Background: Diabetes mellitus exacerbates myocardial ischemia/reperfusion (MI/R) injury by incompletely understood mechanisms. Adipocyte dysfunction contributes to remote organ injury. However, the molecular mechanisms linking dysfunctional adipocytes to increased MI/R injury remain unidentified. The current study attempted to clarify whether and how small extracellular vesicles (sEV) may mediate pathological communication between diabetic adipocytes and cardiomyocytes, exacerbating MI/R injury. Methods: Adult male mice were fed a normal or a high-fat diet for 12 weeks. sEV (from diabetic serum, diabetic adipocytes, or high glucose/high lipid-challenged nondiabetic adipocytes) were injected intramyocardially distal of coronary ligation. Animals were subjected to MI/R 48 hours after injection. Results: Intramyocardial injection of diabetic serum sEV in the nondiabetic heart significantly exacerbated MI/R injury, as evidenced by poorer cardiac function recovery, larger infarct size, and greater cardiomyocyte apoptosis. Similarly, intramyocardial or systemic administration of diabetic adipocyte sEV or high glucose/high lipid-challenged nondiabetic adipocyte sEV significantly exacerbated MI/R injury. Diabetic epididymal fat transplantation significantly increased MI/R injury in nondiabetic mice, whereas administration of a sEV biogenesis inhibitor significantly mitigated MI/R injury in diabetic mice. A mechanistic investigation identified that miR-130b-3p is a common molecule significantly increased in diabetic serum sEV, diabetic adipocyte sEV, and high glucose/high lipid-challenged nondiabetic adipocyte sEV. Mature (but not primary) miR-130b-3p was significantly increased in the diabetic and nondiabetic heart subjected to diabetic sEV injection. Whereas intramyocardial injection of a miR-130b-3p mimic significantly exacerbated MI/R injury in nondiabetic mice, miR-130b-3p inhibitors significantly attenuated MI/R injury in diabetic mice. Molecular studies identified AMPKα1/α2, Birc6, and Ucp3 as direct downstream targets of miR-130b-3p. Overexpression of these molecules (particularly AMPKα2) reversed miR-130b-3p induced proapoptotic/cardiac harmful effect. Finally, miR-130b-3p levels were significantly increased in plasma sEV from patients with type 2 diabetes mellitus. Incubation of cardiomyocytes with diabetic patient sEV significantly exacerbated ischemic injury, an effect blocked by miR-130b-3p inhibitor. Conclusions: We demonstrate for the first time that miR-130b-3p enrichment in dysfunctional adipocyte-derived sEV, and its suppression of multiple antiapoptotic/cardioprotective molecules in cardiomyocytes, is a novel mechanism exacerbating MI/R injury in the diabetic heart. Targeting miR-130b-3p mediated pathological communication between dysfunctional adipocytes and cardiomyocytes may be a novel strategy attenuating diabetic exacerbation of MI/R injury.

Objective—: Reduced plasma adiponectin (APN) in diabetic patients is associated with endothelial dysfunction. However, APN knockout animals manifest modest systemic dysfunction unless metabolically challenged. The protein family CTRPs (C1q/TNF-related proteins) has recently been identified as APN paralogs and some CTRP members share APN′s metabolic regulatory function. However, the vasoactive properties of CTRPs remain completely unknown. Methods and Results—: The vasoactivity of currently identified murine CTRP members was assessed in aortic vascular rings and underlying molecular mechanisms was elucidated in human umbilical vein endothelial cells. Of 8 CTRPs, CTRPs 3, 5, and 9 caused significant vasorelaxation. The vasoactive potency of CTRP9 exceeded that of APN (3-fold) and is endothelium-dependent and nitric oxide (NO)-mediated. Mechanistically, CTRP9 increased AMPK/Akt/eNOS phosphorylation and increased NO production. AMPK knockdown completely blocked CTRP9-induced Akt/eNOS phosphorylation and NO production. Akt knockdown had no significant effect on CTRP9-induced AMPK phosphorylation, but blocked eNOS phosphorylation and NO production. Adiponectin receptor 1, but not receptor 2, knockdown blocked CTRP9-induced AMPK/Akt/eNOS phosphorylation and NO production. Finally, preincubating vascular rings with an AMPK-inhibitor abolished CTRP9-induced vasorelaxative effects. Conclusion—: We have provided the first evidence that CTRP9 is a novel vasorelaxative adipocytokine that may exert vasculoprotective effects via the adiponectin receptor 1/AMPK/eNOS dependent/NO mediated signaling pathway.

