| Background and ObjectiveGrowth differentiation factor 11(GDF11)has been reported that protects against myocardial ischemiAReperfusion(IR)injury,but the underlying mechanisms have not been fully clarified.Considering that GDF11 plays a role in the aging/rejuvenation process and that aging is associated with telomere shortening and cardiac dysfunction,we hypothesized that GDF11 might protect against IR injury by activating telomerase.Human plasma GDF11 levels were significantly lower in acute coronary syndrome patients than in chronic coronary syndrome patients.IR mice with myocardial overexpression GDF11(oe-GDF11)exhibited a significantly smaller myocardial infarct size,less cardiac remodeling and dysfunction,fewer apoptotic cardiomyocytes,higher telomerase activity,longer telomeres and higher ATP generation than IR mice treated with an adenovirus carrying a negative control plasmid.Furthermore,mitochondrial biogenesis-related proteins and some antiapoptotic proteins were significantly upregulated by oe-GDF11.These cardioprotective effects of oe-GDF11 were significantly antagonized by BIBR1532,a specific telomerase inhibitor.Similar effects of oe-GDF11 on apoptosis and mitochondrial energy biogenesis were observed in cultured neonatal rat cardiomyocytes,whereas GDF11 silencing elicited the opposite effects to oe-GDF11 in mice.We concluded that telomerase activation by GDF11 contributes to the alleviation of myocardial IR injury through enhancing mitochondrial biogenesis and suppressing cardiomyocyte apoptosis.MethodsThe study was carried out in accordance with the principles of the Helsinki Declaration.Informed consent forms for participants in this study were all provided by patients.STROBE scales were used to evaluate the quality of this study.All procedures were performed in accordance with our Institutional Animal Research Guidelines,which are in line with the Guidelines for the Care and Use of Laboratory Animals(NIH Publication No.85-23,revised 1996).This study was approved by the Ethics Committee of Nanfang Hospital,Southern Medical University.1.Participants and measurement of plasma GDF11The cohort consisted of patients who were diagnosed with coronary artery disease(CAD)and healthy participants collected from Nanfang Hospital between January 2018 and September 2018.Patients with CAD were divided into the chronic coronary syndrome(CCS)group and acute coronary syndrome(ACS)group according to diagnostic criteria.We excluded patients if they 1)were younger than 15 years;2)had a history of other cardiac diseases,including valvular disease and cardiomyopathy;or 3)had a history of severe systemic diseases,such as malignancies,serious infection,and hepatic disease.Hence,the final cohort included 218 participants.Plasma GDF11 and cardiac tropicin I(cTnt-I)concentrations were detected according to the instructions.CSB-EL009344HU(CuSabio)and CSB-E05139H ELISA kit(CuSabio)were used,respectively.2.IR model generation,infarct size and histological examination in mice10-week-old C57/BL6 male mice were anesthetized with a mixture of ketamine(100 mg/kg,intraperitoneally)and thiazide(5 mg/kg,intraperitoneally),the left coronary artery(LCA)was ligated for 45 min,then the ligation was removed and reperfusion allowed for 24 h,as described elsewhere.Sham-operated mice underwent an identical surgical operation without ligation.Myocardial ischemia was evaluated based on ST segment elevation on electrocardiogram(ECG).According to different purposes,the animals were sacrificed with 2%isoflurane inhalation and cervical spine dislocation at 1 and 28 days postoperatively.Twenty-four hours after surgery,the hearts of mice were harvested and cut into pieces.Myocardial infarction was stained with 1%triphenyltetrazolium chloride(TTC)(Sigma Aldrich,USA)at 37℃ for 20 min.Evans Blue was injected into the aorta to determine the risk area(AAR).Myocardial infarct size was measured using Image J software.The heart was resected,washed with phosphate buffered saline(PBS),fixed with 4%paraformaldehyde,and embedded in paraffin.4-6 μm sections were prepared.Masson’s trichrome staining was utilized to evaluate myocardial fibrosis.For troponin T staining,paraffin sections obtained as described above were rinsed 3 times in PBS,blocked in buffer(10%bovine serum albumin;BSA;20 min),and rinsed 3 times in PBS.Slides