| Background：Diabetes mellitus (DM) and its related severe chronic complications has become amajor public health problem worldwide. Cardiovascular diseases have been recognized asthe leading cause of morbidity and mortality in the diabetic population and more than70%of deaths in patients with diabetes are caused by cardiovascular diseases. Hallmarks ofdiabetic cardiovascular complications include cardiac diastolic with or without systolicdysfunction, myocardial hypertrophy, fibrosis and microangiopathy. Myocardial injury isone of the key components of cardiovascular complications of DM, which is closelyassociated with cardiac structural anomaly and dysfunction, ultimately leading to heartfailure. Moreover, diabetic individuals frequently present increased vulnerability tomyocardial ischemia/reperfusion injury and the prognosis is much worse than non-diabeticpatients. Therefore, it is of significance to investigate the underlying mechanisms ofglucose cardiotoxicity in order to reduce cardiovascular morbidity and mortality indiabetes.Among the various signaling mechanisms identified to link the adverse effects ofhyperglycemia with diabetic complications, the chronic activation of PKC, that functions in a wide variety of cellular pathophysiology, has received increasing attention as a coreeffecter that direct the toxicity of hyperglycemia to cell apoptosis, hypertrophy, fibrosis,and oxidative stress. However, molecular mechanisms controlling myocardial PKCactivity in the context of hyperglycemia is not completely understood. A-kinase anchoringproteins (AKAPs) belong to a family of proteins that share the ability to bind theregulatory subunit of protein kinase A (PKA). AKAPs bind multiple signaling proteins andhave subcellular targeting domains that allow them to greatly impact cellular signaling.AKAPs localize, specify, amplify, and accelerate signal transduction within the cell bybringing signaling proteins together in space and time. AKAP5gene products (AKAP79inhuman and AKAP150in rodent) bind and integrate cAMP, calcium and phospholipidsignaling through PKA, PKC and the Ca2+/calmodulin-dependent protein phosphatasecalcineurin. In arterial smooth myocytes，PKC is targeted to the plasma membrane throughthe direct binding of AKAP79/150and potentiates channel opening by directphosphorylation of L-type Ca2+channel, which regulates vascular tone. However, there isno evidence addressing the anchoring of PKC by AKAP150in cardiomyocytes, and apossible role of AKAP150in modulating myocardial PKC activation in diabetes.The present study was designed to assess the role of AKAP150in cardiacglucotoxicity and to investigate the underlying regulatory mechanism underlying theactivation of PKC signaling.Aims1. To identify myocardial injury induced by diabetes and the effect of hyperglycemiaon the AKAP150expression and PKC activation;2. To determine the role of AKAP150in the diabetic myocardial injury in vivo and invitro;3. To determine the underlying regulatory mechanism of AKAP150in the diabeticmyocardial injury in vitro. Specifically, AKAP150induces cardiomyocytes oxidativestress and apoptosis through PKC/p47phox/ROS pathway, and promotes intracellularcalcium concentration via PKC/PLB/SERCA pathway, with preferentialco-localization withPKC and β.Methods1. Male Sprague–Dawley (SD) rats weighing between220-250g were used to establish diabetic animal model via intraperitoneal injection of streptozotocin (STZ,35mg/kg/day in0.9%saline, Sigma, USA) for3days. Animals were considered diabeticwithblood glucose levels≥16.7mmol/Lat1week after STZ injection.2. Neonatal rat cardiomyocytes (NRCM) were isolated and cultured in mediumsupplemented with25mmol/L glucose.3. AKAP150knockdown was established via intramyocardial injection in vivo andtransfection in vitro of adenovirus carrying AKAP150targeted shRNA.4. Cardiac function was examined by animal echocardiography.5. TUNEL staining, Caspase3activity assay and annexin V/PI flow cytometr y wereperformed to evaluate cell apoptosis in vivo and in vitro.6. Superoxide assay, dihydroethidine (DHE) staining and lipid peroxidation assay wereused to assess oxidative stress.7. NADPH activity assay was used to assess NADPH oxidase activity.8. Fluo-3AM assay kit was used to determine intracellular calcium concentration.9. Non-radioactive PKC assay was used to measure PKC activity.10. SERCA2ELISA assay kit was used to assess SERCA2activity.11. The mRNA expression of AKAP150, PKC, p47phox, NF-κB mRNA were examined byRT-PCR analysis.12. The protein expression or activity of AKAP150, PKC, p47phox, NF-κB, PLB wereexamined by Western blot analysis.13. Immunofluorescent co-expression and immunoprecipitation were used to identify thebinding and co-localizaiton of AKAP150and PKC.Results1. Compared with normal hearts, diabetic rats exhibited significantly compromised leftventricular diastolic dysfunction and increased myocardial apoptosis. Compared withcontrol NRCMs, high glucose induced remarkable increase in ROS production,cardiomoyocytes apoptosis and intracellular calcium concentration. The expression ofAKAP150, PKC and PKC phosphorylation significantly increased in diabeticmyocardium from both diabetic animals and high glucose cultured NRCMs.Expression and phosphorylation level of p47phoxand p65NF-κB, two knownphosphorylation targets of PKC and key regulatory subunits of NF-κB and Nox respectively, also increased significantly.2. AKAP150knockdown alleviated diabetes-induced left ventricular diastolicdysfunction and myocardial apoptosis.3. AKAP150knockdown reduced high glucose-induced oxidative stress and apoptosis,inhibits NADPH oxidase activity and decreased intracellular calcium concentration inNRCMs.4. AKAP150knockdown reduced high glucose-induced activition of PKC, as well asphosphorylationof p47phoxand NF-κB.5. AKAP150knockdown increased phosphorylation of PLB and the activity of SERCA2.6. Immunofluorescent co-expression and immunoprecipitation results demonstrated thatAKAP150co-localized with PKC in high glucose treated cells, and AKAP150inhibition reversed the increased membrane expression of PKC induced by highglucose.7. Immunofluorescent co-expression showed high levels of PKC and β co-expressingwith AKAP150in NRCMs with high glucose treatment, in contrast to minimal PKCγco-localization with AKAP150.8. PKC and β specific inhibitor safingol and LY333531significantly reduced highglucose-induced increases in ROS production, apoptosis, and intracellular calcium inNRCMs.Conclusions:1. Diabetes induced left ventricular diastolic dysfunction, increased oxidative stress,myocardial apoptosis and intracellular calcium concentration. Cardiac AKAP150expression and PKC activity were upregulated in response to hyperglycemic stimuli.2. AKAP150knockdown both in vivo and in vitro significantly reduced highglucose-induced cardomyocytes injury, and suppressed the activation of PKCsignaling pathway.3. AKAP150induced cardiomyocytes oxidative stress and apoptosis throughPKC/p47phox/ROS pathway, and promoted intracellular calcium concentration viaPKC/PLB/SERCA2pathway, with preferential co-localizationwithPKC and β. |
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