Background: Myocardial insulin resistance is a hallmark of diabetic cardiac injury. However, the underlying molecular mechanisms remain unclear. Recent studies demonstrate that the diabetic heart is resistant to other cardioprotective interventions, including adiponectin and preconditioning. The “universal” resistance to multiple therapeutic interventions suggests impairment of the requisite molecule(s) involved in broad prosurvival signaling cascades. Cav (Caveolin) is a scaffolding protein coordinating transmembrane signaling transduction. However, the role of Cav3 in diabetic impairment of cardiac protective signaling and diabetic ischemic heart failure is unknown. Methods: Wild-type and gene-manipulated mice were fed a normal diet or high-fat diet for 2 to 12 weeks and subjected to myocardial ischemia and reperfusion. Insulin cardioprotection was determined. Results: Compared with the normal diet group, the cardioprotective effect of insulin was significantly blunted as early as 4 weeks of high-fat diet feeding (prediabetes), a time point where expression levels of insulin-signaling molecules remained unchanged. However, Cav3/insulin receptor-β complex formation was significantly reduced. Among multiple posttranslational modifications altering protein/protein interaction, Cav3 (not insulin receptor-β) tyrosine nitration is prominent in the prediabetic heart. Treatment of cardiomyocytes with 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride reduced the signalsome complex and blocked insulin transmembrane signaling. Mass spectrometry identified Tyr ⁷³ as the Cav3 nitration site. Phenylalanine substitution of Tyr ⁷³ (Cav3 ^Y73F ) abolished 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride–induced Cav3 nitration, restored Cav3/insulin receptor-β complex, and rescued insulin transmembrane signaling. It is most important that adeno-associated virus 9–mediated cardiomyocyte-specific Cav3 ^Y73F reexpression blocked high-fat diet–induced Cav3 nitration, preserved Cav3 signalsome integrity, restored transmembrane signaling, and rescued insulin-protective action against ischemic heart failure. Last, diabetic nitrative modification of Cav3 at Tyr ⁷³ also reduced Cav3/AdipoR1 complex formation and blocked adiponectin cardioprotective signaling. Conclusions: Nitration of Cav3 at Tyr ⁷³ and resultant signal complex dissociation results in cardiac insulin/adiponectin resistance in the prediabetic heart, contributing to ischemic heart failure progression. Early interventions preserving Cav3-centered signalsome integrity is an effective novel strategy against diabetic exacerbation of ischemic heart failure.

Background: The proliferative capacity of cardiomyocytes in adult mammalian hearts is far too low to replace the cells that are lost to myocardial infarction. Both cardiomyocyte proliferation and myocardial regeneration can be improved via the overexpression of a constitutively active variant of YAP5SA (Yes‐associated protein, 5SA [active] mutant), but persistent overexpression of proliferation‐inducing genes could lead to hypertrophy and arrhythmia, whereas off‐target expression in fibroblasts and macrophages could increase fibrosis and inflammation. Methods and Results: Transient overexpression of YAP5SA or GFP (green fluorescent protein; control) was targeted to cardiomyocytes via our cardiomyocyte‐specific modified mRNA translation system ( ^YAP5SA CM‐SMRTs or ^GFP CM‐SMRTs, respectively). YAP5SA‐cardiomyocyte specificity was confirmed via in vitro experiments in cardiomyocytes and cardiac fibroblasts that had been differentiated from human induced‐ pluripotent stem cells and in human umbilical‐vein endothelial cells, and the regenerative potency of ^YAP5SA CM‐SMRTs was evaluated in a mouse myocardial infarction model. In cultured human induced‐pluripotent stem cells‐cardiomyocytes, YAP was abundantly expressed for 3 days after ^YAP5SA CM‐SMRTs administration and was accompanied by increases in the expression of markers for proliferation, before declining to near‐background levels after day 7, whereas GFP fluorescence remained high from days 1 to 3 after ^GFP CM‐SMRTs treatment and then slowly declined. GFP fluorescence was also observed in human induced‐pluripotent stem cells‐cardiac fibroblasts and human umbilical‐vein endothelial cells on day 1 after ^GFP CM‐SMRTs administration but declined substantially by day 3. In the mouse myocardial infarction model, echocardiographic assessments of left‐ventricular ejection fraction and fractional shortening were significantly greater, whereas infarct sizes were significantly smaller in ^YAP5SA CM‐SMRTs–treated mice than in vehicle‐treated control animals, and ^YAP5SA CM‐SMRTs appeared to promote cardiomyocyte proliferation. Conclusions: The CM‐SMRTs can be used to transiently and specifically overexpress YAP5SA in cardiomyocytes, and this treatment strategy significantly promoted cardiomyocyte proliferation and myocardial regeneration in a mouse myocardial infarction model.