were incubated overnight(4℃)with a primary antibody against cardiac troponin T(1:100,mouse monoclonal,Santa Cruz,CA,USA).The slides were rinsed with PBS the next day for 3 times and diluted with Alexa Fluor 488 or 555(1:100 dilution;Santa Cruz,CA,USA)for 1 h at room temperature.The slides were washed 3 times in PBS and stained with DAPI for 3 min to label nuclei.3.AnoxiAReoxygenation(AR)of neonatal rat cardiomyocytes(NRCMs)and cell viability assayThe cells were placed in either a hypoxic or normoxic environment.Normoxic conditions of 5%CO2 and 21%O2 were created at 37℃ in a normoxic incubator.To induce anoxia,the NRCMs were cultured in a humidified condition at 37℃ in a hypoxic chamber maintained at 5%CO2 and 1%O2 for 3 h.After anoxia,the NRCMs were cultured under normoxic conditions for 2 h reoxygenation.Cells cultured under normoxic conditions were not subjected to AR and served as controls.MTT assay(ABCAM,AB211091)was used to determine cell viability according to manufacturer’s instructions.4.Transmission election microscopy(TEM)Mouse hearts were fixed with 3%glutaraldehyde,flushed with metharsenate buffer,osmium tetroxide and suffix 1%0.1 M SC buffer for 1 h.After rinsing,tissue specimens were dehydrated with graded ethyl alcohol and then permeated with 100%acetone and embedded resin for 2 days.Following polymerization at 60℃ overnight,the blocks were then sections.Thin sections were cut and stained with UA replacement stain.The mitochondrial content was determined by quantifying the number and size of each mitochondrion per field using Image J software.5.Construction of adenovirus carrying a GDF11 or short hairpin-GDF11 plasmidA CMV-MCS-EGFP vector carrying the adenovirus(Ad)-GDF11 plasmid(oe-GDF11),HU6-MCS-CMV-EGFP vectors carrying the Ad-GDF11-shRNA(sh-1:5’-GCCTGAGGACTTCTTGGAA-3’;sh-2:5’-GCAGATCTTACGACTGAAA-3’;sh-3:5’-GCCGATATCCTCTCACAGT-3’)plasmid and negative controls were generated by a commercial company(Genechem Company,Shanghai,China).Ad particles were multipoint injected into the left ventricles of the mice 72 h before IR surgery or added to cultured NRCMs(multiplicity of infection=200).Infection efficiency were evaluated by fluorescence microscopy.6.Measurement of TL and telomerase activityPCR method for TL:DNA samples were extracted from mouse heart tissues and NRCMs with the PureLinkTM Genomic DNA Mini Kit(k1820-01,Invitrogen),The mean TL of cardiomyocytes was assessed by a modified monochrome multiplex quantitative PCR method.The relative TL is expressed as the T/S ratio(the ratio of the number of duplicated telomere copies to the number of copies of a single gene).Q-FISH method for TL:After deparaffinization,tissues were postfixed in 4%formaldehyde.Tissues were incubated in pepsin solution(Sigma Aldrich)at 37℃for 15 min.The tissue sections were mounted on slides and dehydrated in ethanol.After 10 min of air drying,0.5 mg/ml PNA probe(Panagene)were added to each slide.The slides were incubated for 3 min at 90℃ and for an additional 2 h at room temperature in the dark.Then,the slides were incubated with DAPI(Sigma Aldrich).Confocal images were obtained in a stacked manner using a Leica SP5-MP confocal microscope.Telomere signal intensity was quantified using Definiens software.According to the protocol of quantitative telomerase activity qPCR kit(KGA1028R,KEYGEN),the telomerase activity was quantified by the improved fluorescence telomere repeat amplification method.7.Mitochondrial DNA(mt-DNA)mt-DNA was extracted using a mitochondrial DNA isolation kit(K280-50,Biovision)according to the company’s recommended protocol.Real-time PCR was used to detect the copy number of mt-DNA primers.The ratio of mt-DNA to 18S rRNA was calculated as mt-DNA copy number in each group.8.Apoptosis determinationApoptosis is mediated by terminal deoxyribonucleotide transferase mediated by TDT-mediated DUTP Nick terminal labeling(TUNEL)with a kit based on the company’s protocol(Roche,Germany).The percentage of TUNEL positive cells in the total nucleus was calculated as the percentage of apoptotic cells.9.Statistical analysisQuantitative data are expressed as mean(SDM).Student unpaired double-tailed t test was used to compare the two groups.One-way ANOVA was used to compare>3 groups of parameters,followed by Bonferroni correction for posthoc multiple comparisons.Linear correlations