Leptin Increases Mitochondrial OPA1 Via GSK3-mediated OMA1 Ubiquitination To Enhance Therapeutic Effects Of Mesenchymal Stem Cell Transplantation

BackgroundCardiomyocytes insulted by ischemia would undergo to apoptosis and result in cardiac ventricular dilation and reduction of ejection fraction after myocardial infarction(MI).The cardiomyocytes would be replaced by cardiac-fibroblasts because of limited proliferation of cardiomyocytes,which lead to ischemic cardiomyopathy and heart failure resulted from non-contracted collagen scar tissue.The effectiveness of bone marrow mesenchymal stem cells(MSCs)on myocardial infarction have been reported in many non-human primate animal and clinical researches.Because of their inherent properties that include low immunogenicity,multipotentiality,and maintenance of"sternness",mesenchymal stem cells(MSCs)are potential vectors of choice for regenerative medicine.However,the few surviving transplantation of the cells after delivery to MI hearts is poor and this limit therapeutic efficacy.Our group previously has demonstrated that the survival of hypoxia-preconditioning hMSCs has been enhanced significantly.Leptin may play a role in the enhanced survival of hMSCs through global gene expression profile.Mitochondrial integrity and morphology determine cellular death and diseases by preventing the release of diverse pro-apoptotic factors,and excessive fragmentation of mitochondria would promote cellular death.Indeed,mitochondrial fusion is a control point for apoptotic processes,and cellular death is tightly linked to mitochondrial dysfunction.Originally identified as a peptidase secreted by adipocytes,leptin plays an important role in regulating metabolic normalization,neuroendocrine and immune homeostasis.Growing evidences suggest that the potential of leptin may contribute to mitochondrial changes.On exposure to leptin,mitochondrial dynamics and function improve in MCF-7 cells and ob/ob mice.Additionally,leptin was reported to protect cardiomyocytes form apoptosis.Thus,we speculated that leptin conferred the anti-apoptotic ability of hMSCs by maintain of mitochondrial integrity and dynamics.Part 1:The influence of leptin on hMSCs survival response to ischemia in vivo and in vitro.Aims:To investigate the effect of leptin on hMSCs survival response to ischemia in animal model in vivo and mimic ischemia in vitro.Methods and Results:In C57BL/6J mouse-MI model,hMSCs labeled by GFP and infected by lentivirus overexpressing leptin were transplanted into the peri-infarct zone of mouse-MI heart.C57BL/6J mice were divided into three groups(20 mice per group):Ⅰ)C57BL/6J MI-mice treated with leptin overexpressed hMSCs(hMSCiep);Ⅱ)C57BL/6J MI-mice treated with equal numbers of hMSCs infected by empty vector(hMSCvec);Ⅲ)C57BL/6J MI-mice treated with equal vomune of DMEM.Compared to vector treated hMSCs,hMSCiep group exhibited improved viability,leading to enhanced angiogenesis in vivo analyses at day 3 post-MI as evidenced by GFP and TUNEL co-immunostaining(P=0.01).After day 28 post-MI,hMSCiep treated mice performed increased heart function and reduced MI-infracted area measured by two dimensional echocardiography and Sirius red staining respectively(p<0.05).Moreover,we analyzed the markers of angiogenesis in vivo at day 28 post-MI,suggesting that enhanced paracrine efficiency of hMSCs pretreated with leptin to promote angiogenesis to improve heart function after myocardiac infarction.During the hostile micro-environment caused by MI,we cultured hMSCs exposed to glucose and serum deprivation under hypoxia(GSDH)to mimic ischemia.After pretreatment of leptin(50 ng/ml)for 24 h,hMSCs were subjected to GSDH for another 24 h.hMSCs pretreated with leptin exhibited reduced Annexin V/PI staining and TUNEL staining as well as low level of cleaved caspase 3 compared with solvent of leptin controls,suggesting that leptin protected hMSCs from apoptosis.The tube formation of human umbilical vein endothelial cells(HUVECs)in Matrigel assay demonstrated a significantly increased endothelial tube formation of HUVECs in conditioned medium of hMSCs-Leppre groups as compared with hMSCs-Ctrlpre or leptin alone but not in DMEM alone and control-alone groups,consisting with the result of the enhanced paracrine efficiency of hMSCs.Part 2:The mechanism of effectiveness of leptin on mitochondria in hMSCsAims:To investigate the mechanism of effectiveness of leptin on mitochondrial morphology and dynamics through analyzing the regulators of mitochondrial dynamicsMethods and Results:After pretreated with leptin for 24 h,hMSCs were induce to apoptosis under GSDH for another 24 h.We analyzed and quantified mitochondrial ultrastructure using transmission electron microscopy(TEM).hMSCs pretreated with leptin cultured demonstrated dense and elongated tubular mitochondria with swollen cristae dispersed within the cells,whereas sparse,lessened and punctate mitochondria were generally observed in the control group.However,a lower oxygen consumption rate(OCR)was found when mitochondrial respiratory function was measured in the hMSCs pretreated with leptin group compared with controls.Compared to control group,the increased protein expression level of Optic atrophy 1(OPA1)but no change in the mRNA level in hMSCs pretreated with leptin.OPA1 is a mitochondrial inner membrane protein that regulates fusion and cristae structure in the inner mitochondrial membrane.Different OPA1 isoforms are known to perform counter-regulatory roles in maintaining mitochondrial dynamics,and long-OPA1 isoforms(L-OPA1)regulate the mitochondrial fusion,whereas short-OPA1 isoforms(S-OPA1)are correlated with mitochondrial fission.The L-OPA1 were accumulated in hMSCs pretreated with leptin,indicating that leptin promoted mitochondria to fusion consisted with the results of TEM.And the protection of leptin was abrogated by targeting OPA1 with a selective siRNA.Furthermore,we showed that OMA1,a mitochondrial protease that cleaves OPA1,decreased in a leptin-dependent manner.Pretreatment of cells with MG 132,an inhibitor of the proteasome,prevented leptin-induced OMA1 degradation,implicating the ubiquitinatin/proteasome system as part of the protective leptin pathway.GSK3 inhibitor(SB216763)also reduced degradation of OMA1,indicating a GSK3-dependent step in OMA1 ubiquitination.Conclusion:During the hostile micro-environment caused by MI,(a)leptin can maintain mitochondrial integrity and prolong surviving hMSCs;(b)leptin-mediated mitochondria requires phosphorylation of GSK3 as a prerequisite for ubiquitination-depended degradation of OMA1 and attenuation of long-OPA1 cleavage.We suggest that leptin targeting of the GSK3/OMA1/OPA1 signaling pathway can optimize transplantated hMSCs therapy for cardiovascular disease in particular myocardial infarction.