NADPH oxidase 2 mediates cardiac sympathetic denervation and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced cardiomyopathy.

Doxorubicin has been used extensively as a potent anticancer agent, but its clinical use is limited by its cardiotoxicity. However, the underlying mechanisms remain to be fully elucidated. In this study, we tested whether NADPH oxidase 2 (Nox2) mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced heart failure. Nox2 knockout (KO) and wild-type (WT) mice were randomly assigned to receive a single injection of doxorubicin (15 mg/kg, i.p.) or saline. WT doxorubicin mice exhibited the decreases in survival rate, left ventricular (LV) wall thickness and LV fractional shortening and the increase in the lung wet-to-dry weight ratio 1 week after the injections. These alterations were attenuated in Nox2 KO doxorubicin mice. In WT doxorubicin mice, myocardial oxidative stress was increased, myocardial noradrenergic nerve fibers were reduced, myocardial expression of PGP9.5, GAP43, tyrosine hydroxylase and norepinephrine transporter was decreased, and these changes were prevented in Nox2 KO doxorubicin mice. Myocyte autophagy was increased and myocyte size was decreased in WT doxorubicin mice, but not in Nox2 KO doxorubicin mice. Nox2 mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy-both of which contribute to cardiac atrophy and failure after doxorubicin treatment.

Enhanced oxidative stress mediates pathological autophagy and necroptosis in cardiac myocytes in pressure overload induced heart failure in rats.

In cardiac myocytes in vitro, hydrogen peroxide induces autophagic cell death and necroptosis. Oxidative stress, myocyte autophagy and necroptosis coexist in heart failure (HF). In this study, we tested the hypothesis that excessive oxidative stress mediates pathological autophagy and necroptosis in myocytes in pressure overload-induced HF. HF was produced by chronic pressure overload induced by abdominal aortic constriction (AAC) in rats. Rats with AAC or sham operation were randomised to orally receive an antioxidant N-acetylcysteine (NAC) or placebo for 4 weeks. Echocardiography was performed for the assessments of left ventricular (LV) structure and function. AAC rats exhibited decreased LV fractional shortening (FS) at 4 weeks after surgery. NAC treatment attenuated decreased LV FS in AAC rats. In AAC rats, myocardial level of 8-hydroxydeoxyguanosine assessed by immunohistochemical staining, indicative of oxidative stress, was increased, LC3 II protein, a marker of autophagy, Beclin1 protein and Atg4b, Atg5, Atg7 and Atg12 mRNA expression were markedly increased, RIP1, RIP3 and MLKL expression, indicative of necroptosis, was increased, and all of the alterations in AAC rats were prevented by the NAC treatment. NAC treatment also attenuated myocyte cross-sectional area and myocardial fibrosis in AAC rats. In conclusion, NAC treatment prevented the increases in oxidative stress, myocyte autophagy and necroptosis and the decrease in LV systolic function in pressure overload-induced HF. These findings suggest that enhanced oxidative stress mediates pathological autophagy and necroptosis in myocytes, leading to LV systolic dysfunction, and antioxidants may be of value to prevent HF through the inhibition of excessive autophagy and necroptosis.

Sphingosine-1-phosphate induces myocyte autophagy after myocardial infarction through mTOR inhibition.

Sphingosine-1-phosphate (S1P)/S1P receptor 1 signaling exerts cardioprotective effects including inhibition of myocyte apoptosis. However, little is known about the effect of S1P treatment on myocyte autophagy after myocardial infarction (MI). In the present study, we tested the hypothesis that S1P induces myocyte autophagy through inhibition of the mammalian target of rapamycin (mTOR), leading to improvement of left ventricular (LV) function after MI. Sprague-Dawley rats underwent MI or sham operation. The animals were randomized to receive S1P (50 µg/kg/day, i.p.) or placebo for one week. H9C2 cardiomyocytes cultured in serum- and glucose-deficient medium were treated with or without S1P for 3 h. MI rats exhibited an increase in LV end-diastolic dimension (EDD) and decreases in LV fractional shortening (FS) and the maximal rate of LV pressure rise (+dP/dt). S1P treatment attenuated the increase in LV EDD and decreases in LV FS and +dP/dt. In the MI placebo group, the LC3 II/I ratio, a marker of autophagy, was increased, and increased further by S1P treatment. S1P also enhanced the autophagy-related proteins Atg4b and Atg5 after MI. Similarly, in cultured cardiomyocytes, autophagy was increased under glucose and serum deprivation, and increased further by S1P treatment. The effect of S1P on myocyte autophagy was associated with mTOR inhibition after MI or in cultured cardiomyocytes under glucose and serum deprivation. S1P treatment prevents LV remodeling, enhances myocyte autophagy and inhibits mTOR activity after MI. These findings suggest that S1P treatment induces myocyte autophagy through mTOR inhibition, leading to the attenuation of LV dysfunction after MI.

