The Role And Mechanisms Of CD38 Over-expressed In Myocardial Autophagic Flux Blockade Under Hypoxia/ischemia Conditions

Background:Our previous studies indicated that early serious burn injury led to hypoxia/ischemia(H/I)cardiac damage together with elevated expression of CD38,autophagy activation and autophagic flux blockade,however,the relationship among CD38,autophgaic flux and H/I-cardiac damage was unclear.Thus,exploring the pathophysiological significance of CD38 overexpression might help us unravel the possible cardiac injury and the molecular mechanisms that influence autophagic flux after hypoxia/ischemia injury and provide us the therapeutic potential to ameliorate cardiac injury after serious burn injury.Meanwhile,acute hypoxia/ischemia-related diseases,such as myocardial infarction(MI),or chronic heart failure,such as tetralogy of Fallot(TOF),always led to cardiac damage,but the detailed mechanisms that led to H/I related cardiac damage was still unclear and how individual CM sensed the force pattern imposed by hypoxia/ischemia and subsequently transduced the signaling to activating cell death were remained elusive.Therefore,investigating the potential role of CD38 overexpression in cardiac under H/I comditions was essential.CD38,one of nicotinamide adenine dinucleotide(NAD)-consuming enzymes(NADases),is widely found in many organs of body.Stress-related NAD decline may be caused by increase in NADases and may lead to cell metabolic disorder,reactive oxygen species(ROS)flooding,all which can active the autophagy.Autophagy is an intracellular lysosomal degradative process that involves cellular homeostasis and survival through quality control of amino acid pools and energy metabolism.Autophagic flux is a process which include four steps: initiation,engulfment of cargo by a double-membrane structure,formation of autophagolysosome via the fusion of autophagosome and lysosome,and the final process of lysosomal degradation by acid hydrolases resulting in the recycling of lysosomal contents.It will loss the protect function of autophagy and even cause cell type II programmed cell death when one of the steps is impaired.While the role of CD38 in mediating autophagic flux and H/I-associated cardiomyocyte(CM)death is not fully understood to date.(1)To explore the widely significance of CD38 overexpression in various heart diseases,we collected heart specimen of TOF,constructed MI and 25%TBSA burned mice model to obtain different pattern of heart samples.Subsequently,to further confirm the effect of aberrant overexpression on autophagic flux and cardiac damage in vivo,we generated CD38 knockout mice.The purpose of this study was to provide therapeutic potential in the prevention and treatment of H/I-related cardiac dysfunction.Materials and Methods:1.Fresh right ventricular tissues were collected from 16 TOF patients with normal blood oxygen(oxygenation index(OI)? 300;n = 8)or hypoxemia(OI ? 240;n = 8)in the Department of Cardiovascular Surgery,Xinqiao Hospital,Chongqing,China.Myocardial specimens were divided into two groups depending on the levels of OI upon admission.In addition,MI and 25% of the total body surface area(25%TBSA)sever burned mouse models were induced and the left ventricular(LV)tissues of heart samples were obtained.All of these heart tissues were used to detect the level of CD38 expression by Real-Time PCR(RT-PCR)and immunoblotting.2.CD38 KO mice(backcrossed 12 generations to C57BL/6J)were obtained from Prof.Frances E.Lund from university of Alabama at Birmingham.The strain name of CD38 KO mice is B2.129P2-CD38tm1 Lnd.CD38 KO mice applied in this study was a systemic knock-out mouse rather than cardiomyocytes-specific KO mouse and identified according to the method supplied by the Prof.Frances E.Lund.We explored the cardiac phenotypes of wild type(WT)and CD38 KO mice(8 weeks)using different approaches,including echocardiography analysis,commercial kits detection of creatine kinase-MB(CK-MB),cardiac troponin T(c TnT)and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL),transmission electron microscope(TEM)and general observation,to elucidate the role of CD38 overexpression in H/I related cardiac damage.In addition,to further assess the effect of CD38 overexpression on H/I related cardiac damage in vitro,an overexpression adenovirus encoding mice CD38 to call back the protein level of CD38,was generated and transfected to primary neonatal mouse CMs with or without H/I stimulation(12 h).Lactic dehydrogenase(LDH),Cell Counting Kit 8(CCK-8)and cell apoptosis were measured by related commercial kits detection to determine the degree of cardiac damage under H/I condition.3.To detect autophagic