The Role And Mechanism Of Orai1 Channel In Diabetic Cardiomyocyte Hypertrophy

Diabetic cardiomyopathy is defined as a disease of clinical ventricular dysfunction that occurs in diabetic patients without coronary atherosclerosis and hypertension.In the early stage,diabetic cardiomyopathy is characterized by structural and functional abnormalities,including left ventricular hypertrophy,fibrosis,and abnormal cellular signals.These pathophysiological changes initially caused heart failure associated with subclinical diastolic dysfunction with normal ejection fraction,and eventually turned into heart failure with reduced ejection fraction,thereby increasing patient mortality.The leading causes of DCM include hyperglycemia,insulin resistance,elevated levels of free fatty acids,tissue inflammatory cell infiltration,increased oxidative stress,activation of the renin-angiotensin-aldosterone system,and active sympathetic nervous system.In particular,dysregulation of calcium homeostasis,which decreased calcium pump activity and sarcoplasmic reticulum Ca2+leakage,are considered to be important factors in the development of diastolic dysfunction.Ca2+are important second messengers.Intracellular Ca2+levels regulate cell metabolism,muscle contraction,and cell signaling.Usually,in the heart"excitation-contraction coupling",Ca2+enter the cytoplasm through a voltage-sensitive L-type calcium channel after depolarization of the muscle fiber membrane,triggers the release of Ca2+from the sarcoplasmic reticulum,bind to Ca2+-binding troponin C,and finally leads to the myofibrillar contraction.During diastole,most of Ca2+ is transported back to the sarcoplasmic reticulum,and the remaining ones are pumped out by NCX and PMCA.However,in diabetic cardiomyopathy,proteins that maintain intracellular Ca2+homeostasis are likely to be damaged,thereby increasing the duration of action potentials and prolonging the diastolic phase.In the heart of type 2 diabetic mice,scholars record the increase of Ca2+in the resting state of cells,prolonged intracellular Ca2+decay,slowed calcium transients,decreased sarcoplasmic reticulum Ca2+pump,and impaired sarcoplasmic reticulum Ca2+reuptake.These studies suggest that Ca2+homeostasis in damaged cardiomyocytes plays a key role in the development of diastolic dysfunction in early diabetic cardiomyopathy.However,these results do not explain the cardiomyocyte hypertrophy in diabetic cardiomyopathy.As an important second messenger in the cell,Ca2+can form an "excitatory-transcription coupling" process in addition"excitation-contraction coupling".This part of the calcium ion is mainly composed of Orail protein,which mediates Ca2+influx and activates CaMKII/HDAC or CaN/NFAT signaling pathway.And then it activates various transcription factors,including MEF2,GATA-4,NFAT,cAMP response element binding protein and serum reaction factor and so on.Based on the research above,we proposed hypothesis:in cardiomyocytes under diabetes microenvironment changed,caused increasing expression of Orail channel,increased Ca2+influx,activated CaN/NFAT signaling pathway and promoted cardiomyocyte hypertrophy related gene expression.If Orail is down-regulated,myocardial cell hypertrophy can be alleviated,and DCM ventricular diastolic dysfunction can be reversed.1.Cardiomyocyte function changes and abnormal calcium regulation in diabetes.Objective:Establish rat models of type 2 diabetes,observe the functional changes of diabetic cardiomyocytes and abnormal calcium regulation.Methods:①Using Purina5008 feed to ZL and ZDF rats,establish a diabetic rat model,regularly monitor blood glucose and body weight,and test the heart function of the two groups of rats after B-ultrasound measurment at the 22nd week and measure the length of the tibia,take care the heart tissue.②HE staining of heart tissue to detect pathological changes,and heart tissue was subjected to WGA immunofluorescence staining to detect myocardial cell size.③The cardiomyocytes of the two groups were isolated and the ICA-L currents were detected by whole cell patch clamp.The changes of calcium transients in the cardiomyocytes of the two groups were detected by laser confocal microscopy.④Western blot was used to detect the expression of ANP and B-MHC in the two groups.The expression levels of ion channel related proteins Oral 1,CaV1.2,PLN,SERCA2a and NCX were detected.⑤Western blot was used to detect the signal channel related proteins CaN,NFATc3 and NFATc4.,CamK Ⅱ expression level.RESULTS:①The blood glucose and body weight of the ZDF group were higher than the control group.