| Background and aims:Acute myocardial infarction(AMI)is a common and serious ischemic heart disease with high morbidity and mortality.In the past few decades,with the advent of reperfusion therapy,the survival rate of patients with AMI has significantly increased,however,the mortality rate is still as high as 40%after operation.CD38 is a multifunctional transmembrane protein originally discovered in lymphocytes and subsequently shown to be expressed in a variety of cell types.CD38 is involved in the degradation of nicotinamide adenine dinucleotide(NAD+)in mammalian cells.CD38 deficiency significantly increased the level of intracellular NAD+.As an important coenzyme in mammalian cells,NAD+is involved in many important physiological functions,such as oxidation-reduction,metabolism and DNA repair,etc.Our previous study showed that deletion of CD38 gene or supplementation with exogenous NAD+could effectively protect the heart from ischemia reperfusion(IR)injury.The periods of the acute injury and subsequent myocardial repair in AMI are complex pathophysiological processes involving in a variety of cells,such as myocardial cells,macrophages of different origin,fibroblasts and endothelial cells.As the largest cell population in the heart(about 75%of the total cell volume),myocardial cells are the most severely damaged cells in myocardial infarction.Macrophages,which have important immunomodulatory effects,play an important role in different stages of AMI.In this study,cardiomyocyte-specific CD38 deficiency mice and macrophage-specific CD38 deficiency mice were used to investigate the effects and mechanisms of CD38 in different cell types on the occurrence and repair process of myocardial injury caused by ischemic heart disease.Our study will provide a new strategy and experimental basis for clinical prevention and treatment of AMI.Methods:1.Preparation and identification of cardiomyocyte or macrophage-specific CD38 deficiency mice:The Flox sites were inserted on both sides of exons 2 and 3 of the CD38 gene to prepare CD38flox/flox mice,which were hybridized with Mlc-Cre mice and Lyz-Cre mice to obtain cardiomyocyte specific CD38 deficiency mice(CD38flox/floxMlc-Cre mice)and monocyte derived macrophage specific CD38 deficiency mice(CD38flox/floxLyz-Cre mice),respectively.Q-PCR and Western blot were used to identify mouse genotypes and detect the knockout efficiency of CD38 in cardiac tissue and macrophages.2.Establishment of acute myocardial infarction model:CD38flox/floxMlc-Cre,CD38flox/floxLyz-Cre,and CD38flox/floxmale mice with 6-8 weeks age were used.The mice were anesthetized with isoflurane and fixed to a thermostatic heating plate.The chest cavity was sterilized with alcohol,and the hair was removed from the chest cavity using a hair removal cream.The chest cavity was opened 1-2 cm with scissors,and the ribs at positions 3-4 of mice were separated with blunt instrument to expose the heart.The left anterior descending coronary artery was quickly ligated with a 7-0 sterile line.Once the bottom of the heart turned white,the chest cavity was quickly sutured with 4-0 sterile line.The control group was the mice with sham operation.Only the chest was opened to expose the left anterior descending coronary artery without ligation.3.Effects of tissue specific deficiency of CD38 gene on acute myocardial infarction injury and phenotypic analysis:1)The model mice were examined by echocardiography on the 3rd,7th and 28th days respectively.The main indexes were as follows:Ejection fraction(EF),shortening fraction(FS),left ventricular end-diastolic diameter(LVIDd)and left ventricular end-systolic diameter(LVIDs);2)The survival rate of mice were observed within 28 days after modeling;3)The content of LDH in serum,heart/body weight ratio and heart/tibia length ratio were detected on day 28 after modeling;4)Masson staining and Sirius red staining were used to detect collagen deposition in the myocardial infarct area and the infarct edge.4.Mechanism analysis:The effects of cardiomyocyte specific deletion of CD38 on hypoxic-induced cardiomyocytes apoptosis,mitochondrial function and fibroblast transformation were studied in vivo and in vitro,respectively.1)The cardiac tissue proteins and mitochondrial proteins were extracted from mice on the 7th day after acute myocardial infarction.In addition,the expressions of proapoptotic gene Bax and anti apoptotic gene BCL2 were detected in the cytoplasm and mitochondria.The protein expressions of Pro-Caspase3 and Cleaved Caspase3 in the cytoplasm were also examined.Moreover,the changes of mitochondrial function-related proteins,including mitochondrial fusion proteins Mfn1 and Mfn2 and mitochondrial fission protein Drp-1 were detected.On the 28th day after the model,the mouse heart tissues were sliced and stained to analyze the collagen deposition in the infarct area and the infarct edge.Western blot and immunofluorescence were used to detect the phenotypic transformation of fibroblasts to myofibroblasts,such as α-SMA and TGF-β etc.2)The rat cardiomyocyte line H9c2 with CD38 knockdown was constructed.Firstly,hypoxia