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...

It is well established that the aging heart exhibits left ventricular (LV) diastolic dysfunction and changes in mechanical properties, which are thought to be due to alterations in the extracellular matrix. We tested the hypothesis that the mechanical properties of cardiac myocytes significantly change with aging, which could contribute to the global changes in LV diastolic dysfunction. We used atomic force microscopy (AFM), which determines cellular mechanical property changes at nanoscale resolution in myocytes, from young (4 mo) and old (30 mo) male Fischer 344 x Brown Norway F1 hybrid rats. A measure of stiffness, i.e., apparent elastic modulus, was determined by analyzing the relationship between AFM indentation force and depth with the classical infinitesimal strain theory and by modeling the AFM probe as a blunted conical indenter. This is the first study to demonstrate a significant increase (P < 0.01) in the apparent elastic modulus of single, aging cardiac myocytes (from 35.1 +/- 0.7, n = 53, to 42.5 +/- 1.0 kPa, n = 58), supporting the novel concept that the mechanism mediating LV diastolic dysfunction in aging hearts resides, in part, at the level of the myocyte.

Background:The cysteine desulfurase Nfs1 provides sulfur for Fe-S cluster biogenesis in mitochondria. Results: Frataxin or a mutant Fe-S scaffold protein (Isu1 Sup ) with frataxin-bypassing ability stimulates cysteine binding to Nfs1. Conclusion: Frataxin or Isu1 Sup likely induces a conformational change in Nfs1, exposing substrate-binding sites. Significance: Data presented here may help develop a drug for treating Friedreich ataxia associated with frataxin deficiency.

In this study we have shown that the histone variant H2A.z is up-regulated during cardiac hypertrophy. Upon its knockdown with RNA interference, hypertrophy and the underlying increase in growth-related genes, protein synthesis, and cell size were down-regulated. During attempts to understand the mode of regulation of H2A.z, we found that overexpression of silent information regulator 2alpha (Sir2␣) specifically induced down-regulation of H2A.z via NAD-dependent activity. This effect was reversed by the proteasome inhibitor epoxomicin, suggesting a Sir2␣-mediated ubiquitin/proteasome-dependent mechanism for degradation of H2A.z. An increase in Sir2␣ also resulted in a dose-dependent reduction of the response to hypertrophic stimuli, whereas its inhibition resulted in enhanced hypertrophy and apoptosis. We have shown that Sir2␣ directly deacetylates H2A.z. Mutagenesis proved that lysines 4, 7, 11, and 13 do not play a role in the stability of H2A.z, whereas Lys-15 was indispensable. Meanwhile, Lys-115 and conserved, ubiquitinatable Lys-121 are critical for Sir2␣-mediated degradation. Fusion of the C terminus of H2A.z (amino acids 115-127) to H2A.x or green fluorescence protein conferred Sir2␣-inducible degradation to the former protein only. Because H2A.x and H2A.z have conserved N-tails, this implied that both the C and N termini are critical for mediating the effect of Sir2␣. In short, the results suggest that H2A.z is required for cardiac hypertrophy, where its stability and the extent of cell growth and apoptosis are moderated by Sir2␣. We also propose that Sir2␣ is involved in deacetylation of H2A.z, which results in ubiquitination of Lys-115 and Lys-121 and its degradation via a ubiquitin/proteasomedependent pathway.H2A.z is an essential histone variant. Disruption of H2A.z in mice is embryonic lethal at a very early stage (E 4.5) (1). Likewise, H2A.z is vital for the development of Drosophila melanogaster (2) and is indispensable in Tetrahymena thermophila (3). In yeast, disruption of H2A.z results in slow growth and formamide sensitivity and is not rescued by the major H2A but is rescued by T. thermophila H2A.z variant (4). These reports provide evidence that H2A.z has a vital and non-redundant role and that it is conserved among species.One of the mechanisms for transcriptional regulation involves the replacement of core histones with more specialized variants. A report by Abbott et al. (5) shows that nucleosomes harboring H2A.z are destabilized, being less compact than nucleosomes harboring the core histone H2A. This is also supported by the crystal structure of a nucleosome core containing H2A.z that exhibits subtle differences compared with the H2A nucleosomes, which account for instability between the H2A.z-H2B dimer and the H3-H4 tetramer (6). This is consistent with a role for H2A.z in nucleosomal remodeling to reduce its structural compactness, thereby facilitating the binding of transcription factors. It should be noted though that this might not be the sole function of H2A.z in the cell. It has...

Mitochondria transport and utilize iron for the synthesis of haem and Fe–S clusters. Although many proteins are known to be involved in these processes, additional proteins are likely to participate. To test this hypothesis, in the present study we used a genetic screen looking for yeast mutants that are synthetically lethal with the mitochondrial iron carriers Mrs3 and Mrs4. Several genes were identified, including an isolate mutated for Yfh1, the yeast frataxin homologue. All such triple mutants were complemented by increased expression of Rim2, another mitochondrial carrier protein. Rim2 overexpression was able to enhance haem and Fe–S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds. Conversely Rim2 depletion impaired haem and Fe–S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds, indicating a unique requirement for this mitochondrial transporter for these processes. Rim2 was previously shown to mediate pyrimidine exchange in and out of vesicles. In the present study we found that isolated mitochondria lacking Rim2 exhibited concordant iron defects and pyrimidine transport defects, although the connection between these two functions is not explained. When organellar membranes were ruptured to bypass iron transport, haem synthesis from added iron and porphyrin was still markedly deficient in Rim2-depleted mitochondrial lysate. The results indicate that Rim2 is a pyrimidine exchanger with an additional unique function in promoting mitochondrial iron utilization.