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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.

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 (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.

We have shown previously that diallyl trisulfide (DATS) ameliorates mitochondrial fission and oxidative stress in a hyperglycemia-induced endothelial apoptosis and diabetic mouse model. The aim of this study was to investigate whether DATS mitigates Ang II-induced vascular smooth muscle cell (VSMC) phenotypic switching and vascular remodeling, and if so, to determine the underlying molecular events. Male C57BL/6 mice were used to establish a vascular remodeling model by continuous 2-week Ang II infusion using a subcutaneous osmotic pump. Animals were intraperitoneally injected with DATS or vehicle. Physiological parameters, vascular morphology, and molecular markers were assessed. For in vitro studies, VSMCs were pretreated with or without DATS for 1 h, then were stimulated with Ang II, and mitochondrial morphology and phenotypic switching of VSMCs were also measured. In primary mouse VSMCs, we found that Drp1-dependent mitochondrial fission regulated mitochondrial reactive oxygen species (mtROS) generation, which eventually promoted Ang II-induced VSMC proliferation, migration, and phenotypic switching. Moreover, Ang II was found to up-regulate the Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which regulated mitochondrial fission and VSMC phenotypic switching by phosphorylating Drp1. However, the biological effect of Ang II was abrogated by DATS. Consistent with the effects in VSMCs, we found that DATS markedly alleviated mitochondrial fission, VSMC differentiation, and vessel wall thickening in an animal model of Ang II-induced vascular remodeling, which was regulated by the ROCK1/Drp1 signal. Our findings showed that DATS mitigated Ang II-induced vascular remodeling by suppressing Drp1-mediated mitochondrial fission in an ROCK1-dependent manner.

Sepsis-induced myocardial dysfunctions are associated with high morbidity and mortality. Selenium, an essential trace element, has been reported to exert anti-inflammation, anti-oxidative stress, and anti-apoptosis. However, the protective effects of selenium on LPS-induced heart injury are still poorly illustrated. Therefore, in the present study, we sought to explore the effects of selenium pretreatment on LPS-induced myocardial injury in mice. We firstly found that selenium pretreatment significantly improved markers of myocardial injury and alleviated LPS-induced myocardial dysfunctions. Moreover, selenium supplementation reduced pro-inflammatory cytokines expression, decreased oxidative stress, and inhibited myocardial apoptosis. In addition, selenium supplementation inactivated the Sting pathway. In conclusion, our study suggests that selenium exerts protective effects on LPS-induced myocardial injury, and the underlying molecular mechanism may be related to the inactivation of Sting pathway, implying a potential therapy for sepsis-induced myocardial dysfunctions.

Increased oxidative stress and myocyte autophagy co-exist in cardiac remodeling. However, it is unclear whether oxidative stress mediates maladaptive myocyte autophagy in pathological ventricular remodeling. In this study, we tested the hypothesis that antioxidants prevent maladaptive myocyte autophagy in pressure overload-induced left ventricular (LV) remodeling. Sprague-Dawley rats underwent abdominal aortic constriction (AAC) or sham operation. The animals were randomized to receive an antioxidant N-acetylcysteine (NAC), an autophagy inhibitor 3-methyladenine (3-MA) or placebo treatment for 2 weeks. We measured LV structure and function by echocardiography and hemodynamics, myocyte autophagy and oxidative stress assessed by 8-hydroxy-2-deoxyguanosine (8-OHdG). AAC rats exhibited increased LV hypertrophy assessed by LV wall thickness and myocyte cross-sectional area. NAC prevented LV hypertrophy in AAC rats. There were no significant differences in LV fractional shortening, end-diastolic dimension and the maximal rate of LV pressure rise among the groups. AAC rats showed an increase in myocardial 8-OHdG that was prevented by NAC. The expression of LC3 II protein, a marker of autophagy, was increased at 2 weeks after AAC. Immunohistochemical scores further confirmed the increase in LC3 expression in AAC rats. The expression of autophagic proteins Beclin1 and Atg12 and ERK activity were also increased in AAC rats. NAC prevented the increases in LC3 II protein, LC3 scores, Beclin1, Atg12 and ERK activity in AAC rats. Inhibition of autophagy by 3-MA prevented LV hypertrophy after pressure overload. These findings suggest that antioxidants may be of value to prevent pressure overload-induced cardiac remodeling through inhibition of maladaptive myocyte autophagy.

