The Role And Mechanism Research Of Mitochondrial AKAP1 In Diabetic Cardiomyopathy

Background:In 2019,the 9th edition of Diabetes Atlas released by the International Diabetes Federation shows,China has a high prevalence of diabetes in the world.The number of patients with diabetes ranks first in the world,about 116 million.Cardiovascular complications are the most common complications of diabetes.According to statistics,about 50% of patients with diabetes die from cardiovascular complications.The heart is an organ with high energy requirements,and mitochondria are the most productive organelles in cardiomyocytes.Most ATP is produced by mitochondrial oxidative phosphorylation.Mitochondria,as a major provider of ATP and a major source of ROS,are closely related to the occurrence and development of diabetic cardiomyopathy.The main features of diabetic cardiomyopathy are mitochondrial dysfunction and oxidative stress.However,the specific mechanisms of mitochondrial dysfunction and oxidative stress in diabetic cardiomyopathy have not yet been elucidated.A-kinase anchoring proteins are a family of functionally similar proteins whose main function is to recruit c AMP-dependent protein kinase A to specific subcellular structures.A-kinase anchoring protein 121(AKAP1)is located on the mitochondrial outer membrane and is an important regulator of mitochondrial respiration.Previous studies have found that AKAP1 undergoes ubiquitination degradation in ischemic myocardium,which leads to excessive increase of mitochondrial autophagy and aggravates myocardial ischemic injury.The above evidence strongly suggests that AKAP1 has myocardial protection.However,the role and molecular mechanism of AKAP1 in diabetic cardiomyopathy have not been reported.Our pre-experimental results show that the expression of AKAP1 in heart of STZ-induced diabetic mice is significantly reduced,suggesting that AKAP1 may be involved in the development of diabetic cardiomyopathy.Based on this,this study uses constructed Akap1 knockout mice and primary neonatal rat cardiomyocytes to explore the role of AKAP1 in diabetic cardiomyopathy from the overall animal and cell level and clarify its specific mechanisms to provide new ideas for the prevention and treatment of diabetic cardiomyopathy.Objectives:1.To investigate whether AKAP1 is involved in the occurrence and development of diabetic cardiomyopathy.2.To elucidate the molecular mechanism of AKAP1 influencing the occurrence and development of diabetic cardiomyopathy.Methods:Part 1: Akap1 knockout exacerbates diabetes-induced cardiac dysfunctionEight-week-old Akap1 knockout mice and their wild-type mice were injected intraperitoneally with STZ to construct a diabetic mouse model.The control group was injected with citric acid buffer.All mice of the same genotype were randomly assigned into four groups: wild type group(WT),Akap1 knockout group(KO),diabetes group(DM),and Akap1 knockout diabetes group(KO/DM).After modeling,the mice were kept for another 12 weeks,and their blood glucose and body weight were measured regularly.The expression of AKAP1 in the heart of WT group and DM group was detected at 6 and 12 weeks after modeling.Twelve weeks later,echocardiography was used to detect cardiac function in all mice;Millar catheter was used to detect hemodynamic changes in mice;Masson staining was used to analyze myocardial fibrosis.Part 2: Akap1 knockout aggravates myocardial mitochondrial dysfunction and apoptosis in diabetic miceFresh mouse hearts were collected and made into histological samples.Transmission electron microscope was used to observe and analyze morphological characteristics of myocardial mitochondria in mice;Mito SOX staining was used to analyze mitochondrial ROS level with a confocal microscope;TUNEL staining was used to analyze cardiomyocyte apoptosis.For cell experiments,primary cardiomyocytes from neonatal SD rat(1-2 days)were isolated and cultured.After Akap1 was knocked down,high glucose(33 mmol/L glucose)culture was continued for 48 hours.JC-1 staining was used to analyze mitochondrial membrane potential;Seahorse experiment was used to analyze mitochondrial respiratory function related indicators;Annexin V-FITC/PI staining was used to analyze primary cardiomyocyte apoptosis.Part 3: Akap1 knockout inhibits mitochondrial localization of NADH-ubiquinone oxidoreductase 75 k Da subunit(NDUFS1)and subsequently inhibits mitochondrial complex Ⅰ activityCo-immunoprecipitation method was used to capture the AKAP1 interacting protein.After silver staining,differential bands were cut and analyzed by mass spectrometry.Immunofluorescence was used to co-localize of AKAP1 and its interacting protein.Mito Tracker staining was used to localize mitochondria.Small interfering RNA was designed and synthesized to knock down Akap1 and its interacting protein,respectively.Western blotting was used to detect protein expression.Mitochondrial complex Ⅰ activity detection kit was used to analyze complex Ⅰ activity.Part 4: Upregulation of AKAP1 promotes mitochondrial localization of NDUFS1 and alleviates diabetic