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Aims: This study aims to explore the efficacy of punicalagin (PG) on diabetic cardiomyopathy (DCM), with a specific focus on the mechanisms underlying the effects of PG on mitochondrial fusion/fiss...

Diabetes disrupts mitochondrial function and often results in diabetic cardiomyopathy (DCM). Paeonol is a bioactive compound that has been reported to have pharmacological potential for cardiac and mitochondrial protection. This study aims to explore the effects of paeonol on mitochondrial disorderes in DCM and the underlying mechanisms. We showed that paeonol promoted Opa1-mediated mitochondrial fusion, inhibited mitochondrial oxidative stress, and preserved mitochondrial respiratory capacity and cardiac performance in DCM in vivo and in vitro. Knockdown of Opa1 blunted the above protective effects of paeonol in both diabetic hearts and high glucose-treated cardiomyocytes. Mechanistically, inhibitor screening, siRNA knockdown and chromatin immunoprecipitation experiments showed that paeonol-promoted Opa1-mediated mitochondrial fusion required the activation of Stat3, which directly bound to the promoter of Opa1 to upregulate its transcriptional expression. Moreover, pharmmapper screening and molecular docking studies revealed that CK2α served as a direct target of paeonol that interacted with Jak2 and induced the phosphorylation and activation of Jak2-Stat3. Knockdown of CK2α blunted the promoting effect of paeonol on Jak2-Stat3 phosphorylation and Opa1-mediated mitochondrial fusion. Collectively, we have demonstrated for the first time that paeonol is a novel mitochondrial fusion promoter in protecting against hyperglycemia-induced mitochondrial oxidative injury and DCM at least partially via an Opa1-mediated mechanism, a process in which paeonol interacts with CK2α and restores its kinase activity that subsequently increasing Jak2-Stat3 phosphorylation and enhancing the transcriptional level of Opa1. These findings suggest that paeonol or the promotion of mitochondrial fusion might be a promising strategy for the treatment of DCM., Graphical abstract Image 1, Highlights • Paeonol is identified as a novel mitochondrial fusion promoter against diabetic cardiomyopathy. • Paeonol promotes Opa1-mediated mitochondrial fusion in a Stat3-dependent transcriptional manner. • CK2α serves as a direct target of Paeonol that directly activates the Jak2–Stat3 signaling pathway.

This study aims to explore the efficacy of punicalagin on diabetic cardiomyopathy, with a specific focus on the mechanisms underlying the effects of punicalagin on mitochondrial fusion/fission dynamics. Diabetes was induced by a single intraperitoneal injection of streptozotocin intraperitoneally in Sprague-Dawley rats. Cardiac structural and functional abnormalities were ameliorated in diabetic rats receiving punicalagin administration as evidenced by increased ejection fraction and attenuated myocardial fibrosis and hypertrophy. Punicalagin promoted mitochondrial fusion and enhanced mitochondrial function in both diabetic hearts and high glucose-treated cardiomyocytes. Punicalagin-induced increases in Opa1 expression were responsible for the above mitochondrial enhancements, and these changes could be blocked by knockdown of Opa1. Inhibitor screening and chromatin immunoprecipitation analysis showed that Stat3 directly regulated the transcriptional expression of Opa1 by binding to its promoter and was responsible for punicalagin-induced Opa1-mediated mitochondrial fusion. Moreover, pharmmapper screening and molecular docking studies revealed that punicalagin embedded into the activity pocket of PTP1B and inhibited the activity of PTP1B. Overexpression of PTP1B blocked the promoting effect of punicalagin on Stat3 phosphorylation and Opa1-mediated mitochondrial fusion. Collectively, our study has provided the first evidence that punicalagin protects against diabetic cardiomyopathy and uncovered the precise mechanisms on how punicalagin promotes Opa1-mediated mitochondrial fusion under hyperglycemic condition, a process in which punicalagin interacts with PTP1B and inhibits its activity that in turn increasing Stat3 phosphorylation and then enhancing the transcriptional expression of Opa1. Disclosure M. Ding: None. M. Li: None. F. Fu: None. C. Liu: None. Funding National Natural Science Foundation of China (81970316, 82070387)

