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...

During phagocytosis, gp91phox , the catalytic subunit of the phagocyte NADPH oxidase, becomes activated to produce superoxide, a precursor of microbicidal oxidants. Currently increasing evidence suggests that nonphagocytic cells contain similar superoxide-producing oxidases, which are proposed to play crucial roles in various events such as cell proliferation and oxygen sensing for erythropoiesis. Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4. The NOX4 protein of 578 amino acids exhibits 39% identity to gp91 phox with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase. The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADHand NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA. The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys. In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells. In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth. The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney.

Cell killing is a critical pharmacological activity of imatinib to eradicate Bcr/Abl + leukemias. We found that imatinib kills Bcr/Abl + leukemic cells by triggering the Bcl-2-regulated apoptotic pathway. Imatinib activated several proapoptotic BH3-only proteins: bim and bmf transcription was increased, and both Bim and Bad were activated posttranslationally. Studies using RNAi and cells from gene-targeted mice revealed that Bim plays a major role in imatinib-induced apoptosis of Bcr/Abl + leukemic cells and that the combined loss of Bim and Bad abrogates this killing. Loss of Bmf or Puma had no effect. Resistance to imatinib caused by Bcl-2 overexpression or loss of Bim (plus Bad) could be overcome by cotreatment with the BH3 mimetic ABT-737. These results demonstrate that Bim and Bad account for most, perhaps all, imatinib-induced killing of Bcr/Abl + leukemic cells and suggest previously undescribed drug combination strategies for cancer therapy.