Apocynin prevents reduced myocardial nerve growth factor, contributing to amelioration of myocardial apoptosis and failure.

Reduced nerve growth factor (NGF) is associated with cardiac sympathetic nerve denervation in heart failure (HF) which is characterized by increased oxidative stress. Apocynin is considered an antioxidant agent which inhibits NADPH oxidase activity and improves reactive oxygen species scavenging. However, it is unclear whether apocynin prevents reduced myocardial NGF, leading to improvement of cardiac function in HF. In this study, we tested the hypothesis that apocynin prevents reduced myocardial NGF, contributing to amelioration of myocardial apoptosis and failure. Rabbits with myocardial infarction (MI) or sham operation were randomly assigned to receive apocynin or placebo for 4 weeks. MI rabbits exhibited left ventricular (LV) dysfunction, and elevation in oxidative stress, as evidenced by a decreased reduced-to-oxidized glutathione ratio and an increased 4-hydroxynonenal expression, and reduction in NGF and NGF receptor tyrosine kinase A (TrKA) expression in the remote non-infarcted myocardium. Apocynin treatment ameliorated LV dysfunction, reduced oxidative stress, prevented decreases in NGF and TrKA expression and reduced cardiomyocyte apoptosis after MI. In cultured H9C2 cardiomyocytes, hypoxia or hydrogen peroxide decreased NGF expression, and apocynin normalized hypoxia-induced reduction of NGF. Recombinant NGF attenuated hypoxia-induced apoptosis. Apocynin prevented hypoxia-induced apoptosis, and the suppressive effect of apocynin on apoptosis was abolished by NGF receptor TrKA inhibitor K252a. We concluded that apocynin prevented reduced myocardial NGF, leading to attenuation of cardiomyocyte apoptosis and LV remodelling and dysfunction in HF after MI. These findings suggest that strategies to prevent NGF reduction by inhibition of oxidative stress may be of value in amelioration of LV dysfunction in HF.

The NADPH oxidase inhibitor apocynin improves cardiac sympathetic nerve terminal innervation and function in heart failure.

NEW FINDINGS: What is the central question of this study? Does NADPH oxidase activation mediate cardiac sympathetic nerve denervation and dysfunction in heart failure. What is the main findings and its importance? Cardiac sympathetic nerve terminal density and function were reduced in heart failure after myocardial infarction in rabbits. The NADPH oxidase inhibitor apocynin prevented the reduction in cardiac sympathetic nerve terminal density and function in heart failure. This suggest that NADPH oxidase activation mediates cardiac sympathetic nerve terminal abnormalities in heart failure. NADPH oxidase may be a potential therapeutic target for cardiac sympathetic denervation and dysfunction in heart failure. ABSTRACT: Congestive heart failure (CHF) is characterized by cardiac sympathetic nerve terminal abnormalities, as evidenced by decreased noradrenaline transporter (NAT) density and cardiac catecholaminergic and tyrosine hydroxylase (TH) profiles. These alterations are associated with increased reactive oxygen species (ROS). NADPH oxidase is a major source of ROS in CHF. In this study, we tested the hypothesis that NADPH oxidase activation mediates cardiac sympathetic nerve terminal abnormalities in CHF. CHF was produced by myocardial infarction (MI) in rabbits. Rabbits with MI or a sham operation were randomized to orally receive an NADPH oxidase inhibitor, apocynin (6 mg kg-1  day-1 ), or placebo for 30 days. MI rabbits exhibited left ventricular dilatation, systolic dysfunction, and increases in NADPH oxidase activity and 4-hydroxynonenal expression in the remote non-infarcted myocardium, all of which were prevented by treatment with apocynin. Cardiac catecholaminergic histofluorescence profiles and immunostained TH and PGP9.5 expression were decreased, and the decreases were ameliorated by apocynin treatment. NAT, TH and PGP9.5 protein and mRNA expression were reduced and the reduction was mitigated by apocynin treatment. The effects of apocynin were confirmed by utilizing the NADPH oxidase inhibitor diphenyleneiodonium in a separate experiment. In conclusion, the NADPH oxidase inhibitor apocynin attenuated increased myocardial oxidative stress and decreased cardiac sympathetic nerve terminals in CHF after MI in rabbits. These findings suggest that the activation of NADPH oxidase mediates cardiac sympathetic nerve terminal abnormalities in CHF, and the inhibition of NADPH oxidase may be beneficial for the treatment of heart failure.