NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis.
ABSTRACT: Cardiac failure occurs when the heart fails to adapt to chronic stresses. Reactive oxygen species (ROS)-dependent signaling is implicated in cardiac stress responses, but the role of different ROS sources remains unclear. Here we report that NADPH oxidase-4 (Nox4) facilitates cardiac adaptation to chronic stress. Unlike other Nox proteins, Nox4 activity is regulated mainly by its expression level, which increases in cardiomyocytes under stresses such as pressure overload or hypoxia. To investigate the functional role of Nox4 during the cardiac response to stress, we generated mice with a genetic deletion of Nox4 or a cardiomyocyte-targeted overexpression of Nox4. Basal cardiac function was normal in both models, but Nox4-null animals developed exaggerated contractile dysfunction, hypertrophy, and cardiac dilatation during exposure to chronic overload whereas Nox4-transgenic mice were protected. Investigation of mechanisms underlying this protective effect revealed a significant Nox4-dependent preservation of myocardial capillary density after pressure overload. Nox4 enhanced stress-induced activation of cardiomyocyte hypoxia inducible factor 1 and the release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. These data indicate that cardiomyocyte Nox4 is a unique inducible regulator of myocardial angiogenesis, a key determinant of cardiac adaptation to overload stress. Our results also have wider relevance to the use of nonspecific antioxidant approaches in cardiac disease and may provide an explanation for the failure of such strategies in many settings.
Project description:Although many animal studies indicate insulin has cardioprotective effects, clinical studies suggest a link between insulin resistance (hyperinsulinemia) and heart failure (HF). Here we have demonstrated that excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents. Chronic pressure overload induced hepatic insulin resistance and plasma insulin level elevation. In contrast, cardiac insulin signaling was upregulated by chronic pressure overload because of mechanical stretch-induced activation of cardiomyocyte insulin receptors and upregulation of insulin receptor and Irs1 expression. Chronic pressure overload increased the mismatch between cardiomyocyte size and vascularity, thereby inducing myocardial hypoxia and cardiomyocyte death. Inhibition of hyperinsulinemia substantially improved pressure overload-induced cardiac dysfunction, improving myocardial hypoxia and decreasing cardiomyocyte death. Likewise, the cardiomyocyte-specific reduction of insulin receptor expression prevented cardiac ischemia and hypertrophy and attenuated systolic dysfunction due to pressure overload. Conversely, treatment of type 1 diabetic mice with insulin improved hyperglycemia during pressure overload, but increased myocardial ischemia and cardiomyocyte death, thereby inducing HF. Promoting angiogenesis restored the cardiac dysfunction induced by insulin treatment. We therefore suggest that the use of insulin to control hyperglycemia could be harmful in the setting of pressure overload and that modulation of insulin signaling is crucial for the treatment of HF.
Project description:<h4>Aims</h4>Chronic pressure or volume overload induce concentric vs. eccentric left ventricular (LV) remodelling, respectively. Previous studies suggest that distinct signalling pathways are involved in these responses. NADPH oxidase-4 (Nox4) is a reactive oxygen species-generating enzyme that can limit detrimental cardiac remodelling in response to pressure overload. This study aimed to assess its role in volume overload-induced remodelling.<h4>Methods and results</h4>We compared the responses to creation of an aortocaval fistula (Shunt) to induce volume overload in Nox4-null mice (Nox4-/-) vs. wild-type (WT) littermates. Induction of Shunt resulted in a significant increase in cardiac Nox4 mRNA and protein levels in WT mice as compared to Sham controls. Nox4-/- mice developed less eccentric LV remodelling than WT mice (echocardiographic relative wall thickness: 0.30 vs. 0.27, P?<?0.05), with less LV hypertrophy at organ level (increase in LV weight/tibia length ratio of 25% vs. 43%, P?<?0.01) and cellular level (cardiomyocyte cross-sectional area: 323 µm2 vs. 379 ?m2, P?<?0.01). LV ejection fraction, foetal gene expression, interstitial fibrosis, myocardial capillary density, and levels of myocyte apoptosis after Shunt were similar in the two genotypes. Myocardial phospho-Akt levels were increased after induction of Shunt in WT mice, whereas levels decreased in Nox4-/- mice (+29% vs. -21%, P?<?0.05), associated with a higher level of phosphorylation of the S6 ribosomal protein (S6) and the eIF4E-binding protein 1 (4E-BP1) in WT compared to Nox4-/- mice. We identified that Akt activation in cardiac cells is augmented by Nox4 via a Src kinase-dependent inactivation of protein phosphatase 2A.<h4>Conclusion</h4>Endogenous Nox4 is required for the full development of eccentric cardiac hypertrophy and remodelling during chronic volume overload. Nox4-dependent activation of Akt and its downstream targets S6 and 4E-BP1 may be involved in this effect.