②B-ultrasound showed that the ventricular wall of ZDF rats was thickened,the left ventricular volume was decreased,the diastolic function was decreased,and the systolic function was enhanced.③The ratio of the heart/tibia increased.④Western blot showed that the expression of ANP and β-MCH in ZDF rats increased.The WGA stain showed that the surface area of myocardial cells in ZDF rats increased.HE staining showed that the myocardial fine arrangement of ZDF rats was disordered and obvious hypertrophy,nuclear deep staining;⑤whol cell patch clamp results showed that the ICa-L current of ZDF rats was weakened;⑥calcium transient results showed that the calcium transient amplitude of ZDF rats decreased significantly;calcium transient peak time and recovery Time prolonged;⑦Western blot results showed that the expression of CaN was up-regulated in ZDF rats,nuclear translocation was observed in NFATc3,CamKII was not changed,and GATA4 phosphorylation was up-regulated.CONCLUSION:In the animal model of diabetes:(1)Ventricular wall thickening,diastolic function decline,contractile force enhanced,ANP,and β-MHC expression increased.(2)The expression of Orail is increased,the current of ICa-L is weakened,and the calcium transient is abnormal,suggesting calcium imbalance in cardiomyocytes(3)Activation of the CaN/NFATc3 pathway may serve as a potential mechanism for diabetic hypertrophy2.To explore the mechanism of Orail mediating cardiomyocyte hypertrophy in high glucose.OBJECTIVE:In a condition of high glucose,Orail-mediated calcium influx in primary cardiomyocytes promotes hypertrophy of cardiomyocytes through activation of the CaN/NFATc3 pathwayMETHODS:①High glucose medium was used to stimulate primary cardiomyocytes to establish cardiomyocyte hypertrophy model.Western blot was used to detect hypertrophy protein ANP,channel protein Orail level changes,and myocardial cell volume and morphology were observed under light microscope.Detection of intracellular calcium changes in myocardial cells by laser confocal microscopy;③Infection of cardiomyocytes by adenovirus or lentivirus,high expression or down-regulation of myocardial cells Orail.BTP2 specifically blocks SOC channels;④Western blot analysis of CaN/NFATc3 signaling pathway-related protein expression levels.RESULTS:Under the stimulation of high glucose medium,the volume of cardiomyocytes increased under light microscope,and the expression of ANP and Orail protein was increased by Western blot.②Laser confocal microscopy showed that increased Ca2+concentration in Orail-mediated influx in myocardial cells of high glucose group;③Adenovirus-infected cells overexpress Orail,which activates the CaN/NFATc3 signaling pathway and further promotes cardiomyocyte hypertrophy Lentivirus-infected cells knockout Orail or BTP2-specific blockade of SOC channels inhibit the activation of CaN/NFATc3 signaling pathway,thereby inhibiting cardiomyocyte hypertrophy.CONCLUSION:(1)In vitro studies have shown that inhibition of SOC channels or knockout of Orail gene can inhibit high glucose-induced cardiomyocyte hypertrophy;overexpression of Orial gene can promote hypertrophy of cardiomyocytes in the condition of high glucose。(2)In vitro studies,we found that Orail-mediated calcium influx increases,may directly participate in the "excitation-transcription" coupling,and activate the expression of downstream hypertrophic genes.(3)In vitro studies,we found that inhibition of SOC channels or knockout of Orail gene can inhibit the activation of high glucose-induced CaN/NFATc3 pathway;overexpression of Orail gene can promote the activation of high glucose-induced CaN/NFATc3 pathway.SUMMARY:In vivo experiments,structural changes and functional changes of the heart can be observed in the the type 2 diabetic rat model.Myocardial cells were observed to have a weak ICa-L current and abnormal calcium transients in electrophysiologically Further experiments showed that the expression of Orail in cardiomyocytes was increased,the activation of CaN/NFATc3 signaling pathway and the expression of downstream hypertrophic related proteins were increased in diabetic conditon.In vitro,it was found that high glucose can induce cardiomyocyte hypertrophy,promote the increase of Orail expression,mediate Ca2+influx,promote the activation of CaN/NFATc3 signaling pathway and expression of hypertrophy related proteins Blocking SOC channel by using BTP2 or knocking down Orail by sgRNA inhibited high glucose-induced CaN/NFATc3 activation and cadiomyocyte hypertrophy.In contrast,over expression of Orail promotes the expression of hypertrophic proteins.