Reductive Stress Causes Pathological Cardiac Remodeling and Diastolic Dysfunction.

Aims: Redox homeostasis is tightly controlled and regulates key cellular signaling pathways. The cell's antioxidant response provides a natural defense against oxidative stress, but excessive antioxidant generation leads to reductive stress (RS). This study elucidated how chronic RS, caused by constitutive activation of nuclear erythroid related factor-2 (caNrf2)-dependent antioxidant system, drives pathological myocardial remodeling. Results: Upregulation of antioxidant transcripts and proteins in caNrf2-TG hearts (TGL and TGH; transgenic-low and -high) dose dependently increased glutathione (GSH) redox potential and resulted in RS, which over time caused pathological cardiac remodeling identified as hypertrophic cardiomyopathy (HCM) with abnormally increased ejection fraction and diastolic dysfunction in TGH mice at 6 months of age. While the TGH mice exhibited 60% mortality at 18 months of age, the rate of survival in TGL was comparable with nontransgenic (NTG) littermates. Moreover, TGH mice had severe cardiac remodeling at ∼6 months of age, while TGL mice did not develop comparable phenotypes until 15 months, suggesting that even moderate RS may lead to irreversible damages of the heart over time. Pharmacologically blocking GSH biosynthesis using BSO (l-buthionine-SR-sulfoximine) at an early age (∼1.5 months) prevented RS and rescued the TGH mice from pathological cardiac remodeling. Here we demonstrate that chronic RS causes pathological cardiomyopathy with diastolic dysfunction in mice due to sustained activation of antioxidant signaling. Innovation and Conclusion: Our findings demonstrate that chronic RS is intolerable and adequate to induce heart failure (HF). Antioxidant-based therapeutic approaches for human HF should consider a thorough evaluation of redox state before the treatment.

Disruption of nuclear factor (erythroid-derived-2)-like 2 antioxidant signaling: a mechanism for impaired activation of stem cells and delayed regeneration of skeletal muscle.

Recently we have reported that age-dependent decline in antioxidant levels accelerated apoptosis and skeletal muscle degeneration. Here, we demonstrate genetic ablation of the master cytoprotective transcription factor, nuclear factor (erythroid-derived-2)-like 2 (Nrf2), aggravates cardiotoxin (CTX)-induced tibialis anterior (TA) muscle damage. Disruption of Nrf2 signaling sustained the CTX-induced burden of reactive oxygen species together with compromised expression of antioxidant genes and proteins. Transcript/protein expression of phenotypic markers of muscle differentiation, namely paired box 7 (satellite cell) and early myogenic differentiation and terminal differentiation (myogenin and myosin heavy chain 2) were increased on d 2 and 4 postinjury but later returned to baseline levels on d 8 and 15 in wild-type (WT) mice. In contrast, these responses were persistently augmented in Nrf2-null mice suggesting that regulation of the regeneration-related signaling mechanisms require Nrf2 for normal functioning. Furthermore, Nrf2-null mice displayed slower regeneration marked by dysregulation of embryonic myosin heavy chain temporal expression. Histologic observations illustrated that Nrf2-null mice displayed smaller, immature TA muscle fibers compared with WT counterparts on d 15 after CTX injury. Improvement in TA muscle morphology and gain in muscle mass evident in the WT mice was not noticeable in the Nrf2-null animals. Taken together these data show that the satellite cell activation, proliferation, and differentiation requires a functional Nrf2 system for effective healing following injury.-Shelar, S. B., Narasimhan, M., Shanmugam, G., Litovsky, S. H., Gounder, S. S., Karan, G., Arulvasu, C., Kensler, T. W., Hoidal, J. R., Darley-Usmar, V. M., Rajasekaran, N. S. Disruption of nuclear factor (erythroid-derived-2)-like 2 antioxidant signaling: a mechanism for impaired activation of stem cells and delayed regeneration of skeletal muscle.

Impaired transcriptional activity of Nrf2 in age-related myocardial oxidative stress is reversible by moderate exercise training.