Project description:The heart relies on complex mechanisms that provide adequate myocardial oxygen supply in order to maintain its contractile function. At the cellular level, oxygen undergoes one electron reduction to superoxide through the action of different types of oxidases (e.g. xanthine oxidases, uncoupled nitric oxide synthases, NADPH oxidases or NOX). Locally generated oxygen-derived reactive species (ROS) are involved in various signaling pathways including cardiac adaptation to different types of physiological and pathophysiological stresses (e.g. hypoxia or overload). The specific effects of ROS and their regulation by oxidases are dependent on the amount of ROS generated and their specific subcellular localization. The NOX family of NADPH oxidases is a main source of ROS in the heart. Seven distinct Nox isoforms (NOX1-NOX5 and DUOX1 and 2) have been identified, of which NOX1, 2, 4 and 5 have been characterized in the cardiovascular system. For the purposes of this review, we will focus on the effects of NADPH oxidase 4 (NOX4) in the heart.
Project description:Nox-dependent signaling plays critical roles in the development of heart failure, cardiac hypertrophy, and myocardial infarction. NADPH oxidase 4 (Nox4) as a major source of oxidative stress in the heart offers a new therapeutic target in cardiovascular disease. In the present work, a novel flavonoid was isolated from Zanthoxylum bungeanum. Its structure was elucidated as Quercetin-3-O-(6''-O-?-l-rhamnopyransoyl)-?-d-glucopyranoside-7-O-?-d-glucopyranoside (ZYZ-772) for the first time. ZYZ-772 exhibited significant cardio-protective property against CoCl? induced H9c2 cardiomyocyte cells injury. In CoCl? stimulated cardiomyocyte injury, ZYZ-772 inhibited expression of Nox4, and alleviated ROS overproduction. Importantly, ROS triggered MAPKs phosphorylation and P53 signaling mediated apoptosis were restored by ZYZ-772. Our findings present the first piece of evidence for the therapeutic properties of ZYZ-772 in preventing cardiomyocyte injury, which could be attributed to the suppression of Nox4/MAPKs/P53 axis. This will offer a novel therapeutic strategy for the treatment of cardiac ischemia disease.
Project description:Regular exercise has widespread health benefits. Fundamental to these beneficial effects is the ability of the heart to intermittently and substantially increase its performance without incurring damage, but the underlying homeostatic mechanisms are unclear. We identify the ROS-generating NADPH oxidase-4 (Nox4) as an essential regulator of exercise performance in mice. Myocardial Nox4 levels increase during acute exercise and trigger activation of the transcription factor Nrf2, with the induction of multiple endogenous antioxidants. Cardiomyocyte-specific Nox4-deficient (csNox4KO) mice display a loss of exercise-induced Nrf2 activation, cardiac oxidative stress and reduced exercise performance. Cardiomyocyte-specific Nrf2-deficient (csNrf2KO) mice exhibit similar compromised exercise capacity, with mitochondrial and cardiac dysfunction. Supplementation with an Nrf2 activator or a mitochondria-targeted antioxidant effectively restores cardiac performance and exercise capacity in csNox4KO and csNrf2KO mice respectively. The Nox4/Nrf2 axis therefore drives a hormetic response that is required for optimal cardiac mitochondrial and contractile function during physiological exercise.
