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.

NADPH oxidase 2 inhibitor GSK2795039 prevents doxorubicin-induced cardiac atrophy by attenuating cardiac sympathetic nerve terminal abnormalities and myocyte autophagy.

Doxorubicin is widely used for the treatment of human cancer, but its clinical use is limited by a cumulative dose-dependent cardiotoxicity. However, the mechanism of doxorubicin-induced cardiac atrophy and failure remains to be fully understood. In this study, we tested whether the specific NADPH oxidase 2 (Nox2) inhibitor GSK2795039 attenuates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, leading to the amelioration of cardiac atrophy and dysfunction in chronic doxorubicin-induced cardiomyopathy. Mice were randomized to receive saline, doxorubicin (2.5 mg/kg, every other day, 6 times) or doxorubicin plus GSK2795039 (2.5 mg/kg, twice a day, 9 weeks). Left ventricular (LV) total wall thickness and LV ejection fraction were decreased in doxorubicin-treated mice compared with saline-treated mice and the decreases were prevented by the treatment of the specific Nox2 inhibitor GSK2795039. The ratio of total heart weight to tibia length and myocyte cross-sectional area were decreased in doxorubicin-treated mice, and the decreases were attenuated by the GSK2795039 treatment. In doxorubicin-treated mice, myocardial Nox2 and 4-hydroxynonenal levels were increased, myocardial expression of GAP43, tyrosine hydroxylase and norepinephrine transporter, markers of sympathetic nerve terminals, was decreased, and these changes were prevented by the GSK2795039 treatment. The ratio of LC3 II/I, a marker of autophagy, and Atg5, Atg12 and Atg12-Atg5 conjugate proteins were increased in doxorubicin-treated mice, and the increases were attenuated by the GSK2795039 treatment. These findings suggest that inhibition of Nox2 by GSK2795039 attenuates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, thereby ameliorating cardiac atrophy and dysfunction after chronic doxorubicin treatment.

GSK2795039 prevents RIP1-RIP3-MLKL-mediated cardiomyocyte necroptosis in doxorubicin-induced heart failure through inhibition of NADPH oxidase-derived oxidative stress.

Doxorubicin (DOX), which is widely used for the treatment of cancer, induces cardiomyopathy associated with NADPH oxidase-derived reactive oxygen species. GSK2795039 is a novel small molecular NADPH oxidase 2 (Nox2) inhibitor. In this study, we investigated whether GSK2795039 prevents receptor-interacting protein kinase 1 (RIP1)-RIP3-mixed lineage kinase domain-like protein (MLKL)-mediated cardiomyocyte necroptosis in DOX-induced heart failure through NADPH oxidase inhibition. Eight-week old mice were randomly divided into 4 groups: control, GSK2795039, DOX and DOX plus GSK2795039. H9C2 cardiomyocytes were treated with DOX and GSK2795039. In DOX-treated mice, the survival rate was reduced, left ventricular (LV) end-systolic dimension was increased and LV fractional shortening was decreased, and these alterations were attenuated by the GSK2795039 treatment. GSK2795039 inhibited not only myocardial NADPH oxidase subunit gp91phox (Nox2) protein, but also p22phox, p47phox and p67phox proteins and prevented oxidative stress 8-hydroxy-2'-deoxyguanosine levels in DOX-treated mice. RIP3 protein and phosphorylated RIP1 (p-RIP1), p-RIP3 and p-MLKL proteins, reflective of their respective kinase activities, markers of necroptosis, were markedly increased in DOX-treated mice, and the increases were prevented by GSK2795039. GSK2795039 prevented the increases in serum lactate dehydrogenase and myocardial fibrosis in DOX-treated mice. Similarly, in DOX-treated cardiomyocytes, GSK2795039 improved cell viability, attenuated apoptosis and necrosis and prevented the increases in p-RIP1, p-RIP3 and p-MLKL expression. In conclusion, GSK2795039 prevents RIP1-RIP3-MLKL-mediated cardiomyocyte necroptosis through inhibition of NADPH oxidase-derived oxidative stress, leading to the improvement of myocardial remodeling and function in DOX-induced heart failure. These findings suggest that GSK2795039 may have implications for the treatment of DOX-induced cardiomyopathy.

Antioxidant N-acetylcysteine inhibits maladaptive myocyte autophagy in pressure overload induced cardiac remodeling in rats.

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.

Oxidative stress impairs myocyte autophagy, resulting in myocyte hypertrophy.

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.

The role of endogenous reactive oxygen species in cardiac 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.