Emerging evidence indicates that nuclear receptors play a critical regulatory role in cardiovascular physiology/pathology. Recently, farnesoid-X-receptor (FXR), a member of the metabolic nuclear receptor superfamily, has been demonstrated to be expressed in vascular cells, with important roles in vascular physiology/pathology. However, the potential cardiac function of FXR remains unclear. We investigated the cardiac expression and biological function of FXR. Farnesoid-X-receptor was detected in both isolated neonatal rat cardiac myocytes and fibroblasts. Natural and synthetic FXR agonists upregulated cardiac FXR expression, stimulated myocyte apoptosis, and reduced myocyte viability dose- and time-dependently. Mechanistic studies demonstrated that FXR agonists disrupted mitochondria, characterized by mitochondrial permeability transition pores activation, mitochondrial potential dissipation, cytochrome c release, and both caspase-9 and -3 activation. Such mitochondrial apoptotic responses were abolished by siRNA-mediated silencing of endogenous FXR or pharmacological inhibition of mitochondrial death signalling. Furthermore, low levels of FXR were detected in the adult mouse heart, with significant (∼2.0-fold) upregulation after myocardial ischaemia/reperfusion (MI/R). Pharmacological inhibition or genetic ablation of FXR significantly reduced myocardial apoptosis by 29.0–53.4%, decreased infarct size by 23.4–49.7%, and improved cardiac function in ischaemic/reperfused myocardium. These results demonstrate that nuclear receptor FXR acts as a novel functional receptor in cardiac tissue, regulates apoptosis in cardiomyocytes, and contributes to MI/R injury.

Apoptosis contributes to myocardial ischemia/reperfusion (MI/R) injury, and both thioredoxin (Trx) and nitric oxide have been shown to exert antiapoptotic effectsin vitro. Recent evidence suggests that this particular action of Trx requires S-nitrosation at Cys-69. The present study sought to investigate whether or not exogenously applied Trx reduces MI/R injuryin vivoand to which extent this effect depends on S-nitrosation. Adult mice were subjected to 30 min of MI and treated with either vehicle or human Trx (hTrx, 2 mg/kg, i.p.) 10 min before reperfusion. Native hTrx was incorporated into myocardial tissue as shown by immunostaining, and reduced MI/R injury as evidenced by decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, DNA fragmentation, caspase-3 activity, and infarct size. When hTrx was partially S-nitrosated by preincubation withS-nitrosoglutathione, its cardioprotective effect was markedly enhanced. Treatment with hTrx significantly reduced p38 mitogen-activated protein kinase (MAPK) activity, and this effect was also potentiated by S-nitrosation. To further address the role of S-nitrosation for the overall antiapoptotic effect to Trx, the action ofEscherichia coliTrx (eTrx) was investigated in the same model. Whereas eTrx inhibited MI/R-induced apoptosis to a degree similar to hTrx, S-nitrosation of this protein, which lacks Cys-69, failed to further enhance its antiapoptotic action. Collectively, our results demonstrate that systemically applied Trx is taken up by the myocardium to exert potent cardioprotective effectsin vivo, offering interesting therapeutic avenues. In the case of hTrx, these effects are further potentiated by S-nitrosation, but this posttranslational modification is not essential for protection.

Background—: Obesity and diabetes mellitus adversely affect postischemic heart remodeling via incompletely understood mechanisms. C1q/tumor necrosis factor–related protein-3 (CTRP3) is a newly identified adipokine exerting beneficial metabolic regulation, similar to adiponectin. The aim of the present study was to determine whether CTRP3 may regulate postischemic cardiac remodeling and cardiac dysfunction, and, if so, to elucidate the underlying mechanisms. Methods and Results—: Male adult mice were subjected to myocardial infarction (MI) via left anterior descending coronary artery occlusion. Both the effect of MI on endogenous CTRP3 expression/production and the effect of exogenous CTRP3 (adenovirus or recombinant CTRP3) replenishment on MI injury were investigated. MI significantly inhibited adipocyte CTRP3 expression and reduced the plasma CTRP3 level, reaching a nadir 3 days after MI. CTRP3 replenishment improved survival rate ( P <0.05), restored cardiac function, attenuated cardiomyocyte apoptosis, increased revascularization, and dramatically reduced interstitial fibrosis (all P <0.01). CTRP3 replenishment had no significant effect on cardiac AMP-activated protein kinase phosphorylation but significantly increased Akt phosphorylation and expression of hypoxia inducing factor-1α and vascular endothelial growth factor. Surprisingly, treatment of human umbilical vascular endothelial cells with CTRP3 did not directly affect nitric oxide production or tube formation. However, preconditioned medium from CTRP3-treated cardiomyocytes significantly enhanced human umbilical vascular endothelial cell tube formation, an effect blocked by either pretreatment of cardiomyocytes with a PI3K inhibitor or pretreatment of human umbilical vascular endothelial cells with a vascular endothelial growth factor inhibitor. Finally, the protective effect of adipocyte-conditioned medium against hypoxia-induced cardiomyocyte injury is significantly blunted when CTRP3 is knocked down. Conclusion—: CTRP3 is a novel antiapoptotic, proangiogenic, and cardioprotective adipokine, the expression of which is significantly inhibited after MI.