Growth differentiation factor 11 attenuates cardiac ischemia reperfusion injury via enhancing mitochondrial biogenesis and telomerase activity

Figures

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  Fig. 1. Plasma GDF11 levels in patients with CAD. A Plasma GDF11 levels in the healthy control (HC) group (n = 74), patients with chronic coronary syndrome (CCS) (n = 70) and patients with acute coronary syndrome (ACS) (n = 74); *p < 0.05 vs. the HC group; #p < 0.05 vs. the CCS group; one-way ANOVA with Tukey’s multiple comparisons test. B Correlation between plasma GDF11 levels and plasma cTn-I levels in the ACS group; p < 0.01, Pearson correlations. C Correlation between plasma GDF11 levels and age in the non-CAD group; p < 0.01, Pearson correlations. GDF11 growth differentiation factor 11, cTn-I cardiac troponin I, CAD coronary artery disease.

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  Fig. 2. GDF11 was downregulated in rodent cardiomyocytes following IR or AR. A Changes in the ST segment on ECG in response to myocardial IR in mice. B GDF11 mRNA levels in sham and IR mice at 24 h after surgery. C Representative western blots of GDF11 in sham and IR groups. D GDF11 protein levels in sham and IR mice at 24 h after surgery. E GDF11 mRNA levels in NRCMs subjected to AR. F Representative western blots of GDF11 in NRCMs subjected to AR. G GDF11 protein levels in NRCMs subjected to AR. *p < 0.05 vs. the sham group, n = 5 in each group. GDF11 growth differentiation factor 11, ECG electrocardiogram, IR ischemia/reperfusion, AR anoxia/reoxygenation, NRCMs neonatal rat cardiomyocytes.

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  Fig. 3. Effects of GDF11 on the myocardial IS, survival and cardiac remodeling in mice subjected to IR. Adenovirus carrying GDF11 (oe-GDF11) or adenovirus carrying sh-GDF11 were injected into the myocardium. A Representative pictures of Evans blue and triphenyl tetrazolium chloride (TTC) double-stained myocardial sections from mice subjected to 45 min of ischemia/24 h of reperfusion. Scale bar = 2 mm. B AAR in the four groups at 24 h after surgery; n = 5, one-way ANOVA. C Myocardial IS in the four groups at 24 h after surgery; n = 5, one-way ANOVA followed by Bonferroni’s correction for post hoc multiple comparisons. D Four-week survival curves of the six groups. &p < 0.05 vs. the sham group; #p < 0.05 vs. the oe-GDF11 + IR group, Kaplan–Meier survival analysis. E Myocardial scar size at 4 weeks after surgery; n = 10, one-way ANOVA followed by Bonferroni’s correction for post hoc multiple comparisons. F The heart weight (HW)/body weight (BW) ratio at 4 weeks after surgery; n = 10–15, one-way ANOVA followed by Bonferroni’s correction for post-hoc multiple comparisons. G The HW/TiL ratio at 4 weeks after surgery; n = 10–15, one-way ANOVA followed by Bonferroni’s correction for post hoc…

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  Fig. 4. Effects of overexpressing or silencing GDF11 on mitochondrial damage induced by IR in mice. A Representative pictures of the ultrastructure of myocardial tissue under a transmission electron microscope. Yellow arrows: fat droplets; red arrows: vacuolated mitochondria (mt); blue arrows: broken myocardial fibril. B The number of mitochondria per field. C The mean area of a mitochondrion. D The distance between (btw) mitochondrial cristae. E The number of vacuoles per field. F The number of lipid droplets per field. G The mitochondrial DNA (mtDNA) copy number. H The proportion of mitochondrial protein relative to total myocardial protein. I ATP production. J mRNA expression of ATP5b, CYCS, and COX5a. K Representative images of western blots for PGC-1α and TFAM. L Semiquantitative analysis of the protein expression of PGC-1α and TFAM. &p < 0.05 vs. the sham group; *p < 0.05 vs. the IR group; #p < 0.05 vs. the oe-GDF11 + IR group; n = 5, one-way ANOVA followed by Bonferroni’s correction for post hoc multiple comparisons in (B–I) and (K). GDF11 growth differentiation factor 11, IR ischemia/reperfusion, oe-GDF11 GDF11 overexpression, sh-GDF11 short hairpin RNA of GDF11.