Ischemic post-conditioning (IPO) activates Akt signaling to confer cardioprotection. The responsiveness of diabetic hearts to IPO is impaired. We hypothesized that decreased cardiac SIRT1, a positive regulator of Akt, may be responsible for the impaired responsiveness of diabetic hearts to IPO-mediated cardioprotection. High-fat diet and streptozotocin-induced diabetic mice were subjected to myocardial ischemia/reperfusion (MI/R, 30 min ischemia and 180 min reperfusion) or IPO (three cycles of 10 s of reperfusion and ischemia at the onset of reperfusion). Adenoviral vectors encoding GFP or SIRT1 (Ad-SIRT1) were administered by direct injection into the left ventricular. Our results showed that IPO activated the Akt signaling pathway and reduced MI/R injury in non-diabetic hearts but not in diabetic hearts, in which reduced expression of SIRT1 and increased Akt acetylation were observed. Delivery of Ad-SIRT1 into the diabetic hearts reduced Akt acetylation and restored the cardioprotective effects of IPO by modulating Akt signaling pathway. In contrast, cardiac-specific SIRT1 knockout increased Akt acetylation and blunted the cardioprotective effects of IPO. In in vitro study, transfection with wild-type SIRT1 but not inactive mutant SIRT1 reduced the expression of Akt acetylation and restored the protective effects of hypoxic post-conditioning in high glucose-incubated cardiomyocytes. Moreover, the cardiomyocytes transfected with constitutive Akt acetylation showed repressed Akt phosphorylation and blunted protective effects against hypoxia/reoxygenation injury. These findings demonstrate that the reduction of SIRT1 blunts the protective effects of IPO by impairing Akt signaling pathway and that SIRT1 up-regulation restores IPO-mediated cardioprotection in diabetic mice via deacetylation-dependent activation of Akt signaling pathway.

Aim Imbalanced mitochondrial dynamics including suppressed mitochondrial fusion has been observed in diabetic hearts. However, it is still unknown whether mitochondrial fusion promoter is an effective protection to diabetic hearts. This study was designed to explore the efficacy of mitochondrial fusion promoter on diabetic cardiomyopathy (DCM). Methods Male Sprague-Dawley rats were injected with streptozotocin (STZ, 65 mg/kg/d) intraperitoneally to induce diabetes. Seven weeks after vehicle or STZ injection, control or diabetic rats were treated with the vehicle or a mitochondrial fusion promoter-M1 (2 mg/kg/d) intraperitoneally for 6 weeks. Moreover, M1 was administrated to the primary cardiomyocytes cultured in normal glucose medium (NG, 5.5 mmol/L) or high glucose (HG, 33 mnol/L). Results Administration of M1 significantly promoted mitochondrial fusion and attenuated the reduction in optic atrophy 1 (Opa1) expression in diabetic hearts. Importantly, M1 treatment attenuated oxidative stress, improved mitochondrial function and alleviated DCM in diabetic rats. In HG-treated cardiomyocytes, M1 treatment consistently increased the expression of Opa1, promoted mitochondrial fusion, enhanced mitochondrial respiratory capacity and reduced mitochondria-derived superoxide production, all of which were blunted by Opa1 siRNA knockdown. In addition, selective upregulation of Opa1 alone can also promote mitochondrial fusion, improve mitochondrial function and inhibit mitochondria-derived superoxide production in HG-cultured cardiomyocytes. Conclusion Our findings show for the first time that mitochondrial fusion promoter M1 effectively balances mitochondrial dynamics and protects against diabetic cardiomyopathy (DCM) via an Opa1-dependent way, suggesting that promoting mitochondrial fusion might be a potential therapeutic strategy for DCM.

Ischemic post-conditioning (IPO) activates Akt signaling to confer cardioprotection. The responsiveness of diabetic hearts to IPO is impaired. We hypothesized that decreased cardiac SIRT1, a positive regulator of Akt, may be responsible for the impaired responsiveness of diabetic hearts to IPO-mediated cardioprotection. High-fat diet and streptozotocin-induced diabetic mice were subjected to myocardial ischemia/reperfusion (MI/R, 30 minutes ischemia and 180 minutes reperfusion) or IPO (three cycles of 10 s of reperfusion and ischemia at the onset of reperfusion). Adenoviral vectors encoding GFP or SIRT1 (Ad-SIRT1) were administered by direct injection into the left ventricular. IPO activated the Akt signaling pathway and reduced MI/R injury in nondiabetic hearts but not in diabetic hearts, in which reduced expression of SIRT1 and increased Akt acetylation were observed. Delivery of Ad-SIRT1 into the diabetic hearts reduced Akt acetylation and restored the cardioprotective effects of IPO. In in vitro study, transfection with wild type SIRT1 but not inactive mutant SIRT1 reduced the expression of Akt acetylation and restored the protective effects of hypoxic post-conditioning in high glucose-incubated primary cardiomyocytes. Moreover, the cardiomyocytes transfected with Akt acetylation-defective mutant (K20R) showed increased Akt phosphorylation and enhanced protective effects against hypoxia/reoxygenation injury. These findings demonstrate that SIRT1 up-regulation restores IPO-mediated cardioprotection in diabetic mice via deacetylation-dependent activation of Akt signaling pathway. It is suggested that targeting up-regulation of SIRT1 may be a potential therapeutic strategy to restore myocardial responsiveness to post-conditioning in diabetic patients. Disclosure M. Ding: None. K. Zeng: None. M. Yu: None. C. Liu: None. F. Fu: None. Funding National Natural Science Foundation of China (81600235, 81670354); Natural Science Foundation of Shaanxi (2018JM7061)