Aging promotes accumulation of reactive oxygen/nitrogen species (ROS/RNS) in cardiomyocytes, which leads to contractile dysfunction and cardiac abnormalities. These changes may contribute to increased cardiovascular disease in the elderly. Inducible antioxidant pathways are regulated by nuclear erythroid 2 p45-related factor 2 (Nrf2) through antioxidant response cis-elements (AREs) and are impaired in the aging heart. Whereas acute exercise stress (AES) activates Nrf2 signaling and promotes myocardial antioxidant function in young mice (~2 months), aging mouse (>23 months) hearts exhibit significant oxidative stress as compared to those of the young. The purpose of this study was to investigate age-dependent regulation of Nrf2-antioxidant mechanisms and redox homeostasis in mouse hearts and the impact of exercise. Old mice were highly susceptible to oxidative stress following high endurance exercise stress (EES), but demonstrated increased adaptive redox homeostasis after moderate exercise training (MET; 10m/min, for 45 min/day) for ~6 weeks. Following EES, transcription and protein levels for most of the ARE-antioxidants were increased in young mice but their induction was blunted in aging mice. In contrast, 6-weeks of chronic MET promoted nuclear levels of Nrf2 along with its target antioxidants in the aging heart to near normal levels as seen in young mice. These observations suggest that enhancing Nrf2 function and endogenous cytoprotective mechanisms by MET, may combat age-induced ROS/RNS and protect the myocardium from oxidative stress diseases.

Disruption of Nrf2/ARE signaling impairs antioxidant mechanisms and promotes cell degradation pathways in aged skeletal muscle.

Age-associated decline in antioxidant potential and accumulation of reactive oxygen/nitrogen species are primary causes for multiple health problems, including muscular dystrophy and sarcopenia. The role of the nuclear erythroid-2-p45-related factor-2 (Nrf2) signaling has been implicated in antioxidant gene regulation. Here, we investigated the loss-of-function mechanisms for age-dependent regulation of Nrf2/ARE (Antioxidant Response Element) signaling in skeletal muscle (SM). Under basal physiological conditions, disruption of Nrf2 showed minimal effects on antioxidant defenses in young (2months) Nrf2-/- mice. Interestingly, mRNA and protein levels of NADH Quinone Oxidase-1 were dramatically (*P<0.001) decreased in Nrf2-/- SM when compared to WT at 2months of age, suggesting central regulation of NQO1 occurs through Nrf2. Subsequent analysis of the Nrf2-dependent transcription and translation showed that the aged mice (>24months) had a significant increase in ROS along with a decrease in glutathione (GSH) levels and impaired antioxidants in Nrf2-/- when compared to WT SM. Further, disruption of Nrf2 appears to induce oxidative stress (increased ROS, HNE-positive proteins), ubiquitination and pro-apoptotic signals in the aged SM of Nrf2-/- mice. These results indicate a direct role for Nrf2/ARE signaling on impairment of antioxidants, which contribute to muscle degradation pathways upon aging. Our findings conclude that though the loss of Nrf2 is not amenable at younger age; it could severely affect the SM defenses upon aging. Thus, Nrf2 signaling might be a potential therapeutic target to protect the SM from age-dependent accumulation of ROS by rescuing redox homeostasis to prevent age-related muscle disorders such as sarcopenia and myopathy.

Acute exercise stress activates Nrf2/ARE signaling and promotes antioxidant mechanisms in the myocardium.

Oxidative stress has been implicated in the pathogenesis of cardiovascular diseases, including myocardial hypertrophy and infarction. Although impairment of antioxidant defense mechanisms has been thought to provoke oxidative stress-induced myocardial dysfunction, it has been difficult to clearly demonstrate. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive, basic leucine zipper protein that regulates the transcription of several antioxidant genes. We previously reported that sustained activation of Nrf2 upregulates transcription of a number of endogenous antioxidants in the heart. Here, we show that acute exercise stress (AES) results in activation of Nrf2/ARE (antioxidant response element) signaling and subsequent enhancement of antioxidant defense pathways in wild-type (WT) mouse hearts, while oxidative stress, along with blunted defense mechanisms, was observed in Nrf2-/- mice. We also find that AES is associated with increased trans-activation of ARE-containing genes in exercised animals when compared to age-matched sedentary WT mice. However, enhanced oxidative stress in response to AES was observed in Nrf2-/- mice due to lower basal expression and marked attenuation of the transcriptional induction of several antioxidant genes. Thus, AES induces ROS and promotes Nrf2 function, but disruption of Nrf2 increases susceptibility of the myocardium to oxidative stress. Our findings suggest the basis for a nonpharmacological approach to activate Nrf2/ARE signaling, which might be a potential therapeutic target to protect the heart from oxidative stress-induced cardiovascular complications.