myocardial injuryMouse hearts were injected with Adeno-associated virus carrying Akap1(AAV9-Akap1)and its control virus(AAV9-GFP).Mouse models of diabetes were constructed and divided into four groups: diabetic group(DM/AAV9-GFP),Akap1 knockout diabetic group(KO/DM/AAV9-GFP),diabetic overexpression group(DM/AAV9-Akap1),Akap1 knockout diabetic overexpression group(KO/DM/AAV9-Akap1).After feeded for 12 weeks,the cardiac function of the mice was detected by ultrasound;the hemodynamic changes of the mice were detected by Millar catheter;the morphological characteristics of the myocardial mitochondria were observed by transmission electron microscopy;the mitochondrial respiratory function related indicators were analyzed by Seahorse experiment;and TUNEL staining was used to analyze cell apoptosis.In addition,the molecular mechanism was further verified in primary cardiomyocyte.Results:Part 1: Akap1 knockout exacerbates diabetes-induced cardiac dysfunctionWestern blotting showed that compared with WT,the expression level of AKAP1 in the heart of DM group mice decreased significantly after 6 weeks of modeling,and the decline was more significant after 12 weeks of modeling.q PCR and immunohistochemistry also confirmed the same results.Compared with WT,ultrasound results showed that the left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS)of WT mice were significantly decreased,indicating cardiac systolic dysfunction.Compared with DM,the LVEF and LVFS of KO/DM were further decreased.The left ventricle diameter of KO/DM increased significantly.The hemodynamic analysis showed similar results.In addition,compared with the WT group,the myocardial fibrosis in DM was obvious,and the Akap1 knockout further aggravated the level of fibrosis.Part 2: Akap1 knockout aggravates myocardial mitochondrial dysfunction and apoptosis in diabetic miceTransmission electron microscopy showed that compared with WT,the myocardial mitochondrial electron density of diabetic mice was decreased,mitochondrial cristaes are disordered,cristae number is reduced,and Akap1 knockout further aggravated mitochondrial morphological abnormalities.Oxygen consumption results showed that the mitochondrial respiratory function of primary cardiomyocytes cultured in high glucose was decreased,while the knockdown of Akap1 further dereased the mitochondrial respiratory function.JC-1 staining showed that mitochondrial membrane potential of primary cardiomyocytes cultured in high glucose decreased,while the knockdown of Akap1 further decreased mitochondrial membrane potential.In addition,Mito SOX staining of myocardial tissue sections showed that Akap1 knockout increased mitochondrial ROS production in diabetic mice;TUNEL staining of tissue sections and Annexin V-FITC/PI staining of primary cardiomyocytes suggested that Akap1 knockout aggravated cardiomyocyte apoptosis.Part 3: Akap1 knockout inhibits mitochondrial localization of NDUFS1 and subsequently inhibits mitochondrial complex Ⅰ activityThe results of co-immunoprecipitation and silver staining showed that the molecular weight of AKAP1 interacting protein was in the range of 70-100 k Da.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis revealed that NDUFS1 is an important interacting protein of AKAP1.Akap1 knockdown increased NDUFS1 retention in the cytoplasm and reduced expression in mitochondria.In addition,in diabetic mice,knockout of Akap1 significantly reduced mitochondrial complex Ⅰ activity.Part 4: Upregulation of AKAP1 promotes mitochondrial localization of NDUFS1 and alleviates diabetic myocardial injuryCompared with the GFP group,AKAP1 overexpression significantly improved mouse cardiac function.Masson staining showed that AKAP1 overexpression significantly reduced myocardial fibrosis in diabetic mice.In addition,upregulating of AKAP1 in primary cardiomyocytes cultured in high glucose significantly restored mitochondrial morphology,enhanced mitochondrial respiratory function,improved mitochondrial membrane potential,reduced mitochondrial oxidative damage and cardiomyocyte apoptosis caused by high glucose.Further experiments found that AKAP1’s cardiomyocyte protection can be blocked by Ndufs1 knockdown.Western blotting showed that AKAP1 myocardial overexpression promoted the translocation of NDUFS1 from the cytoplasm to mitochondria,and thus restored mitochondrial complex Ⅰ activity to a certain extent.Conclusions:1.The expression of AKAP1 is decreased in the heart of STZ-induced diabetic mice.Under normal conditions,Akap1 gene knockout has no significant effect on cardiac function in mice,but in the case of diabetes,Akap1 knockout aggravates cardiac dysfunction.2.Akap1 knockout exacerbates diabetic cardiomyopathy by aggravating cardiomyocyte mitochondrial respiratory dysfunction and increasing ROS-mediated apoptosis in diabetic mice.3.Cardiac overexpression of AKAP1 can promote the entry of NDUFS1 into mitochondria,improve mitochondrial complex Ⅰ activity,enhance mitochondrial respiratory function,reduce oxidative damage,reduce apoptosis,and thus alleviate cardiac dysfunction in diabetic mice.