between selected variables were assessed by the least-squares method.Kaplan-Meier survival analysis was used to evaluate the overall survival of mice at 4 weeks,and log-rank test was used for comparison between groups.All analyses were performed with GraphPad Prism 7.0 software(GraphPad Software Inc.,San Diego,CA,US),and P<0.05 was considered to be statistically significant.10.EchocardiographyEchocardiography was performed 4 weeks postoperatively using the Vevo 2100 system(Fuji Visual Ultrasound,Ontario,Canada).The inhalational flow of isoflu-rane was adjusted to anaesthetize the mice while maintaining their heart rate at 450-550 beats/min.The left ventricular end-diastolic and systolic diameters(LVEDD,LVESD),left ventricular ejection fraction,and segmental shorting(LVEF,LVFS)were measured at the left ventricular papillary muscle level in M-mode images at the short axis view.Results1.Plasma GDF11 is decreased in patients with CAD and in mice heart subjected to myocardial IRThe plasma concentrations of GDF11 in both CCS and ACS groups were significantly lower than those in healthy control group(P<0.01)and were lower in the ACS group than in the CCS group(P<0.01).There was a significant negative correlation between plasma GDF11 and troponin I level in the ACS group,and a significant negative correlation between plasma GDF11 and age in the control group(r=-0.58 and 0.56,respectively,both P<0.01).The mRNA and protein levels of myocardial GDF11 in IR group were significantly lower than those in sham operation group.Similar results were found in AR cultured NRCMs.2.GDF11 reduces myocardial infarct size and attenuates cardiac remodelingTo further investigate the influence of GDF11 on IR injury,we constructed an adenovirus carrying oe-GDF11 or sh-GDF11 and confirmed the gene and protein expression levels of GDF11 in the mouse heart and NRCMs.The protein levels of GDF11 were increased by approximately 2-fold and decreased by approximately 60-70%in response to treatment with oe-GDF11 and sh-GDF11,respectively.IR mice treated with oe-GDF11 had a significantly smaller infarct size than control IR mice,while silencing of GDF11 increased the infarct size.The four-week mortality due to IR was 18.3%,while it was 11.2%and 24.3%in oe-GDF11-and sh-GDF11-treated IR mice,respectively(log-rank P<0.05).These results suggest that GDF11 has a protective effect on IR injury.Histological examination was performed 4 weeks after surgery.Compared with untreated IR mice,oe-GDF 11-treated IR mice had a significantly smaller infarct scar size,heart weight(HW)/body weight(BW)ratio,HW/tibia length ratio,lung weight(LW)/BW ratio and LW/tibia length ratio,while opposite results were obtained in sh-GDF11-treated IR mice.Similarly,ECGs showed that oe-GDF11-treated IR mice had significantly smaller left ventricular dimensions,a higher left ventricular ejection fraction and higher fractional shortening.However,sh-GDF11 treated mice had larger left ventricular size,lower left ventricular ejection fraction and lower shortening fraction.These findings indicate that GDF11 has an inhibitor effect on cardiac remodeling and heart failure.3.GDF11 alleviates mitochondrial damage induced by IRIn the sham operation group,the mitochondria were compact and the myocardial fibrils were orderly and complete.IR mice exhibited fewer mitochondria as well as myocardial fibril rupture or disappearance and manifestations of mitochondrial injury,including relaxation and swelling of mitochondria;rupture of the mitochondrial outer membrane;increases in mitochondrial cristae distance and the number of vacuolation;and lipid droplets.However,this damage was significantly attenuated by treatment with oe-GDF 11 and exacerbated by sh-GDF 11.We further evaluated mt-DNA copy number and mitochondrial protein content.There was a significant decrease in mt-DNA copy number and mitochondrial protein content in the IR group compared with the sham group.However,mt-DNA copy number and mitochondrial protein content in oe-GDF 11 treatment group were higher than those in IR group,and sh-GDF 11 treatment could weaken this change.We also examined several key enzymes involved in oxidative phosphorylation,namely,ATP synthase,cytochrome C and cytochrome oxidase.The mRNA expression levels of these enzymes were significantly down-regulated during myocardial IR response,and