Increasing evidence has implicated the important role of mitochondrial pathology in diabetic cardiomyopathy (DCM), while the underlying mechanism remains largely unclear. The aim of this study was to investigate the role of mitochondrial dynamics in the pathogenesis of DCM and its underlying mechanisms. Methods: Obese diabetic (db/db) and lean control (db/+) mice were used in this study. Mitochondrial dynamics were analyzed by transmission electron microscopy in vivo and by confocal microscopy in vitro. Results: Diabetic hearts from 12-week-old db/db mice showed excessive mitochondrial fission and significant reduced expression of Mfn2, while there was no significant alteration or slight change in the expression of other dynamic-related proteins. Reconstitution of Mfn2 in diabetic hearts inhibited mitochondrial fission and prevented the progression of DCM. In an in-vitro study, cardiomyocytes cultured in high-glucose and high-fat (HG/HF) medium showed excessive mitochondrial fission and decreased Mfn2 expression. Reconstitution of Mfn2 restored mitochondrial membrane potential, suppressed mitochondrial oxidative stress and improved mitochondrial function in HG/HF-treated cardiomyocytes through promoting mitochondrial fusion. In addition, the down-regulation of Mfn2 expression in HG/HF-treated cardiomyocytes was induced by reduced expression of PPARα, which positively regulated the expression of Mfn2 by directly binding to its promoter. Conclusion: Our study provides the first evidence that imbalanced mitochondrial dynamics induced by down-regulated Mfn2 contributes to the development of DCM. Targeting mitochondrial dynamics by regulating Mfn2 might be a potential therapeutic strategy for DCM.

Identify the molecular basis linking diabetes with MI/R injury is scientifically important and may provide effective therapeutic approaches. Recent studies indicated that enhanced Dynamin-related protein 1 (Drp1)-mediated mitochondrial fission is involved in both diabetic hearts and ischemic hearts. We hypothesized that inhibition of Drp1-mediated mitochondrial fission may be effective to reduce MI/R injury in diabetic hearts. High-fat diet and streptozotocin-induced diabetic mice were subjected to MI/R or sham operation. Mdivi-1 (1.2 mg/kg), a small molecule inhibitor of Drp1 or vehicle was administrated 15 min before the onset of reperfusion. Outcome measures included mitochondrial morphology, mitochondrial function, myocardial injury, cardiac function and oxidative stress. Mitochondrial fission was significantly increased following MI/R as evidenced by enhanced translocation of Drp1 to mitochondria and decreased mitochondrial size. Delivery of Mdivi-1 into diabetic mice markedly inhibited Drp1 translocation to the mitochondria and reduced mitochondrial fission following MI/R. Inhibition of Drp1-mediated mitochondrial fission increased mitochondrial complex I/II/III/IV activities and ATP content and improved cardiac function following MI/R (n = 8, P < 0.01). Moreover, inhibition of Drp1 reduced myocardial infarct size (28.7 ± 4.2% vs. 45.3 ± 4.7% n = 8, P < 0.01) and serum cardiac troponin and lactate dehydrogenase activities. These cardioprotective effects were associated with decreased cardiomyocyte apoptosis and malondialdehyde production and increased activities of antioxidant enzyme manganese superoxide dismutase (n = 8, P < 0.01). These data suggest that Drp1-mediated mitochondrial fission may be a potential novel therapeutic target for diabetic cardiac complications. Disclosure M. Ding: None. J. Feng: None. Z. Li: None. F. Fu: None.