Autophagy is implicated in the maintenance of cardiac homeostasis. Autophagy is activated in heart failure, in which reactive oxygen species (ROS) are increased. Exogenous ROS have been shown to induce cardiomyocyte autophagy alterations. However, little is known about the influences of physiological levels of endogenous ROS on cardiomyocyte autophagy. In the present study, we tested the hypothesis that endogenous ROS in cardiomyocytes play an important role in inducing autophagy. Cultured H9C2 cardiomyocytes or Sprague-Dawley rats were treated with the antioxidant N-acetyl-cysteine (NAC) or the superoxide dismutase mimic tempol under the basal or nutrient deprivation conditions. The autophagic flux was assessed by the lysosomal inhibitor chloroquine. In H9C2 cardiomyocytes, under a basal condition, NAC or tempol increased the ratio of LC3 II/I proteins and reduced LC3 II autophagic flux. Under nutrient deprivation, NAC increased the LC3 II/I ratio and reduced LC3 II autophagic flux. In vivo studies in rats, NAC treatment increased the LC3 II/I ratio and p-Akt protein expression in myocardium. We concluded that the antioxidants reduced autophagic flux in cardiomyocytes under the basal or nutrient deprivation conditions, suggesting that endogenous ROS promote autophagy flux under physiological conditions, and this effect is mediated, at least in part, through Akt inhibition.

NEW FINDINGS What is the central question of this study? Does oxidative stress induce impairment of autophagy that results in myocyte hypertrophy early after pressure overload? What is the main finding and its importance? In cultured myocytes, hydrogen peroxide decreased autophagy and increased hypertrophy, and inhibition of autophagy enhanced myocyte hypertrophy. In rats with early myocardial hypertrophy after pressure overload, myocyte autophagy was progressively decreased. The antioxidant N-acetyl-cysteine or the superoxide dismutase mimic tempol prevented the decrease of myocyte autophagy and attenuated myocyte hypertrophy early after pressure overload. These findings suggest that oxidative stress impairs myocyte autophagy that results in myocyte hypertrophy. ABSTRACT Insufficient or excessive myocyte autophagy is associated with left ventricular (LV) hypertrophy. Reactive oxygen species mediate myocyte hypertrophy in vitro and pressure overload-induced LV hypertrophy in vivo. In the present study, we tested the hypothesis that oxidative stress induces an impairment of autophagy that results in myocyte hypertrophy. H9C2 cardiomyocytes pretreated with the autophagy inhibitor 3-methyladenine were exposed to 10 and 50 μm hydrogen peroxide (H2 O2 ) for 48 h. Male Sprague-Dawley rats underwent abdominal aortic constriction (AAC) or sham operation. The animals were killed 24, 48 or 72 h after surgery. In a separate group, the AAC and sham-operated rats randomly received the antioxidant N-acetyl-cysteine or the superoxide dismutase mimic tempol for 72 h. In H9C2 cardiomyocytes, H2 O2 decreased the ratio of microtubule-associated protein light chain 3 (LC3) II to LC3 I and increased P62 and phosphorylated ERK (p-ERK) proteins and myocyte surface area. 3-Methyladenine further increased H2 O2 -induced p-ERK expression. In rats after AAC, the heart to body weight ratio was progressively increased, the LC3 II/I ratio was progressively decreased, p62 and p-ERK expression was increased, and expression of Beclin1, Atg5 and Atg12 was decreased. N-Acetyl-cysteine or tempol prevented the decreases in the LC3 II/I ratio and Beclin1 and Atg5 expression and attenuated the increases in LV wall thickness, myocyte diameter and brain natriuretic peptide expression in AAC rats. In conclusion, oxidative stress decreases Beclin1 and Atg5 expression that results in impairment of autophagy, leading to myocyte hypertrophy. These findings suggest that antioxidants or restoration of autophagy might be of value in the prevention of early myocardial hypertrophy after pressure overload.

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.

Background/Aims: The alterations in myocyte autophagy after myocardial infarction (MI) and the underlying mechanisms have not been fully understood. In this study, we investigated the temporal changes of myocyte autophagy in the remote non-infarcted myocardium in rabbits after MI and the relationships between alterations of myocyte autophagy and left ventricular (LV) remodeling and myocardial oxidative stress. Methods: Rabbits were assigned to MI or sham operation. Rabbits with MI or sham were randomly assigned to receive chloroquine, an autophagy inhibitor, antioxidant vitamins C and E or placebo for 4 weeks. H9C2 cardiomyocytes were subjected to hypoxia or hydrogen peroxide (H2O2) treatment. Results: MI rabbits exhibited progressive increases of LV end-diastolic dimension (EDD), and decreases of LV fractional shortening (FS) and dP/dt over 8 weeks. Myocyte autophagy assessed by the scores of LC3 and Beclin1 expression was progressively decreased at 1, 4 and 8 weeks after MI. The ratio of LC3 II/I and Beclin1 and Atg5 proteins were also decreased at 4 weeks after MI. There was a negative correlation between autophagy and LV EDD and a positive correlation between autophagy and LV FS and dP/dt. The autophagy inhibitor chloroquine worsened LV remodeling after MI. Decreased myocyte autophagy was associated with increased myocardial 4-hydroxynonenal. Antioxidant vitamins C and E prevented the decrease in myocyte autophagy after MI. In cultured H9C2 cardiomyocytes, the LC3 II/I ratio was decreased at 4 and 8 h after exposure to hypoxia, and the change was associated with increased 8-hydroxy-2-deoxyguanosine. A low concentration of H2O2 decreased the LC3 II/I ratio. Conclusion: Progressive reduction in myocyte autophagy in the remote non-infarcted myocardium was associated with myocardial oxidative stress and LV remodeling after MI. Antioxidants prevented the reduction in myocyte autophagy after MI, suggesting that oxidative stress mediates reduction in myocyte autophagy that contributes to post-MI remodeling.