flux(initiation,engulfment of cargo,fusion of autophagosome and lysosome,lysosomal degradation)in heart by immunobloting,immunofluorescence and TEM,LV heart tissues of WT and CD38 KO mice were obtained at the 8 weeks of age before and after burn.While in vitro experiments,to further demonstrate the effect of CD38 overexpression on cardiac autophagic flux,we treated primary cultured CM under H/I stimulation with or without Ad-CD38 transfection,and analyzed the above parameters with same methods.Lysosomal pH was determined by staining with Lyso Sensor(Green)and LysoTracker(red)in CMs to evaluate the lysosome degradation.4.To detect whether CD38 involves in H/I-related myocardial injury though blockade of autophagic flux,Chloroquine(CQ)was intraperitoneal injected into WT and CD38 KO mice and echocardiography analysis and commercial kits detection(CK-MB,cTnT and TUNEL)were used to analysis the cardiac function and cardiac damage.In vitro experiments,WT and CD38 KO primary neonatal mouse CMs was incubated with CQ and generated with or without H/I stimulation(12 h).Lactic dehydrogenase(LDH),Cell Counting Kit 8(CCK-8)and cell apoptosis were measured by related commercial kits detection to determine the degree of cardiac damage under H/I condition.5.To screen autophagy-related differential genes between WT mice and CD38 KO mice by mRNA microarray and immunobloting,LV heart tissues of WT and CD38 KO mice were obtained at the 8 weeks of age before and after burn.To further assess the regulation of CD38 overexpression on the autophagy-related differential genes,an CD38 overexpression adenovirus was transfected to CD38 KO primary neonatal mouse CMs with or without H/I stimulation(12 h).Lactic dehydrogenase(LDH),Cell Counting Kit 8(CCK-8)and cell apoptosis were measured by related commercial kits detection to determine the degree of cardiac damage under H/I condition.6.To detect the mechanism of the obstructed autophagic flux caused by CD38 overexpression,we first raised a hypothesis that CD38 dictates autophagic flux inhibition through a transcriptional inhibition pathway under H/I conditions and used many approaches,including immunobloting,immunofluorescence,luciferase reporter assay and chromatin immunoprecipitation sequencing(Chip-Seq)in vitro experiments to confirm it.Results:1.Using immunoblotting,immunofluorescence and RT-PCR analysis,we observed markedly increased expression levels of CD38 in myocardiac tissues from TOF patients whose OI was below 240 compared with the one whose OI was above 300.Consistent results were obtained in the comparison between mice in the MI group and sham-operated mice.We also analyzed CD38 expression in myocardiac tissues from C57BL/6J mice after severe burn or sham operation.Elevated CD38 mRNA level and protein expression were observed in severely burned mice.Furthermore,the upregulation of CD38 after H/I injury was confirmed in cultured CMs by using immunoblotting analysis,immunofluorescence staining and RT-qPCR.2.To elucidate the physiological function of overexpression of CD38 in vivo,we get mouse strain that knock out CD38 from Prof.Frances E.Lund from university of Alabama at Birmingham.We found little expression of CD38 in heart,which indicated a successful mouse model.Twenty CD38 KO male mice(8-week-old)and twenty WT male mice(8-week-old)were randomly selected to construct the model of approximately 25% of the total body surface area(TBSA)burn injury.Cardiac function and myocardial enzymes were compared between CD38 KO mice and WT mice.Knockout of CD38 significantly inhibited myocardial enzyme releasing and improved both the systolic and diastolic cardiac function.The number of TUNEL-positive cells was less in CD38 KO mice compared with in WT mice.We also examined the ultrastructure of myocardiac tissue by TEM.Compared with WT burned mice,improved myofilamental and mitochondrial structure were found in the heart of CD38 KO mice.Meanwhile,more accumulational autophagosomes and fewer autophagolysosomes were found in myocardiac tissue of WT burned mice than in CD38 KO burned mice.Consistantly,in vitro experiments,compared with WT CMs,CD38 KO CMs showed a reduced release of LDH and increased viability of CMs under H/I condition.Predicatively,the protective effect was lost when CD38 was upregulated by Ad-CD38 in CD38 KO CMs.3.LV heart tissues of 8 week-old WT and CD38 KO mice with or without burned treatment were examined.We found obvious increases in dephosphorylation of mTOR/P70S6 K and phosphorylation of AMPK/ULK1 after burn,but the degree of activation of the autophagic initiation pathway had no statistically difference between CD38 KO burned mice and WT burned mice.RT-PCR and