GDF11 mitigates high glucose-induced cardiomyocytes apoptosis by inhibiting the ALKBH5-FOXO3-CDR1as/Hippo signaling pathway.

Diabetic cardiomyopathy remains a formidable health challenge with a high mortality rate and no targeted treatments. Growth differentiation factor 11 (GDF11) has shown promising effects on cardiovascular diseases; however, its role and the underlying mechanism in regulating diabetic cardiomyopathy remain unclear. In this study, we developed mouse models of diabetic cardiomyopathy using leptin receptor-deficient (db/db) mice and streptozocin-induced C57BL/6 mice. The diabetic cardiomyopathy model mice exhibited apparent structural damage in cardiac tissues and a significant increase in the expression of apoptosis-related proteins. Notably, we observed a significant decreased expression of GDF11 in the myocardium of mice with diabetic cardiomyopathy. Moreover, GDF11 cardiac-specific knock-in mice (transgenic mice) exhibited improved cardiac function and reduced apoptosis. Moreover, exogenous administration of GDF11 mitigated high glucose-induced cardiomyocyte apoptosis. Mechanistically, we demonstrated that GDF11 alleviated high glucose-induced cardiomyocytes apoptosis by inhibiting the activation of the alkylation repair homolog 5 (ALKBH5)-forkhead box group O3a (FOXO3)-cerebellar degeneration-related protein 1 transcript (CDR1as)/Hippo signaling pathway. Consequently, this novel mechanism effectively counteracted myocardial cell apoptosis, providing valuable insights into potential therapeutic strategies for clinical diabetic cardiomyopathy.

Long non-coding RNA LHX1-DT regulates cardiomyocyte differentiation through H2A.Z-mediated LHX1 transcriptional activation.

Long non-coding RNAs (lncRNAs) play widespread roles in various processes. However, there is still limited understanding of the precise mechanisms through which they regulate early stage cardiomyocyte differentiation. In this study, we identified a specific lncRNA called LHX1-DT, which is transcribed from a bidirectional promoter of LIM Homeobox 1 (LHX1) gene. Our findings demonstrated that LHX1-DT is nuclear-localized and transiently elevated expression along with LHX1 during early differentiation of cardiomyocytes. The phenotype was rescued by overexpression of LHX1 into the LHX1-DT-/- hESCs, indicating LHX1 is the downstream of LHX1-DT. Mechanistically, we discovered that LHX1-DT physically interacted with RNA/histone-binding protein PHF6 during mesoderm commitment and efficiently replaced conventional histone H2A with a histone variant H2A.Z at the promoter region of LHX1. In summary, our work uncovers a novel lncRNA, LHX1-DT, which plays a vital role in mediating the exchange of histone variants H2A.Z and H2A at the promoter region of LHX1.

Evaluation of eleven kiwifruit genotypes for bicarbonate tolerance and characterization of two tolerance-contrasting genotypes.

Screening bicarbonate-tolerant genotypes is an environmentally-friendly and long-term effective strategy to cope with bicarbonate-induced chlorosis in fruit crops grown on calcareous soils. We investigated eleven genotypes from four kiwifruit species (Actinidia chinensis, A. macrosperma, A. polygama, and A. valvata) for differences in bicarbonate tolerance. We also characterized the physiological and molecular differences in two contrasting genotypes of this group. In the first experiment, bicarbonate-treated plantlets were irrigated with 3.0 g L-1 CaCO3 and 5.04 g L-1 NaHCO3 in peat and perlite medium culture. Based on principal component analysis, weight-based membership function method and cluster analysis, the tested genotypes were classified into three groups: (1) tolerant, including YX, Av-1, Acd, Ap, Av-2, and QM; (2) moderately tolerant, including Av-3, Am, Av-4, and HWD; and (3) sensitive, including only QH. In the second experiment, QH (bicarbonate-sensitive) and YX (bicarbonate-tolerant) were grown in sand culture with 4.0 g L-1 CaCO3 and 0.84 g L-1 or 1.26 g L-1 NaHCO3. Compared with QH, YX showed a better ability to take up iron (Fe) by roots and to transport Fe from roots to shoots in the bicarbonate treatments, probably due to a better capacity to protect from oxidative damage and to excrete protons, and a differential expression of genes associated with Fe uptake and translocation, including HA8, IRT1, YSL3 and NRAMP3. The results can facilitate identifying potential resources for bicarbonate tolerance and breeding new rootstocks, and contribute to the elucidation of the bicarbonate tolerance mechanisms in the genus Actinidia.

GDF11 promotes wound healing in diabetic mice via stimulating HIF-1ɑ-VEGF/SDF-1ɑ-mediated endothelial progenitor cell mobilization and neovascularization.

