NAD(P)H oxidases (Noxs) produce O 2 − and play an important role in cardiovascular pathophysiology. The Nox4 isoform is expressed primarily in the mitochondria in cardiac myocytes. To elucidate the function of endogenous Nox4 in the heart, we generated cardiac-specific Nox4 −/− (c- Nox4 −/− ) mice. Nox4 expression was inhibited in c- Nox4 −/− mice in a heart-specific manner, and there was no compensatory up-regulation in other Nox enzymes. These mice exhibited reduced levels of O 2 − in the heart, indicating that Nox4 is a significant source of O 2 − in cardiac myocytes. The baseline cardiac phenotype was normal in young c- Nox4 −/− mice. In response to pressure overload (PO), however, increases in Nox4 expression and O 2 − production in mitochondria were abolished in c- Nox4 −/− mice, and c- Nox4 −/− mice exhibited significantly attenuated cardiac hypertrophy, interstitial fibrosis and apoptosis, and better cardiac function compared with WT mice. Mitochondrial swelling, cytochrome c release, and decreases in both mitochondrial DNA and aconitase activity in response to PO were attenuated in c- Nox4 −/− mice. On the other hand, overexpression of Nox4 in mouse hearts exacerbated cardiac dysfunction, fibrosis, and apoptosis in response to PO. These results suggest that Nox4 in cardiac myocytes is a major source of mitochondrial oxidative stress, thereby mediating mitochondrial and cardiac dysfunction during PO.

Rationale: NADPH oxidases are a major source of superoxide (O 2 ؊ ) in the cardiovascular system. The function of Nox4, a member of the Nox family of NADPH oxidases, in the heart is poorly understood. Objective: The goal of this study was to elucidate the role of Nox4 in mediating oxidative stress and growth/death in the heart. Methods and Results: Expression of Nox4 in the heart was increased in response to hypertrophic stimuli and aging.Neither transgenic mice with cardiac specific overexpression of Nox4 (Tg-Nox4) nor those with catalytically inactive Nox4 (Tg-Nox4-P437H) showed an obvious baseline cardiac phenotype at young ages. Tg-Nox4 gradually displayed decreased left ventricular (LV) function with enhanced O 2 ؊ production in the heart, which was accompanied by increased apoptosis and fibrosis at 13 to 14 months of age. On the other hand, the level of oxidative stress was attenuated in Tg-Nox4-P437H. Although the size of cardiac myocytes was significantly greater in Tg-Nox4 than in nontransgenic, the LV weight/tibial length was not significantly altered in Tg-Nox4 mice. Overexpression of Nox4 in cultured cardiac myocytes induced apoptotic cell death but not hypertrophy. Nox4 is primarily localized in mitochondria and upregulation of Nox4 enhanced both rotenone-and diphenyleneiodonium-sensitive O 2 ؊ production in mitochondria. Cysteine residues in mitochondrial proteins, including aconitase and NADH dehydrogenases, were oxidized and their activities decreased in Tg-Nox4. Key Words: reactive oxygen species Ⅲ oxidative stress Ⅲ superoxide Ⅲ hypertrophy Ⅲ apoptosis Ⅲ aging R eactive oxygen species (ROS), such as superoxide (O 2 Ϫ ) and H 2 O 2 , play an important role in regulating cell growth and death of cardiac myocytes. [1][2][3] In the heart under pathological conditions, mitochondria are the major source of ROS, which are generated primarily through electron leakage from the electron transport chain. 4 The leakage of electrons is a passive process caused by damage and/or downregulation of mitochondrial proteins, and does not appear to be tightly regulated. 5 ROS are also produced through O 2 Ϫ -producing enzymes, such as NADPH oxidases and xanthine oxidase. Although NADPH oxidases are the major source of O 2 Ϫ production, their contribution to overall increases in ROS and myocardial responses under stress is not fully understood. Thus far, seven members of the NADPH oxidase (Nox) family of proteins (Nox1 to Nox5 and Duox1 and 2) have been identified. 6 -8 All Nox proteins possess 6 membranespanning domains and a cytoplasmic region containing NAD(P)H-and FAD-binding domains in their C-terminal regions. Nox1, -2, -3 and -4 form a heterodimer with p22 phox , another catalytic core component of NADPH oxidases which stabilizes Nox proteins. Nox proteins accept electrons from either NADPH or NADH 8,9 and transfer them to molecular oxygen to generate O 2 Ϫ .Nox4 is ubiquitously expressed in various cell types and tissues, including kidneys, the heart, and blood vessels. 10,11 Distinct from other members of...

Background-Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood. Methods and Results-Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro. Conclusions-These

Thioredoxin 1 (Trx1) facilitates the reduction of signaling molecules and transcription factors by cysteine thiol-disulfide exchange, thereby regulating cell growth and death. Here we studied the molecular mechanism by which Trx1 attenuates cardiac hypertrophy. Trx1 upregulates DnaJb5, a heat shock protein 40, and forms a multiple-protein complex with DnaJb5 and class II histone deacetylases (HDACs), master negative regulators of cardiac hypertrophy. Both Cys-274/Cys-276 in DnaJb5 and Cys-667/Cys-669 in HDAC4 are oxidized and form intramolecular disulfide bonds in response to reactive oxygen species (ROS)-generating hypertrophic stimuli, such as phenylephrine, whereas they are reduced by Trx1. Whereas reduction of Cys-274/Cys-276 in DnaJb5 is essential for interaction between DnaJb5 and HDAC4, reduction of Cys-667/Cys-669 in HDAC4 inhibits its nuclear export, independently of its phosphorylation status. Our study reveals a novel regulatory mechanism of cardiac hypertrophy through which the nucleocytoplasmic shuttling of class II HDACs is modulated by their redox modification in a Trx1-sensitive manner.

