RNA+tunicamycin（TM）group,GRP75 si RNA+TM group.The TM group was treated with 200ng/ml TM for 24 hours.After the above groups of cells are processed,the intracellular reactive oxygen species（ROS）level,mitochondrial membrane potential（MMP）,intracellular and mitochondrial calcium levels are detected with a confocal microscope;the endoplasmic reticulum and mitochondrial morphology are observed by transmission electron microscope;seahorse analysis mitochondrial respiratory control rate;flow cytometry was used to detect cell apoptosis.Detect oxidative stress proteins（Mn-SOD）,endoplasmic reticulum stress proteins（GRP78,CHOP）,mitochondrial-associated endoplasmic reticulum membrane proteins（IP3R1,GRP75,VDAC1）and apoptosis-related proteins（BAX,Bcl-2,Cleaved-caspase3/Caspase3,Cleaved-caspase9/Caspase9）expression by western blotting.CRISPR-Pro conditional Hspa9（GRP75）knockout mice required for animal experiments were constructed by Cyagen Biosciences Inc（Suzhou）.8 week old male mice were selected and divided into 4 groups:Hspa9^flox/floxgroup;Hspa9^flox/flox+TM group;Hspa9^flox/flox/Myh6-Cre⁺group;Hspa9^flox/flox/Myh6-Cre⁺+TM group（TM intraperitoneal injection,once a week for two consecutive weeks）.All animals were subjected to follow-up experiments after two weeks.We evaluate the structure and function of the heart by echocardiography;detect the surface electrocardiogram and hemodynamic changes in each group of mice;detect the cardiac conduction function and the atrial fibrillation induction by endocardial electrophysiological experiments and epicardial electrical conduction mapping to evaluate the atrial electrical remodeling.We observe the cell morphology by HE staining,Masson staining to measure atrial fibrosis to evaluate the atrial structural remodeling in mice.Results:（1）High glucose upregulates the expression of endoplasmic reticulum stress proteins GRP78 and CHOP.At the same time,under high glucose stimulation,intracellular ROS and MMP decreased.The expression of mitochondrial-associated endoplasmic reticulum membrane proteins IP3R1,GRP75,VDAC1 increased,and cell apoptosis increased.Endoplasmic reticulum stress inhibitors can improve these changes.（2）Using the endoplasmic reticulum stress agonist TM significantly increased the expression of GRP78 protein in HL-1 cells.The NC si RNA+TM group showed swelling mitochondria that were accompanied by dilatation and vesiculation of the endoplasmic reticulum.And TM will shorten the distance between the endoplasmic reticulum and the outer mitochondrial membrane.Intracellular and mitochondrial calcium levels were increased significantly,intracellular ROS levels increased,MMP and mitochondrial function were decreased,and apoptosis increased in NC si RNA+TM group.Using GRP75 si RNA to transfect HL-1 cells,the knockdown efficiency up to 60%.In the GRP75 si RNA-treated group,the distance between the endoplasmic reticulum and the outer mitochondrial membrane increased.In the GRP75 si RNA-treated TM group,mitochondrial calcium levels are significantly reduced,intracellular ROS levels are reduced,MMP and mitochondrial function is improved,and apoptosis is reduced.It is suggested that following knockdown GRP75,the calcium transport from the endoplasmic reticulum to the mitochondria is reduced,which reduces the mitochondria calcium overload and cell apoptosis.（3）The GRP75 protein expression in the atrium of Myh6-Cre⁺/Hspa9^flox/floxmice is about 80%lower than that of Hspa9^flox/flox mice.There were no significant differences in left ventricular ejection fraction（LVEF）and E/A values,which are indicators of diastolic function,as well as ECG and hemodynamic indicators among the four groups.The left atrium electrical conduction of the Hspa9^flox/flox group was uniform and showed orderly spread to the surrounding tissue.However,in the Hspa9^flox/flox+TM group,left atrium conduction was disordered,and there were abnormal conduction positions in the wave conduction.The mapping images showed that left atrium conduction velocity was significantly lower in the Hspa9^flox/flox+TM group than in the Hspa9^flox/flox group.The electrophysiological parameters obtained from intracardiac programmed electrical stimulation showed no significant differences for the sinus cycle length（SCL）,atrial-ventricular Wenckebach cycle length conduction（AVWCL）,and sinus node recovery time（SNRT）between the four groups.We observed significantly higher AF incidence in the Hspa9^flox/flox+TM group than in the Hspa9^flox/flox group and AF was not induced in the Myh6-Cre⁺/Hspa9^flox/flox+TM group.Left atrial morphological changes were detected using HE and Masson’s trichrome staining.Compared with the control group,increased inflammatory cell infiltration and extensive interstitial fibrosis of atrial cardiomyocytes were observed in the Hspa9^flox/flox+TM group,and GRP75knockdown attenuated these changes.Conclusion:IP3R1/GRP75/VDAC1 complex plays an important role in high glucose-induced endoplasmic reticulum stress and mitochondrial oxidative stress in atrial myocytes,while GRP75 knockdown can improve mitochondrial dysfunction mediated by mitochondrial calcium overload and reduce endoplasmic reticulum stress-mediated death of cardiomyocytes,thereby ameliorateing atrial remodeling.