Project description:Aims: Oxidative stress is implicated in cardiomyocyte cell death and cardiac remodeling in the failing heart. The role of NADPH oxidase 4 (NOX4) in cardiac adaptation to pressure overload is controversial, but its function in myocardial ischemic stress has not been thoroughly elucidated. This study examined the function of NOX4 in the pathogenesis of ischemic heart failure, utilizing mouse models, cell culture, and human heart samples. Results: Nox4-/- mice showed a protective phenotype in response to permanent left anterior descending coronary artery ligation with smaller infarction area, lower cardiomyocyte cross-sectional area, higher capillary density, and less cell death versus wild-type (WT) mice. Nox4-/- mice had lower activity of soluble epoxide hydrolase (sEH), a potent regulator of inflammation. Nox4-/- mice also showed a 50% reduction in the number of infiltrating CD68+ macrophages in the peri-infarct zone versus WT mice. Adenoviral overexpression of NOX4 in cardiomyoblast cells increased sEH expression and activity and CCL4 and CCL5 levels; inhibition of sEH activity in NOX4 overexpressing cells attenuated the cytokine levels. Human hearts with ischemic cardiomyopathy showed adverse cardiac remodeling, increased NOX4 and sEH protein expression and CCL4 and CCL5 levels compared with control nonfailing hearts. Innovation and Conclusion: These data from the Nox4-/- mouse model and human heart tissues show for the first time that oxidative stress from increased NOX4 expression has a functional role in ischemic heart failure. One mechanism by which NOX4 contributes to ischemic heart failure is by increasing inflammatory cytokine production via enhanced sEH activity.
Project description:Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial.
Project description:BACKGROUND:It has been demonstrated that Tongxinluo (TXL), a traditional Chinese medicine compound, improves ischemic heart disease in animal models via vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The present study aimed to investigate whether TXL protects against pressure overload-induced heart failure in mice and explore the possible mechanism of action. METHODS AND RESULTS:Transverse aortic constriction (TAC) surgery was performed in mice to induce heart failure. Cardiac function was evaluated by echocardiography. Myocardial pathology was detected using hematoxylin and eosin or Masson trichrome staining. We investigated cardiomyocyte ultrastructure using transmission electron microscopy. Angiogenesis and oxidative stress levels were determined using CD31 and 8-hydroxydeoxyguanosine immunostaining and malondialdehyde assay, respectively. Fetal gene expression was measured using real-time PCR. Protein expression of VEGF, phosphorylated (p)-VEGF receptor 2 (VEGFR2), p-phosphatidylinositol 3-kinase (PI3K), p-Akt, p-eNOS, heme oxygenase-1 (HO-1), and NADPH oxidase 4 (Nox4) were measured with western blotting. Twelve-week low- and high-dose TXL treatment following TAC improved cardiac systolic and diastolic function and ameliorated left ventricular hypertrophy, fibrosis, and myocardial ultrastructure derangement. Importantly, TXL increased myocardial capillary density significantly and attenuated oxidative stress injury in failing hearts. Moreover, TXL upregulated cardiac nitrite content and the protein expression of VEGF, p-VEGFR2, p-PI3K, p-Akt, p-eNOS, and HO-1, but decreased Nox4 expression in mouse heart following TAC. CONCLUSION:Our findings indicate that TXL protects against pressure overload-induced heart failure in mice. Activation of the VEGF/Akt/eNOS signaling pathway might be involved in TXL improvement of the failing heart.
Project description:BACKGROUND:The mitochondrial unfolded protein response (UPRmt) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear. OBJECTIVES:The purpose of this study was to: 1) identify conditions that activate the UPRmt in the heart; and 2) study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function. METHODS:Cultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis. RESULTS:Diverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction (N-terminal pro-B-type natriuretic peptide). CONCLUSIONS:These results identify the induction of the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.
Project description:Acute myocardial infarction is the leading cause of cardiovascular?related mortality and chronic heart failure worldwide. As regards treatment, the reperfusion of ischemic tissue generates irreversible damage to the myocardium, which is termed 'cardiac ischemia?reperfusion (IR) injury'. Due to the large number of mitochondria in cardiomyocytes, an increasing number of studies have focused on the roles of mitochondria in IR injury. The primary causes of IR injury are reduced oxidative phosphorylation during hypoxia and the increased production of reactive oxygen species (ROS), together with the insufficient elimination of these oxidative species following reperfusion. IR injury includes the oxidation of DNA, incorrect modifications of proteins, the disruption of the mitochondrial membrane and respiratory chain, the loss of mitochondrial membrane potential (??m), Ca2+ overload, mitochondrial permeability transition pore formation, swelling of the mitochondria, and ultimately, cardiomyocyte necrosis. The present review article discusses the molecular mechanisms of IR injury, and summarizes the metabolic and dynamic changes occurring in the mitochondria in response to IR stress. The mitochondria are strongly recommended as a target for the development of therapeutic agents; however, the appropriate use of agents remains a challenge.