Mechanical trauma (MT) causes myocardial injury and cardiac dysfunction. However, the underlying mechanism remains largely unclear. This study investigated the role of mitochondrial dynamics in post-traumatic cardiac dysfunction and the protective effects of melatonin. Adult male Sprague Dawley rats were subjected to 5-minute rotations (200 revolutions at a rate of 40 rpm) to induce MT model. Melatonin was administrated intraperitoneally 5 minute after MT. Mitochondrial morphology, myocardial injury, and cardiac function were determined in vivo. There was smaller size of mitochondria and increased number of mitochondria per μm2 in the hearts after MT when the secondary myocardial injury was induced. Melatonin treatment at the dose of 30 mg/kg reduced serine 616 phosphorylation of Drp1 and inhibited mitochondrial Drp1 translocation and mitochondrial fission in the hearts of rats subjected to MT, which contributed to the reduction of myocardial injury and the improvement of cardiac function. In vitro, H9c2 cells cultured in 20% traumatic plasma (TP) for 12 hour showed enhanced mitochondrial fission, mitochondrial membrane potential (∆Ψm) loss, mitochondrial cytochrome c release, and decreased mitochondrial complex I-IV activities. Pretreatment with melatonin (100 μmol/L) efficiently inhibited TP-induced mitochondrial fission, ∆Ψm loss, cytochrome c release, and improved mitochondrial function. Melatonin's protective effects were attributed to its role in suppressing plasma TNF-α overproduction, which was responsible for Drp1-mediated mitochondrial fission. Taken together, our results demonstrate for the first time that abnormal mitochondrial dynamics is involved in post-traumatic cardiac dysfunction. Melatonin has significant pharmacological potential in protecting against MT-induced cardiac dysfunction by preventing excessive mitochondrial fission.

Punicalagin (PUN), a major bioactive component in pomegranate juice, has been proven to exert neuroprotective effects against cerebral ischemia/reperfusion (I/R) insult via anti-oxidant properties. This study aims to investigate whether PUN provides cardioprotection against myocardial I/R (MI/R) injury and the underlying mechanisms. PUN (30[Formula: see text]mg/kg/d) or vehicle was intragastrically administered to Sprague-Dawley rats for one week before the operation. MI/R was induced by ligating the left anterior descending coronary artery for 30[Formula: see text]min and subsequent reperfusion for 3[Formula: see text]h. PUN pretreatment conferred cardioprotective effects against MI/R injury by improving cardiac function, limiting infarct size, reducing serum creatine kinase-MB and lactate dehydrogenase activities, and suppressing cardiomyocyte apoptosis. Moreover, PUN pretreatment inhibited I/R-induced myocardial oxidative stress as evidenced by decreased generation of superoxide content and malonaldialdehyde formation and increased antioxidant capability. Furthermore, PUN pretreatment increased adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in I/R hearts. AMPK inhibitor compound c inhibited PUN-enhanced AMPK phosphorylation, and blunted PUN-mediated anti-oxidative effects and cardioprotection. These results indicate for the first time that PUN pretreatment protect against I/R-induced oxidative stress and myocardial injury via activation of AMPK.

Myocardial contractile dysfunction is associated with an increase in mitochondrial fission in patients with diabetes. However, whether mitochondrial fission directly promotes diabetes‐induced cardiac dysfunction is still unknown. Melatonin exerts a substantial influence on the regulation of mitochondrial fission/fusion. This study investigated whether melatonin protects against diabetes‐induced cardiac dysfunction via regulation of mitochondrial fission/fusion and explored its underlying mechanisms. Here, we show that melatonin prevented diabetes‐induced cardiac dysfunction by inhibiting dynamin‐related protein 1 (Drp1)‐mediated mitochondrial fission. Melatonin treatment decreased Drp1 expression, inhibited mitochondrial fragmentation, suppressed oxidative stress, reduced cardiomyocyte apoptosis, improved mitochondrial function and cardiac function in streptozotocin (STZ)‐induced diabetic mice, but not in SIRT1−/− diabetic mice. In high glucose‐exposed H9c2 cells, melatonin treatment increased the expression of SIRT1 and PGC‐1α and inhibited Drp1‐mediated mitochondrial fission and mitochondria‐derived superoxide production. In contrast, SIRT1 or PGC‐1α siRNA knockdown blunted the inhibitory effects of melatonin on Drp1 expression and mitochondrial fission. These data indicated that melatonin exerted its cardioprotective effects by reducing Drp1‐mediated mitochondrial fission in a SIRT1/PGC‐1α‐dependent manner. Moreover, chromatin immunoprecipitation analysis revealed that PGC‐1α directly regulated the expression of Drp1 by binding to its promoter. Inhibition of mitochondrial fission with Drp1 inhibitor mdivi‐1 suppressed oxidative stress, alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. These findings show that melatonin attenuates the development of diabetes‐induced cardiac dysfunction by preventing mitochondrial fission through SIRT1‐PGC1α pathway, which negatively regulates the expression of Drp1 directly. Inhibition of mitochondrial fission may be a potential target for delaying cardiac complications in patients with diabetes.