Non-healing diabetic wounds (DW) are a serious clinical problem that remained poorly understood. We recently found that topical application of growth differentiation factor 11 (GDF11) accelerated skin wound healing in both Type 1 DM (T1DM) and genetically engineered Type 2 diabetic db/db (T2DM) mice. In the present study, we elucidated the cellular and molecular mechanisms underlying the action of GDF11 on healing of small skin wound. Single round-shape full-thickness wound of 5-mm diameter with muscle and bone exposed was made on mouse dorsum using a sterile punch biopsy 7 days following the onset of DM. Recombinant human GDF11 (rGDF11, 50 ng/mL, 10 µL) was topically applied onto the wound area twice a day until epidermal closure (maximum 14 days). Digital images of wound were obtained once a day from D0 to D14 post-wounding. We showed that topical application of GDF11 accelerated the healing of full-thickness skin wounds in both type 1 and type 2 diabetic mice, even after GDF8 (a muscle growth factor) had been silenced. At the cellular level, GDF11 significantly facilitated neovascularization to enhance regeneration of skin tissues by stimulating mobilization, migration and homing of endothelial progenitor cells (EPCs) to the wounded area. At the molecular level, GDF11 greatly increased HIF-1ɑ expression to enhance the activities of VEGF and SDF-1ɑ, thereby neovascularization. We found that endogenous GDF11 level was robustly decreased in skin tissue of diabetic wounds. The specific antibody against GDF11 or silence of GDF11 by siRNA in healthy mice mimicked the non-healing property of diabetic wound. Thus, we demonstrate that GDF11 promotes diabetic wound healing via stimulating endothelial progenitor cells mobilization and neovascularization mediated by HIF-1ɑ-VEGF/SDF-1ɑ pathway. Our results support the potential of GDF11 as a therapeutic agent for non-healing DW.

NAD+ attenuates cardiac injury after myocardial infarction in diabetic mice through regulating alternative splicing of VEGF in macrophages.

Diabetic mellitus (DM) complicated with myocardial infarction (MI) is a serious clinical issue that remained poorly comprehended. The aim of the present study was to investigate the role of NAD+ in attenuating cardiac damage following MI in diabetic mice. The cardiac dysfunction in DM mice with MI was more severe compared with the non-diabetic mice and NAD+ administration could significantly improve the cardiac function in both non-diabetic and diabetic mice after MI for both 7 days and 28 days. Moreover, application of NAD+ could markedly reduce the cardiac injury area of DM complicated MI mice. Notably, the level of NAD+ was robustly decreased in the cardiac tissue of MI mice, which was further reduced in the DM complicated mice and NAD+ administration could significantly restore the NAD+ level. Furthermore, NAD+ was verified to facilitate the angiogenesis in the MI area of both diabetic mice and non-diabetic mice by microfil perfusion assay and immunofluorescence. Additionally, we demonstrated that NAD+ promoted cardiac angiogenesis after myocardial infarction in diabetic mice by promoting the M2 polarization of macrophages. At the molecular level, NAD+ promoted the secretion of VEGF in macrophages and therefore facilitating migration and tube formation of endothelial cells. Mechanistically, NAD+ was found to promote the generation of pro-angionesis VEGF165 and inhibit the generation of anti-angionesis VEGF165b via regulating the alternative splicing factors of VEGF (SRSF1 and SRSF6) in macrophages. The effects of NAD+ were readily reversible on deficiency of it. Collectively, our data showed that NAD+ could attenuate myocardial injury via regulating the alternative splicing of VEGF and promoting angiogenesis in diabetic mice after myocardial infarction. NAD+ administration may therefore be considered a potential new approach for the treatment of diabetic patients with myocardial infarction.

LncRNA-ZFAS1 Promotes Myocardial Ischemia-Reperfusion Injury Through DNA Methylation-Mediated Notch1 Down-Regulation in Mice.

The most devastating and catastrophic deterioration of myocardial ischemia-reperfusion injury (MIRI) is cardiomyocyte death. Here we aimed to evaluate the role of lncRNA-ZFAS1 in MIRI and delineate its mechanism of action. The level of lncRNA-ZFAS1 was elevated in MIRI hearts, and artificial knockdown of lncRNA-ZFAS1 in mice improved cardiac function. Notch1 is a potential target of lncRNA-ZFAS1, and lncRNA-ZFAS1 could bind to the promoter region of Notch1 and recruit DNMT3b to induce Notch1 methylation. Nicotinamide mononucleotide could promote the expression of Notch1 by competitively inhibiting the expression of DNMT3b and improving the apoptosis of cardiomyocytes and cardiac function.

CircRNA CDR1as promotes cardiomyocyte apoptosis through activating hippo signaling pathway in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM), as a major complication of diabetic patients, can cause myocardial metabolic remodeling and lead to severe and irreversible cardiac dysfunction. Previously, we found that the circular RNA cerebellar degeneration-related protein 1 antisense (Circ-CDR1as) independently predicted acute myocardial infarction (AMI) and might be a new indicator marker for this. However, CDR1as was not clearly described in diabetic cardiomyopathy. Therefore, our purpose was to deeply explore the function of CDR1as in DCM. In this study, we found that CDR1as was upregulated in DCM, and knockdown of CDR1as could improve the apoptosis caused by DCM. Mechanistically, CDR1as activates the Hippo signaling pathway by significantly inhibiting Mammalian sterile 20-like kinase 1 (MST1) ubiquitination level. Furthermore, as a transcriptional factor of CDR1as, Forkhead box group O3a (FOXO3) was identified to activate the Hippo signaling pathway. Notably, the total m6A level was downregulated in the cardiac tissue of DCM. Alk B homolog 5 (ALKBH5), a m6A demethylation enzyme, was upregulated in the cardiomyocytes of DCM mice and posttranscriptionally activated FOXO3 by m6A demethylation in an m6A-YTHDF2-dependent manner. Hence, our work reveals the key function of the ALKBH5-FOXO3-CDR1as/Hippo signaling pathway in DCM and provides insight into the critical roles of m6A methylation in DCM.

