Date Published: December 27, 2010
Publisher: Impact Journals LLC
Author(s): Tetsuro Ago, Shouji Matsushima, Junya Kuroda, Daniela Zablocki, Takanari Kitazono, Junichi Sadoshima.
Oxidative stress in mitochondria is believed to promote aging. Although passive leakage of electron from the mitochondrial electron transport chain has been considered as a major source of oxidative stress in the heart and the cardiomyocytes therein, enzymes actively producing reactive oxygen species may also exist in mitochondria. We have shown recently that Nox4, a member of the NADPH oxidase family, is localized on intracellular membranes, primarily at mitochondria, in cardiomyocytes. Mitochondrial expression of Nox4 is upregulated by cardiac stress and aging in the heart, where Nox4 could become a major source of oxidative stress. This raises an intriguing possibility that Nox4 may play an important role in mediating aging of the heart. Here we discuss the potential involvement of Nox4 in mitochondrial oxidative stress and aging in the heart.
Oxidative stress is defined as an excessive accumulation of reactive oxygen species (ROS) beyond the capacity of antioxidants . ROS usually emerge as superoxide (O2−), which is dismutated immediately to hydrogen peroxide (H2O2) by superoxide dismutase (SOD). H2O2 is further converted into water by catalase or several types of peroxidases, including glutathione peroxidases and peroxiredoxin (Prx) (thioredoxin peroxidases) . However, this sequential conversion from O2− to water is not 100% efficient. Residual O2− acts directly as an oxidant or reacts immediately with NO to produce peroxynitrite (ONOO−), a very harmful ROS, in NO-producing cells, such as endothelial cells  and macrophages . H2O2 is stable and diffusible, but it can also be converted to hydroxyradical (OH−), a potent oxidant, by the Fenton reaction (Fe2+ + H2O2 → Fe3+ + OH· + OH−) .
Aerobic cells need antioxidants localized in mitochondria in order to overcome inevitable ROS pro-duction following energy generation. A functional decline in the antioxidants or increased production of ROS in mitochondria causes accumulation of ROS, thereby leading to mitochondrial dysfunction. Accum-ulation of oxidative stress in mitochondria is highly relevant to aging and the development of various aging-related common diseases, including cardiovascular diseases. This hypothesis is referred to as the “free radical theory of aging” . However, the involvement of specific forms of ROS and each antioxidant and/or ROS-producing enzyme in the process of aging remains obscure. Recently, generation of mouse models in which the level of specific ROS and/or enzymes modifying the level of ROS is altered has provided us with valuable information regarding the role of ROS in mediating aging in mammalian hearts.
The NADPH oxidases are membrane-spanning proteins with NAD(P)H and FAD binding domains in their C-terminal tails that produce O2− by transferring an electron from NADPH (or NADH) to molecular oxygen . The NADPH oxidase was thought to be a phagocyte-specific enzyme and to play a critical role in mediating bacterial killing by producing a burst of O2− , until its family protein Nox1 was discovered in smooth muscle cells and colonic epithelium . There are now 7 known proteins in the NADPH oxidase (Nox) family, i.e. Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2, which, with the exception of Nox2, were all identified in this decade [1,10,11]. The initial cloning paper reported that Nox4 was expressed highly in the kidney [26,27]. However, in contrast to other Nox proteins that are expressed only in specific tissues or cells, Nox4 is ubiquitously expressed at high levels, including in cardiovascular systems, such as endothelial cells , smooth muscle cells , and cardiomyocytes [12,30]. As we discuss below, Nox4 has unique characteristics compared to the other members of the Nox family. In particular, its localization on intracellular membranes, including mitochondria, makes it an important candidate for regulating mitochondrial oxi-dative stress during aging in the heart.
Although Nox2, a prototypical NADPH oxidase, is localized primarily on the plasma membrane, Nox4 appears to be localized on intracellular membranes. Although the intracellular localization of Nox4 remains controversial, Nox4 appears to be localized in mitochondria in mesangial cells  and cardiomyocytes , the nucleus  or endoplasmic reticulum  in vascular endothelial cells, and the plasma membrane, especially at focal adhesions, in vascular smooth muscle cells . The subcellular localizations of Nox4 may be truly cell type-dependent. However, different results may also be due to the distinct specificities of Nox4 antibodies used for analyses.
The localization of Nox4 in mitochondria and its upregulation during aging support the hypothesis that Nox4 plays an important role in mediating ROS production during aging and controlling the aging process in the heart. Overexpression of Nox4 induces cellular senescence in fibroblasts [26,27] and apoptosis in cardiomyocytes . In vascular smooth muscle cells, Nox4 upregulation plays a causal role in mediating the accumulation of polyploid cells, a biomarker of aging . Cardiac-specific overexpression of Nox4 in mice exacerbates aging-associated cardiac pheno-types, such as left ventricular dysfunction, apoptosis, and fibrosis, accompanied by mitochondrial oxidative stress and dysfunction .
The effects of Nox4-derived ROS appear to be cell type-dependent. Nox4 stimulates apoptosis in endothelial cells  and cardiomyocytes , while it induces cell proliferation in smooth muscle cells  and cardiac fibroblasts . Although the molecular mechanisms through which Nox4 exerts different effects in different cell types are currently unknown, it is possible that ROS generated by Nox4 affect distinct targets in each cell type. Interestingly, the response of the heart to pressure overload differs substantially between “cardiomyocyte-specific”  and “global”  Nox4 KO mice. Although cardiomyocytes are the major cell type in the heart, the heart also consists of other cell types, including cardiac fibroblasts, endothelial cells, and smooth muscle cells. The absence of Nox4 differentially affects each cell type and, thus, the overall response of the heart differs when Nox4 is deleted only in cardiomyocytes from when it is deleted in every cell type. By inference, the role of Nox4 in regulating aging could differ from tissue to tissue, and the role of Nox4 in regulating lifespan in the whole animal is probably complex. In this regard, the role of endogenous Nox4 in mediating aging in each organ should be addressed with both tissue-specific and systemic Nox4 KO mice.
Unlike other ROS-producing enzymes, the Nox family proteins produce O2− and/or H2O2 purposefully in a regulated manner. Nox4 is localized in the peri-nuclear region, especially in mitochondria, in cardiomyocytes. Due to its close proximity to mitochondrial proteins, ROS generated by Nox4 oxidize mitochondrial proteins, which in turn trigger mitochondrial dysfunction and electron leakage. We speculate that when ROS production via Nox4 is beyond the capacity of antioxidants, ROS are accumulated in mitochondria, thereby triggering the aging process. Aging not only upregulates Nox4 but also downregulates antioxidant mechanisms in mitochondria. We speculate that, as in the failing heart, Nox4 could be an important source of mitochondrial oxidative stress in the aging heart. If Nox4 is shown to be involved in the aging process in the heart, it could be a promising target of pharmacological intervention because aging-induced cardiomyopathy remarkably enhances the patient’s risk of developing heart failure in response to many cardiac conditions, including high blood pressure, ischemia, and diabetes.