immunobloting results revealed that the mRNA and protein level of LC3-II and P62,which was unrelated to CD38,were markedly increased after burn.Using immunofluorescence method,we found that deficiency of CD38 promoted the colocalization of Lamp1(red)with LC3(green),which was completely abrogated by CQ treatment.To further access the regulation of CD38 on autophagic flux,experiments in vitro were performed and we got the results which were consistent with the results of experiments in vivo.Meanwhile,Lysosomal pH was determined by staining with Lyso Sensor(Green)and LysoTracker(red)in CMs.H/I-induced CD38 KO CMs exhibited a similar yellow fluorescence to H/I-induced WT CMs.4.To detect whether CD38 involves in H/I-related myocardial injury though blockade of autophagic flux,Chloroquine(CQ)was intraperitoneal injected to WT and CD38 KO mice and was incubated with WT and CD38 KO neonatal primary CMs with or without H/I stimulation.Systolic and diastolic cardiac function and myocardial enzymes were compared among WT mice,WT+CQ mice,CD38 KO mice,and CD38 KO+CQ mice,CD38 deficiency mediated protection of myocardium was abolished by the blockade of autophagic flux by CQ injection.Consistently,the decline of LDH release and activation of cell ability under H/I condition mediated by CD38 deficiency was abolished by CQ.5.We evaluated the effect of CD38 on autophagosome-lysosome fusion-related genes expression in myocardiac tissue by RT-qPCR.Compared with WT mice,a dramatic increase of Rab7 mRNA was only found in CD38 KO mice of burn group,while PLEKHM1 mRNA was found significant elevation in CD38 KO mice of burn group and sham group.The protein abundance of Rab7 and PLEKHM1 showed similar trends in all the groups.Furthermore,we found that the upregulation of Rab7 and PLEKHM1 mediated by CD38 knockout was attenuated by transduction of Ad-CD38 adenovirus in CD38 KO CMs.To note,the mRNA level of Atg12 was also significantly higher in CD38 KO mice post-burn than in WT mice post-burn.However,the protein abundance of Atg12 was not affected by CD38 knockout.To further explore the role of Rab7 and PLEKHM1 in the regulation of CD38-impaired fusion of autophagosomes and lysosomes,we used adenoviruses to overexpress or knock down Rab7 or PLEKHM1 in vitro.Knockout of CD38 promoted the fusion of autophagosomes and lysosomes and the degradation of LC3-II and P62,which was abolished when Rab7 or PLEKHM1 was knocked down in H/I-induced CD38 KO CMs.The protective effect also disappeared with the knockdown of Rab7 or PLEKHM1 in CMs from CD38 KO mice.We then applied adenoviruses to stimulate the overexpression of Rab7 and PLEKHM1 in H/I-induced WT CMs.Consistently,overexpression of Rab7 and PLEKHM1 together improved autophagosome-lysosome fusion and promoted the degradation of LC3-II and P62,which was not observed when Rab7 or PLEKHM1 was overexpressed alone in H/I-induced WT CMs.Moreover,only simultaneously overexpression of Rab7 and PLEKHM1 could improve cardiac function.These data demonstrated that Rab7 and PLEKHM1 involved together in the CD38-mediated impairment of autophagosome-lysosome fusion and damage of cardiomyocyte under H/I conditions.6.In vitro experiments,we used immunobloting and chemical kit(NAD,Sirt1,sirtuins)and found that CD38 overexpression inhibited Rab7 transcription by a NAD-Sirt1-FOXO1 pathway,but NAD had no effect on PLEKHM1.Then,we performed a luciferase reporter assay and found that CD38 could not be bound to the PLEKHM1 promoter sequence.And Ch IP-seq assays showed that CD38 could be bound to the promoter sequences of some other transcription factors excluding PLEKHM1 gene.Thus,it was likely that CD38 regulated PLEKHM1 by modulating expression of other transcription factors.The indirect transcriptional regulation of PLEKHM1 by CD38 should be further investigated.Conclusions:In summary,findings reported in our study reveal that CD38 overexpression may serve as a springboard for the pathogenesis of multiple hypoxia/ischemia cardiomyopathies including TOF,MI and shock heart after burn,suggesting its therapeutic potentials in hypoxia/ischemia cardiomyopathies.The overexpression of CD38 specifically downregulated the expression of Rab7 and its adaptor protein Pleckstrin homology domain containing protein family member 1(PLEKHM1)through nicotinamide adenine dinucleotide(NAD)-dependent and non-NAD-dependent pathways,respectively.Loss of Rab7/PLEKHM1 impaired the fusion of autophagosomes and lysosomes,resulting in autophagosmes accumulation in myocardium,consequently cardiac dysfunction under H/I conditions.Thus,it was concluded that CD38 mediated autophagic flux blockade and cardiac dysfunction in a Rab7/PLEKHM1 dependent manner.