Heart failure is characterized by cardiac sympathetic nerve terminal abnormalities, including the decreases of norepinephrine transporter (NET) density and cardiac catecholaminergic and tyrosine hydroxylase (TH) profiles. The alterations in cardiac sympathetic nerve terminals in heart failure are

In cardiac myocytes in vitro, a high concentration of hydrogen peroxide (1 mM) induces myocyte autophagy. Oxidative stress and myocyte autophagy coexist in pressure overload-induced heart failure. In this study, we tested the hypothesis that excessive oxidative stress mediates pathological autophagy

What is the central question of this study? We investigated the changes of myocyte autophagy during the stages of myocardial hypertrophy and failure and the relationship between autophagy and oxidative stress. What is the main findings and its importance? Myocyte autophagy is reduced during myocardial hypertrophy and increased during heart failure. Reduced autophagy is correlated with myocyte hypertrophy, and increased autophagy is correlated with myocyte apoptosis. The distinct alterations are associated with oxidative stress. Hydrogen peroxide causes distinct, concentration-dependent changes in autophagy in cultured cardiomyocytes. Oxidative stress may mediate the distinct alterations of myocyte autophagy during cardiac hypertrophy and failure. Myocyte autophagy occurs at basal levels in the heart in normal conditions and increases in heart failure. However, the changes of myocyte autophagy during the stages of myocardial hypertrophy and failure are not fully understood. Little is known about the relationship among myocyte autophagy, hypertrophy, apoptosis and oxidative stress. In the present study, we first examined the changes of myocyte autophagy in mice with chronic pressure overload and the relationships between myocyte autophagy and hypertrophy, apoptosis and oxidative stress. Second, we determined the direct role of hydrogen peroxide on autophagy in cultured cardiomyocytes. Eight-week-old male C57BL/6J mice underwent transverse aortic constriction (TAC) or sham operation. In TAC mice, left ventricular wall thickness was increased at 1 week and increased further at 9 weeks. Left ventricular end-diastolic dimension showed no change at 1 week, but increased at 9 weeks in association with systolic dysfunction. Myocyte autophagy was decreased at 1 week after TAC, and the decrease was correlated with increased myocyte size. Myocyte autophagy was increased at 9 weeks after TAC, and the increase was correlated with increased myocyte apoptosis. The alterations in autophagy after TAC were associated with myocardial oxidative stress. Hydrogen peroxide caused distinct, concentration-dependent changes in autophagy in cultured cardiomyocytes. In conclusion, myocyte autophagy was decreased during myocardial hypertrophy and increased during heart failure. The distinct changes were associated with myocyte hypertrophy, apoptosis and oxidative stress. These findings suggest that oxidative stress may mediate the distinct alterations of myocyte autophagy during myocardial hypertrophy and heart failure.

Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase activity and endoplasmic reticulum (ER) stress are increased after myocardial infarction (MI). In this study, we proposed to test whether activation of the NADPH oxidase in the remote non-infarcted myocardium mediates ER stress and left ventricular (LV) remodeling after MI. Rabbits with MI or sham operation were randomly assigned to orally receive an NADPH oxidase inhibitor apocynin or placebo for 30days. The agents were administered beginning at 1week after surgery. MI rabbits exhibited decreases in LV fractional shortening, LV ejection fraction and the first derivative of the LV pressure rise, which were abolished by apocynin treatment. NADPH oxidase Nox2 protein and mRNA expressions were increased in the remote non-infarcted myocardium after MI. Immunolabeling further revealed that Nox2 was increased in cardiac myocytes in the remote myocardium. The apocynin treatment prevented increases in the Nox2 expression, NADPH oxidase activity, oxidative stress, myocyte apoptosis and GRP78, CHOP and cleaved caspase 12 protein expression in the remote myocardium. The apocynin treatment also attenuated increases in myocyte diameter and cardiac fibrosis. In cultured H9C2 cardiomyocytes exposed to angiotensin II, an important stimulus for post-MI remodeling, Nox2 knockdown with siRNA significantly inhibited angiotensin II-induced NADPH oxidase activation, reactive oxygen species and GRP78 and CHOP protein expression. We conclude that NADPH oxidase inhibition attenuates increased ER stress in the remote non-infarcted myocardium and LV remodeling late after MI in rabbits. These findings suggest that the activation of NADPH oxidase in the remote non-infarcted myocardium mediates increased ER stress, contributing to myocyte apoptosis and LV remodeling after MI.