Background Diabetic patients are more sensitive to myocardial ischemic injury than non-diabetic patients. Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent histone deacetylase making the heart more resistant to ischemic injury. As SIRT1 expression is considered to be reduced in diabetic heart, we therefore hypothesized that up-regulation of SIRT1 in the diabetic heart may overcome its increased susceptibility to ischemic injury. Methods Male Sprague–Dawley rats were fed with high-fat diet and injected with streptozotocin once to induce diabetes. Diabetic rats received injections of adenoviral vectors encoding SIRT1 (Ad-SIRT1) at five myocardial sites. Four days after adenoviral injection, the rats were subjected to myocardial ischemia and reperfusion (MI/R). Outcome measures included left ventricular function, infarct size, cellular death and oxidative stress. Results Delivery of Ad-SIRT1 into the hearts of diabetic rats markedly increased SIRT1 expression. Up-regulation of SIRT1 in diabetic hearts improved cardiac function and reduced infarct size to the extent as in non-diabetic animals following MI/R, which was associated with reduced serum creatine kinase-MB, lactate dehydrogenase activities and cardiomyocyte apoptosis. Moreover, Ad-SIRT1 reduced the increase in the superoxide generation and malonaldialdehyde content and simultaneously increased the antioxidant capability. Furthermore, Ad-SIRT1 increased eNOS phosphorylation and reduced eNOS acetylation in diabetic hearts. NOS inhibitor L-NAME inhibited SIRT1-enhanced eNOS phosphorylation, and blunted SIRT1-mediated anti-apoptotic and anti-oxidative effects and cardioprotection. Conclusions Overexpression of SIRT1 reduces diabetes-exacerbated MI/R injury and oxidative stress via activating eNOS in diabetic rats. The findings suggest SIRT1 may be a promising novel therapeutic target for diabetic cardiac complications.

Background The hormone adiponectin (APN) circulates in plasma as various multimeric complexes. The high-molecular-weight (HMW) isoform has been reported to exert the most favourable metabolic regulatory and vasculoprotective effects. This study determined the circulatory distribution of APN multimers and their relationships with cardiovascular disease (CVD)-related biochemical indicators in patients with hypercholesterolaemia (HC). Methods A total of 148 male age- and BMI-matched patients with HC (80 with CVD and 68 without CVD) and 84 male healthy controls were enrolled. Diabetes mellitus, hypertension, nephropathy and cigarette use constituted exclusion criteria. Results Both HMW and medium-molecular-weight (MMW) forms of APN were significantly increased in HC without CVD (HMW: 4·98 ± 0·87 vs 2·51 ± 0·33 in control, P

Many cardioprotective pharmacological agents failed to exert their protective effects in diabetic hearts subjected to myocardial ischemia/reperfusion (MI/R). Identify the molecular basis linking diabetes with MI/R injury is scientifically important and may provide effective therapeutic approaches. Dynamin-related protein 1 (Drp1)-mediated mitochondrial fission plays an important role in MI/R injury under non-diabetic conditions. Importantly, recent studies indicated that Drp1-mediated mitochondrial fission is enhanced in the myocardium of diabetic mice. The above evidences suggested that Drp1 may be one critical molecule linking diabetes with MI/R injury. We hypothesized that inhibition of Drp1 may be effective to reduce MI/R injury in diabetic hearts. High-fat diet and streptozotocin-induced diabetic mice were subjected to MI/R or sham operation. Mdivi-1 (1.2 mg/kg), a small molecule inhibitor of Drp1 or vehicle was administrated 15 min before the onset of reperfusion. Outcome measures included mitochondrial morphology, mitochondrial function, myocardial injury, cardiac function and oxidative stress. Mitochondrial fission was significantly increased following MI/R as evidenced by enhanced translocation of Drp1 to mitochondria and decreased mitochondrial size. Delivery of Mdivi-1 into diabetic mice markedly inhibited Drp1 translocation to the mitochondria and reduced mitochondrial fission following MI/R. Inhibition of Drp1 in diabetic hearts improved mitochondrial function and cardiac function following MI/R. Moreover, inhibition of Drp1 reduced myocardial infarct size and serum cardiac troponin I and lactate dehydrogenase activities. These cardioprotective effects were associated with decreased cardiomyocyte apoptosis and malondialdehyde production and increased activities of antioxidant enzyme manganese superoxide dismutase. Pharmacological inhibition of Drp1 prevents mitochondrial fission and reduces MI/R injury in diabetic mice. The findings suggest Drp1 may be a potential novel therapeutic target for diabetic cardiac complications.