treatment was performed.At different time points,cardiomyocyte apoptosis and mitochondrial membrane potential were detected by flow cytometry.Western blot and Q-PCR were used to detect the changes of related genes such as BCL2 and Bax.3)The protein expression of Sirt3 in myocardial tissue or cardiomyocytes was detected by Western blot,and the effects of Sirt3 inhibitors on apoptosis and mitochondrial membrane potential were detected in primary cardiomyocytes with CD38 deletion.Results:1.CD38flox/floxMlc-Cre mice and CD38flox/floxLyz-Cre mice were successfully prepared:The target DNA bands of CD38flox/flox mice,CD38flox/floxMlc-Cre mice and CD38flox/floxLyz-Cre mice were 313bp,450bp and 700bp,respectively,which were consistent with the target DNA bands of the transgenic mice.Meanwhile,cardiac tissues of CD38flox/floxMlc-Cre mice were isolated and the knock-out efficiency of cardiomyocytes was detected by Western blot and Q-PCR.Compared with CD38flox/flox mice,CD38 expression in the heart tissue of CD38flox/floxMlc-Cre mice was decreased by more than 90%.Compared with CD38flox/flox mice,the expressions of CD38 in peritoneal macrophages and bone-marrow derived macrophages of CD38flox/floxLyz-Cre mice were reduced by about 70%,and about 80%,respectively.The results suggested that CD38flox/floxMlc-Cre mice and CD38flox/floxLyz-Cre mice were successfully established.2.A mouse model of acute myocardial infarction was successfully established:After ligation of the left anterior descending branch of the coronary artery in mice,cardiac function changes of the sham operation mice and model mice were detected by echocardiography.Our results showed that ejection fraction,shortened fraction,left ventricular end-systolic diameter and left ventricular end-diastolic diameter of the model mice were significantly changed,while there were no significant changes in the sham operation group.At the same time,TTC staining showed that there were severiou myocardial infarction in AMI model.The results showed that we successfully established a mouse model of AMI.3.Phenotypic analysis of acute heart infarction model:Compared with the control group,the survival rate of CD38flox/floxMlc-Cre mice in AMI was significantly increased.And the cardiac function such as ejection fraction,shortening fraction,left ventricular end-systolic diameter and left ventricular end-diastolic diameter were significantly improved,however,the survival rate and cardiac functions of CD38flox/floxLyz-Cre mice were not significantly changed.In addition,cardiomyocyte specific CD38 deficiency significantly ameliorated the increase in LDH content caused by ischemia.We also found both heart/body weight ratio and heart/tibia length ratio were decreased in CD38flox/floxMlc-Cre mice after AMI compared with CD38flox/flox mice.Histological results showed that collagen deposition was significantly increased at infarct area of the heart in AMI,but not at infarct edge.The cardiac function,survival rate and histological staining results of macrophage-specific CD38 deletion mice in AMI were not significantly different from those of control mice.4.Mechanism analysis:1)The results of Western blot showed that CD38flox/floxMlcCre mice significantly increased the transport of anti-apoptotic gene BCL2 from cytoplasm to mitochondria,reduced the apoptosis induced by hypoxia,and decreased cytoplasmic Cleaved Caspase3 expression,while the pro-apoptotic gene Bax had no significant changes in heart tissues at 7th of AMI.2)CD38 deficiency significantly increased the expression of mitochondrial fusion proteins Mfn-1 and Mfn-2,but the mitochondrial fission protein Drp-1 had no significant change.3)Histological staining and protein detection results showed that CD38 deletion significantly increase the expression of myoblast-related genes of infarction area;4)Knockdown of CD38 significantly reduced hypoxia-induced apoptosis and improved hypoxia-induced mitochondrial membrane potential decline.Moreover,CD38 knockdown increased anti-apoptotic gene BCL2 transport to mitochondria and reduced the expression of Cleaved Caspase3 in cytoplasm.Furthermore,we found themitochondrial fusion protein Mfn2 expression was increased under hypoxia.5)CD38 deletion significantly increased the expression of Sirt3 under hypoxia conditions,and Sirt3 inhibitors significantly inhibited the protective effect of CD38 deletion on hypoxia-induced apoptosis and mitochondrial membrane potential.Conclusion:1.Cardiomyocyte specific CD38 knockout significantly improved the cardiac function and survival rate of mice after acute myocardial infarction,but macrophage-specific CD38 knockout had no the protective effect.2.CD38 deletion prevented the death of a large number of cardiomyocytes under hypoxia by increasing the transport of anti-apoptotic gene BCL2 from cytoplasm to mitochondria and improving hypoxia-induced mitochondrial dysfunction.3.The protective effects of CD38 deletion on hypoxic-induced apoptosis of cardiomyocytes were mainly achieved through the activation of NAD+/Sirt3 signaling pathway. |
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