BACKGROUND: APN (adiponectin) and APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) are potent vasculoprotective molecules, and their deficiency (eg, hypoadiponectinemia) contributes to diabetic vascular complications. However, the molecular mechanisms that govern their vasculoprotective genes as well as their alteration by diabetes remain unknown. METHODS: Diabetic medium-cultured rat aortic endothelial cells, mouse aortic endothelial cells from high-fat-diet animals, and diabetic human aortic endothelial cells were used for molecular/cellular investigations. The in vivo concept-prove demonstration was conducted using diabetic vascular injury and diabetic hindlimb ischemia models. RESULTS: In vivo animal experiments showed that APN replenishment caused APPL1 nuclear translocation, resulting in an interaction with HDAC (histone deacetylase) 2, which inhibited HDAC2 activity and increased H3Kac27 levels. Based on transcriptionome pathway-specific real-time polymerase chain reaction profiling and bioinformatics analysis, Angpt1 (angiopoietin 1), Ocln (occludin), and Cav1 (caveolin 1) were found to be the top 3 vasculoprotective genes suppressed by diabetes and rescued by APN in an APPL1-dependent manner. APN reverses diabetes-induced inhibition of Cav1 interaction with APPL1. APN-induced Cav1 expression was not affected by Angpt1 or Ocln deficiency, whereas APN-induced APPL1 nuclear translocation or upregulation of Angpt1/Ocln expression was abolished in the absence of Cav1 both in vivo and in vitro, suggesting Cav1 is upstream molecule of Angpt1/Ocln in response to APN administration. Chromatin immunoprecipitation–qPCR (quantitative polymerase chain reaction) demonstrated that APN caused significant enrichment of H3K27ac in Angpt1 and Ocln promoter region, an effect blocked by APPL1/Cav1 knockdown or HDAC2 overexpression. The protective effects of APN on the vascular system were attenuated by overexpression of HDAC2 and abolished by knocking out APPL1 or Cav1. The double knockdown of ANGPT1/OCLN blunted APN vascular protection both in vitro and in vivo. Furthermore, in diabetic human endothelial cells, HDAC2 activity is increased, H3 acetylation is decreased, and ANGPT1/OCLN expression is reduced, suggesting that the findings have important translational implications. CONCLUSIONS: Hypoadiponectinemia and dysregulation of APPL1-mediated epigenetic regulation are novel mechanisms leading to diabetes-induced suppression of vasculoprotective gene expression. Diabetes-induced pathological vascular remodeling may be prevented by interventions promoting APPL1 nuclear translocation and inhibiting HDAC2.