this effect was blocked by oe-GDF 11 treatment and enhanced by sh-GDF 11 treatment.The protein expression of PGC-1α and TFAM was significantly lower in IR mice than in sham-operated mice,while oe-GDF 11 increased and sh-GDF 11 decreased the expression of PGC-1α and TFAM.4.GDF11 suppresses IR-induced myocyte apoptosisIt has been reported that decreased mitochondrial numbers and mitochondrial dysfunction often aggravate cell apoptosis.There were significantly more total TUNEL-positive cardiomyocyte nuclei in the IR group than in the sham group(35.63±4.71%vs 1.05±0.24%,P<0.01),while oe-GDF11 and sh-GDF11 suppressed and promoted apoptosis,respectively(21.10±3.03%and 40.20±5.23%).Compared with the sham operation group,the expressions of pro-apoptotic proteins Bax and P53 were up-regulated in IR group,while the expressions of Bax and P53 were decreased and increased in oe-GDF11 and sh-GDF11,respectively.Similarly,the antiapoptotic pathway PI3K/Akt/FoxO3a was inhibited in response to IR injury,while oe-GDF11 treatment significantly decreased and sh-GDF11 treatment significantly enhanced the inhibitory effect of IR on PI3K,FoxO3a protein expression and Akt phosphorylation.5.GDF11 activates telomere reverse transcriptase and inhibits telomere shorteningTo determine whether the antiapoptotic effect of GDF11 was attributable to the inhibition of telomere shortening,we measured the TL of cultured NRCMs in response to AR and treatment with GDF11.We observed that the TL was shorter in AR-treated cardiomyocytes than in cells cultured under normoxic conditions.However,oe-GDF11 alleviated the telomere shortening caused by AR,and this effect was blocked by cotreatment with the telomerase inhibitor BIBR1532.Considering that telomerase controls the length of telomeres,we examined the activity of telomerase.The telomerase activity in AR treated cardiomyocytes was significantly lower than that in normoxic treated cardiomyocytes,while the telomerase activity in oe-GDF11 and AR treated cardiomyocytes was significantly higher than that in AR treated cardiomyocytes without oe-GDF11,and this effect was partially offset by co-treatment with BIBR1532The telomerase reverse transcriptase(TERT)protein expression was low in the control cells and AR stimulation further downregulated TERT expression.However,oe-GDF11 activated TERT expression,which the effect was partially antagonized by the telomerase inhibitor BIBR1532.AR stimulation significantly downregulated the levels of the telomerase reverse transcriptase(TERT)and telomeric repeat-binding factor 2(TERF2),protection of telomere 1(POT1)and tripeptidyl peptidase 1(TPP1)but significantly upregulated the levels of telomeric repeat-binding factor 1(TERF1)and DNA-binding transcription factor(RAP 1),and these changes were alleviated by cotreatment with oe-GDF11.Addition of BIBR1532 partially inhibited the effects of oe-GDF11 on the levels of the telomere-related genes.6.GDF11 attenuates AR-induced mitochondrial damage and suppresses myocyte apoptosis in cultured cardiomyocytesIn cultured NRCMs,we examined the effects of oe-GDF11 on mt-DNA copy number,mitochondrial protein content,the mRNA levels of ATP synthase,cytochrome C and cytochrome oxidase,and the protein expression of PGC-1α and TFAM.Compared to control cells cultured under normoxic conditions,cells subjected to AR exhibited significant decreases in mt-DNA copy number and mitochondrial protein content,mRNA levels of ATP synthase,cytochrome C and cytochrome oxidase,and protein levels of PGC-1α and TFAM were significantly down-regulated.These effects were partially reversed by cotreatment with oe-GDF11.The beneficial effects of oe-GDF11 were antagonized by the telomerase inhibitor BIBR1532.Similar to the results obtained in the mouse model of IR,AR increased apoptosis,upregulated the related proapoptotic proteins Bax and P53 and inhibited the antiapoptotic pathway PI3K/Akt/FoxO3a in NRCMs,and these effects were blocked by cotreatment with oe-GDF11.The beneficial effects of oe-GDF11 were antagonized by the telomerase inhibitor BIBR1532.ConclusionsOverexpression of GDF11 alleviates mitochondrial damage by activating telomere reverse transcriptase activity,which protects against myocardial ischemia-reperfusion injury and delays cardiac remodeling. |
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