GDF11 replenishment protects against hypoxia-mediated apoptosis in cardiomyocytes by regulating autophagy.

GDF11 has been reported to play a critical role in rejuvenating hypertrophy heart, skeletal muscle, and blood vessel regeneration in aged mice. Whether GDF11 can regulate autophagy in cardiomyocytes remains largely unknown. Thus, the purpose of the present study was to investigate the effects of GDF11 on cardiomyocyte autophagy induced by hypoxia, in addition to the underlying mechanisms. By using the MTT assay, Flow cytometry assay, LIVE/DEAD® Viability/Cytotoxicity Kit Stains and TUNEL assay, we found that exogenous GDF11 decreased apoptosis caused by prolonged hypoxia in cardiomyocytes. The expression of GDF11 was decreased obviously both in the cardiac tissue of myocardial infarction mice and the hypoxia treated cardiomyocytes. Protein levels of cleaved caspase-3, p-AMPK, SQSTM1, LC3B-I/II and GDF11 were detected by western blot. Autophagosomes and autolysosomes were identified by confocal laser microscopy after transfecting with the mRFP-eGFP-LC3 plasmids. Antibody against GDF11 (anti-GDF11) was used to inhibit the function of GDF11. At the molecular level, exogenous GDF11 increased AMPK function and enhanced autophagy activity. Anti-GDF11 inhibited autophagy and aggravated hypoxia-induced apoptosis in cardiomyocytes. Thus, GDF11 might be a potential target for myocardial infarction therapy.

Topical GDF11 accelerates skin wound healing in both type 1 and 2 diabetic mouse models.

This study aimed to investigate the role of truncated growth differentiation factor 11 (GDF11), in which the recognition site of Furin from wild-type GDF11 was deleted to enhance the cellular stability, in skin wound healing in the setting of diabetes mellitus (DM) and the underlying mechanisms. Our study found that both truncated and natural GDF11s effectively accelerated wound healing processes in both T1DM and T2DM mice with a potency compatible to PDGF, bFGF, and EGF, but being much higher than GDF8. At the cellular level, GDF11 stimulated the proliferation and suppressed HG-induced apoptosis of HSFs. Further study revealed that GDF11 activated the YAP-Smad2/3-CTGF fibrotic signaling pathway by reversing HG-induced upregulation of phosphorylated form of YAP (p-YAP), increases p-Smad2/3 levels, and restoring HG-induced repression of CTGF expression by GDF11. Overall, the study shows that both natural and truncated GDF11s promote the healing process of skin wound in mice of both T1DM and T2DM partly via stimulating dermal fibrosis via the YAP-Smad2/3-CTGF pathway, suggesting it a potential agent for treating skin wound in diabetic population.

lncRNA-ZFAS1 induces mitochondria-mediated apoptosis by causing cytosolic Ca2+ overload in myocardial infarction mice model.

Previously, we have identified ZFAS1 as a potential new long non-coding RNA (lncRNA) biomarker of acute myocardial infarction (MI) and as a sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) inhibitor, causing intracellular Ca2+ overload and contractile dysfunction in a mouse model of MI. In the current study, we aimed to evaluate the effects of ZFAS1 on the apoptosis of cardiomyocytes in the MI mouse model. Knockdown of endogenous ZFAS1 by virus-mediated silencing shRNA or siZFAS1 partially abrogated the ischemia-induced apoptosis of cardiomyocytes. Overexpression of ZFAS1 in normal cardiomyocytes reduced the cell viability, similar to that observed in hypoxia-treated cardiomyocytes. Moreover, ZFAS1 cardiac-specific knock-in mice showed impaired cardiac function, adversely altered Ca2+ homeostasis, repressed expression and activities of SERCA2a, and increased apoptosis. At the subcellular level, ZFAS1 induced mitochondrial swelling and showed a pronounced decrease in mitochondrial membrane potential. At the molecular level, ZFAS1 activated the mitochondria apoptosis pathway, which could be nearly abolished by a calcium chelator. The effects of ZFAS1 were readily reversible upon knockdown of this lncRNA. Notably, ZFAS1-FD (only functional domain) mimicked the effects of full-length ZFAS1 in regulation of cardiomyocyte apoptosis. In conclusion, our study shows that ZFAS1, an endogenous SERCA2a inhibitor, induces mitochondria-mediated apoptosis via cytosolic Ca2+ overload. Therefore, anti-ZFAS1 might be considered a new therapeutic strategy for protecting cardiomyocytes from MI-induced apoptosis.