BACKGROUND: Mortality from acute myocardial infarction (MI) has declined significantly in the past decade for nondiabetic patients. However, both morbidity and mobility of ischemic heart failure (IHF) persistently escalate in the diabetic population via incompletely understood mechanisms. Recent studies demonstrated that small extracellular vesicles (sEVs) released from nondiabetic and diabetic adipocytes (ADps) exert opposite effects on acute myocardial ischemia and reperfusion (MI/R) injury. However, whether and how ADp sEVs may protect against post-MI remodeling and IHF, and more important, whether and how diabetes may impair this protective effect, remain unknown. METHODS: sEVs were isolated from epididymal fat pads of nondiabetic animals and intramyocardially injected in nondiabetic or diabetic hearts subjected to MI (90 minutes of MI followed by 4 weeks of reperfusion). RESULTS: sEV treatment significantly attenuated post-MI cardiac remodeling and improved cardiac function in nondiabetic mice. However, the protection was not observed in diabetic hearts. In adult cardiomyocytes isolated from nondiabetic hearts, sEVs rapidly (15 minutes) activated cell salvage kinases (ERK [extracellular signal-regulated kinase], AMPK [AMP-activated protein kinase], and ACC [acetyl-CoA carboxylase]) and suppressed oxidative stress-induced cell death, suggesting sEV external surface molecules are responsible for the observed cytoprotection. The Exo-Flow (a technology detecting sEV external surface molecules) demonstrated that adiponectin (APN) is enriched on the sEV external surface. The sEVs from APN knockout mice or APN neutralization (NU) antibody pretreated sEVs from the WT mice failed to protect the heart against IHF. Moreover, the cardioprotective effects of sEVs were abolished in APN receptor-1 (AdipoR1)–deficient mice (the primary receptor for APN signaling in the heart) or in mice overexpressing GRK2 (G–protein-coupled receptor kinase 2, a kinase that phosphorylates and inactivates AdipoR1). Finally, diabetes significantly increased cardiac GRK2 expression and AdipoR1 phosphorylation, which prevented sEVs from exerting their beneficial effects. Restoring AdipoR1 function by knock-in a mutated phosphorylation-resistant AdipoR1 (AdipoR1 ^S205A ) via AAV9 (adeno-associated virus 9)-mediated gene delivery to cardiomyocytes rescued ADp sEV cardioprotection in diabetic mice. CONCLUSIONS: Our study reveals that APN is enriched on the ADp-derived external surface of sEVs and is biologically active, playing a critical role in ADp–cardiomyocyte communication. Diabetes disrupts this communication by enhancing GRK2-mediated AdipoR1 phosphorylation, impairing sEV signaling, and exacerbating IHF. These findings provide new insights into the pathophysiology and therapy of IHF in diabetes.

The synergy between radiotherapy and immunotherapy in treating thoracic cancers presents a potent therapeutic advantage, yet it also carries potential risks. The extent and nature of cumulative cardiac toxicity remain uncertain, prompting the need to discern its mechanisms and devise effective mitigation strategies. Radiation alone or in combination with an anti- Programmed cell death protein1 (PD-1) antibody significantly reduced cardiac function in C57BL/6J mice, and this pathologic effect was aggravated by anti-PD-1 (anti-PD-1 + radiation). To examine the cellular mechanism that causes the detrimental effect of anti-PD-1 upon cardiac function after radiation, AC16 human cardiomyocytes were used to study cardiac apoptosis and cardiac autophagy. Radiation-induced cardiomyocyte apoptosis was significantly promoted by anti-PD-1 treatment, while anti-PD-1 combined radiation administration blocked the cardiac autophagic flux. Adenosine 5′-triphosphate (ATP) (a molecule that promotes lysosomal acidification) not only improved autophagic flux in AC16 human cardiomyocytes, but also attenuated apoptosis induced by radiation and anti-PD-1 treatment. Finally, ATP administration in vivo significantly reduced radiation-induced and anti-PD-1-exacerbated cardiac dysfunction. We demonstrated for the first time that anti-PD-1 can aggravate radiation-induced cardiac dysfunction via promoting cardiomyocyte apoptosis without affecting radiation-arrested autophagic flux. ATP enhanced cardiomyocyte autophagic flux and inhibited apoptosis, improving cardiac function in anti-PD-1/radiation combination-treated animals.