GDF11 Enhances Therapeutic Efficacy Of Mesenchymal Stem Cells For Myocardial Infarction Via YME1L-mediated OPA1 Processing

Background:Myocardial infarction?MI?,the most life-threatening diseases with a high death rate worldwide,induces the irreversible loss of cardiomyocytes,scar formation,and may ultimately results in heart failure.Mesenchymal stem cells?MSCs?is a promising strategy for treatment of heart failure after MI,and the preclinical investigations support the concept that donor cells more oriented towards a cardiovascular phenotype favor better repair.However,regardless of the origin of MSCs,low survival of transplanted MSCs in infarcted myocardium limits successful use of MSCs in treating MI.Growth differentiation factor 11?GDF11?,a member of the transforming growth factor-??TGF-??family,has been shown to stimulate proliferation and angiogenesis of stem cells in ischemia/reperfusion models and to be implicated in the regulation of mitochondrial generation or dynamics to participate in mitochondrial quality control of skeletal muscle cells,but little is known about the effect of GDF11 on viability and therapeutic efficacy of MSCs derived from heart for cardiac injury.Maintenance of mitochondrial integrity has a vital role in cellular resistance to apoptosis,and mitochondrial dysfunction results in activation of mitochondrial fission and inactivation of mitochondrial fusion.Therefore,we speculate that GDF11 may protect MSCs against apoptosis by maintaining mitochondrial dynamic stability and integrity under hypoxic condition.Objective:To verify whether GDF11 could protect MSCs under hypoxia and improve the therapeutic effect by improving mitochondrial morphology and function in vivo and vitro models,and further explore the mechanism.Methods and Results:Part 1:Myocardial infarction?MI?model to evaluate the cardiac protective effects of MSCs transduced with lentiviral vector carrying full-length mouse GDF11 tagged with green fluorescent protein?GFP?was performed on male mice?C57BL/6J?.Increased numbers of GFP-positive cells were detected in MSCs ^LV-GDF11group compared with MSCs ^LVgroup at day 3 post-MI as evidenced by GFP immunostaining and RT-PCR.Treatment with MSCs ^LV-GDF11resulted in a better recovery of cardiac function in comparison with that with MSCs ^LVor DMEM after day 28 post-MI measured by echocardiography.These were concomitant with reduced scar size measured by Sirius red staining and more angiogenesis in aspect of increased number of endothelial cells and smooth muscle cells in the remote area at day 28 post MI.All these results supported that GDF11 enhanced therapeutic effects of MSCs for improving heart function after myocardial infarction.After pretreated with recombinant GDF11 for 24h(MSCs^{r GDF11})or overexpressed GDF11(MSCs ^LV-GDF11),MSCs were induced to apoptosis under hypoxia condition for another 48h.Less apoptotic cells were observed in MSCs^{r GDF11}or MSCs ^LV-GDF11as compared with control MSCs.In addition,more tube formation of HUVECs and less apoptosis of H9C2 were observed after these cells were treated with condition medium of MSCs^{r GDF11}or MSCs ^LV-GDF11.Part 2:To investigate the underlying mechanism via which GDF11 improved the activities of MSCs,we first examined mitochondrial morphology by using transmission electron microscopy?TEM?.After exposure to hypoxic stress,mitochondria of MSCs^{r GDF11}were densely populated and maintained elongated tubular morphology,whereas those from control MSCs were roundly shaped.Moreover,MSCs^{r GDF11}showed significantly higher OCR in both basal and maximal levels as compared with control.Both ATP production and mitochondrial membrane potential were greater in MSCs^{r GDF11}than those in control.Optic atrophy 1?OPA1?,a mitochondrial inner membrane protein,was greatly increased under hypoxic condition in response to GDF11 treatment.OPA1can be cleavage from long form?L-OPA1?to short isoform?S-OPA1?.L-OPA1 isoform is mainly responsible for mitochondrial fusion,while the S-OPA1 is involved in the acceleration of mitochondrial fission.In contrast,the protection of GDF11 was abrogated by silencing OPA1 with a selective si RNA.Part 3:Furthermore,GDF11 increased YME1L level through activating Smad2/3pathway,When either ALK5 or Smad2/3 was inhibited and YME1L expression was silenced by si RNA,the anti-apoptosis effect of GDF11 was abrogated with decreased L-OPA1 level.Conclusions:GDF11 could protect MSCs against hypoxia stress-induced damage via enhancing mitochondrial fusion.We suggested that GDF11 can optimize transplanted MSCs therapy for myocardial infarction by targeting the Smad2/3-YME1L-OPA1 signaling pathway.