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  Fig. 5. Effects of overexpressing or silencing GDF11 on IR-induced myocyte apoptosis in mice. A Representative photographs of TUNEL-stained cells. B Quantitative analysis of TUNEL-positive cells. C Representative images and semiquantitative analysis of western blots for Bax, P53, p-P53, C-caspase3, Caspase3, and Bcl2. D Representative images and semiquantitative analysis of western blots for PI3K, Akt, FoxO3a, and p-Akt. &p < 0.05 vs. the sham group; *p < 0.05 vs. the IR group; #p < 0.05 vs. the oe-GDF11 + IR group; n = 5, one-way ANOVA followed by Bonferroni’s correction for post-hoc multiple comparisons in panel (B, D, F). GDF11 growth differentiation factor 11, IR ischemia/reperfusion, oe-GDF11 GDF11 overexpression, sh-GDF11 short hairpin of GDF11.

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  Fig. 6. GDF11 activated telomerase and inhibited telomere shortening in NRCMs under AR. A Representative photographs of telomeres measured by Q-FISH in NRCMs. B Quantitative analysis of the TL of TEL-CY3-positive cells. C TL measured by real-time PCR. D The activity of telomerase. E Representative western blot images and semiquantitative analysis of TERT. F mRNA expression of TERT, TERF2, POT1, TPP1, TERF1, and RAP1. &p < 0.05 vs. the NC group; *p < 0.05 vs. the AR group; #p < 0.05 vs. the oe-GDF + AR group; n = 6 in each group in panel (B) and n = 5 per group in panels (C–G); one-way ANOVA followed by Bonferroni’s correction for post hoc multiple comparisons in panel (B–D, F, G). (B) The telomerase inhibitor BIBR1532; GDF11 growth differentiation factor 11, oe-GDF11 GDF11 overexpression, TL telomere length.

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  Fig. 7. GDF11 inhibited AR-induced mitochondrial damage in NRCMs. A The mitochondrial (mt) DNA copy number. B The proportion of mitochondrial protein relative to total cell protein. C ATP production. D oxygen consumption. E mRNA expression of ATP5b, CYCS, and COX5a. F Representative western blot images and semiquantitative analysis of PGC-1α and TFAM. &p < 0.05 vs. the NC group; *p < 0.05 vs. the IR group; #p < 0.05 vs. the oe-GDF11 + AR group; n = 5, one-way ANOVA followed by Bonferroni’s correction for post-hoc multiple comparisons in panel (A–C, E). AR anoxia/reoxygenation; B the telomerase inhibitor BIBR1532, GDF11 growth differentiation factor 11, oe-GDF11 GDF11 overexpression.

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  Fig. 8. An illustration of the influences of GDF11 on IR/AR injury and the underlying mechanisms. Patients with CAD, especially ACS, and rodent cardiomyocytes subjected to IR or AR insult, exhibit lower circulatory or myocardial GDF11 levels, leading to lower activity of telomerase and shorter TL in the nucleus due to downregulation of TERT, inhibiting mitochondrial energy dynamics and the antiapoptotic PI3K/Akt/FoxO3a pathway and activating the proapoptotic p53/Bax pathway. These changes collectively increase cardiomyocyte apoptosis, enlarge the myocardial infarct size and eventually promote cardiac remodeling. The changes can be antagonized and enhanced by overexpression and silencing of GDF11, respectively, and the beneficial effects of GDF11 can be attenuated by the telomerase inhibitor BIBR1532. ↑: activation, ⊥: inhibition.┆: from other reference.│: experimentally proven.