Modular domains mediating speci®c protein±protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67 phox , which activates the microbicidal phagocyte NADPH oxidase. Each of these speci®cally recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40 phox (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67 phox , which abolishes its interaction with Cdc24p, confers on cells temperaturesensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40 phox , which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.

Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47 phox , a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47 phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47 phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47 phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47 phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47 phox , because protein kinase C treatment of the wild-type p47 phox but not of a mutant protein with the S303͞ 304͞328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47 phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47 phox nor lipid-bindingdefective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47 phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47 phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase. O ne of the most dominant themes in current cell biology is acute and sophisticated targeting of proteins to new cellular locations, e.g., to membranes, the nucleus, and so forth. Recruitment of proteins to cell membranes is often triggered by phosphorylation of the lipid phosphatidylinositol (PtdIns), which can create targeting sites for proteins (1, 2). The phosphorylation or hydrolysis of inositol-containing lipids in cell membranes is currently known to orchestrate numerous complex cellular events (3, 4). A variety of protein modules such as pleckstrin homology and FYVE domains recognize specific phosphoinositides (phosphorylated forms of PtdIns) to recruit proteins to appropriate cell membranes (1, 2).The phagocyte oxidase (phox) homology (PX) domain (5), also known as the phox and Bem1p 2 (PB2) domain (6, 7), occurs in the phox proteins p47 phox and p40 phox in mammals, the polarity establishment protein Bem1p in budding yeast, and a variety of eukaryotic proteins involved in membrane trafficking. We have determined the NMR structure of the PX domain of p47 phox and demonstrated that it interacts with the C-terminal Src homology 3 (SH3) domain of this protein (8). The p47 phox PX domain consists of an antiparallel ␤-sheet formed by three strand...

Summary 5'-AMP-activated protein kinase (AMPK) is a key regulator of metabolism and survival during energy stress. Dysregulation of AMPK is strongly associated with oxidative stress-related disease. However, whether and how AMPK is regulated by intracellular redox status remains unknown. Here we show that the activity of AMPK is negatively regulated by oxidation of Cys130 and Cys174 in its a subunit, which interferes with the interaction between AMPK and AMPK kinases (AMPKK). Reduction of Cys130/Cys174 is essential for activation of AMPK during energy starvation. Thioredoxin1 (Trx1), an important reducing enzyme that cleaves disulfides in proteins, prevents AMPK oxidation, serving as an essential cofactor for AMPK activation. High-fat diet consumption downregulates Trx1 and induces AMPK oxidation, which enhances cardiomyocyte death during myocardial ischemia. Thus, Trx1 modulates activation of the cardioprotective AMPK pathway during ischemia, functionally linking oxidative stress and metabolism in the heart.

Rationale Oxidation of cysteine residues in class II histone deacetylases (HDACs), including HDAC4, causes nuclear exit, thereby inducing cardiac hypertrophy. The cellular source of reactive oxygen species (ROS) responsible for oxidation of HDAC4 remains unknown. Objective We investigated whether Nox4, a major NADPH oxidase, mediates cysteine oxidation of HDAC4. Methods and Results Phenylephrine (PE, 100 μM), an α1 adrenergic agonist, induced upregulation of Nox4 (1.5-fold, p<0.05) within 5 min, accompanied by increases in O2− (3.5-fold, p<0.01) from the nuclear membrane and nuclear exit of HDAC4 in cardiomyocytes (CM). Knockdown of Nox4, but not Nox2, attenuated O2− production in the nucleus and prevented PE-induced oxidation and nuclear exit of HDAC4. After continuous infusion of PE (20 mg/kg/day) for 14 days, wild-type (WT) and cardiac-specific Nox4 knockout (c-Nox4 KO) mice exhibited similar aortic pressures. Left ventricular (LV) weight/tibial length (5.7 ±0.2 vs. 6.4 ±0.2 mg/mm, p<0.05) and CM cross-sectional area (223 ±13 vs. 258 ±12 μm2, p<0.05) were significantly smaller in c-Nox4 KO than in WT mice. Nuclear O2− production in the heart was significantly lower in c-Nox4 KO than in WT mice (4116 ±314 vs. 7057 ±1710 RLU, p<0.05), and cysteine oxidation of HDAC4 was decreased. HDAC4 oxidation and cardiac hypertrophy were also attenuated in c-Nox4 KO mice 2 weeks after transverse aortic constriction. Conclusions Nox4 plays an essential role in mediating cysteine oxidation and nuclear exit of HDAC4, thereby mediating cardiac hypertrophy in response to PE and pressure overload.