Yang Lee

MnSOD Overexpression Reduces Fibrosis and Pro-Apoptotic Signaling in the Aging Mouse Heart

Contractility of the heart is impaired with advancing age via mechanical remodeling, as myocytes are lost through apoptosis and collagenous fibers accumulate. Exercise training confers protection against fibrosis and apoptosis in the aging heart, but the mechanisms remain poorly understood. We recently reported that exercise training elevates Mn isoform of superoxide dismutase (MnSOD) in the aging heart, concomitant with reduction in oxidative stress and fibrosis. Here, we tested the hypothesis that overexpression of MnSOD would be causal in protection against fibrosis and apoptosis in the aging heart. Hearts were extracted from young (8 months) wild-type, young mice overexpressing the Sod2 (MnSOD) gene, old (28 months) wild-type, and old transgenic mice. Left ventricle MnSOD protein levels were elevated in young mice overexpressing the Sod2 (MnSOD) gene and old transgenic mice. MnSODTg mice exhibited lower oxidative stress (total hydroperoxides, 4-hydroxynonenal, and 8-isoprostane) in the old group. Age-related cardiac remodeling and fibrosis was mitigated in MnSOD Tg mice with reductions in extramyocyte space (-65%), collagen-I, and transforming growth factor-β. Pro-apoptotic markers Bax (-38%) and caspase-3 cleavage (-41%) were reduced and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei, DNA laddering) was mitigated in MnSOD Tg hearts compared with old wild-type. We conclude that MnSOD elevation is indeed protective against oxidative stress, fibrosis, and apoptosis in the aging heart.

EUK-134 ameliorates nNOSμ translocation and skeletal muscle fiber atrophy during short-term mechanical unloading

Reduced mechanical loading during bedrest, spaceflight, and casting, causes rapid morphological changes in skeletal muscle: fiber atrophy and reduction of slow-twitch fibers. An emerging signaling event in response to unloading is the translocation of neuronal nitric oxide synthase (nNOSμ) from the sarcolemma to the cytosol. We used EUK-134, a cell-permeable mimetic of superoxide dismutase and catalase, to test the role of redox signaling in nNOSμ translocation and muscle fiber atrophy as a result of short-term (54 h) hindlimb unloading. Fischer-344 rats were divided into ambulatory control, hindlimb-unloaded (HU), and hindlimb-unloaded + EUK-134 (HU-EUK) groups. EUK-134 mitigated the unloading-induced phenotype, including muscle fiber atrophy and muscle fiber-type shift from slow to fast. nNOSμ immunolocalization at the sarcolemma of the soleus was reduced with HU, while nNOSμ protein content in the cytosol increased with unloading. Translocation of nNOS from the sarcolemma to cytosol was virtually abolished by EUK-134. EUK-134 also mitigated dephosphorylation at Thr-32 of FoxO3a during HU. Hindlimb unloading elevated oxidative stress (4-hydroxynonenal) and increased sarcolemmal localization of Nox2 subunits gp91phox (Nox2) and p47phox, effects normalized by EUK-134. Thus, our findings are consistent with the hypothesis that oxidative stress triggers nNOSμ translocation from the sarcolemma and FoxO3a dephosphorylation as an early event during mechanical unloading. Thus, redox signaling may serve as a biological switch for nNOS to initiate morphological changes in skeletal muscle fibers.

Biphasic stress response in the soleus during reloading after hind limb unloading.

INTRODUCTION Extreme disuse and spaceflight elicit rapid skeletal muscle atrophy, accompanied by elevated proinflammatory signaling and impaired stress response proteins (e.g., heat shock proteins (HSP), insulin-like growth factor 1 (IGF-1)). Recovery of muscle mass is delayed during the early stage of reloading after prolonged unloading, with a concomitant impairment of HSP70 and IGF-1. We postulated that proinflammatory signaling and stress response alterations would characterize early and late phases of signaling during reloading. METHODS Twenty-four adult SD rats were divided into the following groups: controls, 28 d of hind limb unloading (HU), HU + early (7 d) reloading (HU-R7), and HU + late (28 d) reloading (HU-R28). RESULTS Soleus mass decreased (-55%) with HU and remained depressed (-41%) at HU-R7. Nuclear factor κB activation and oxidative stress were elevated with HU and remained high during reloading. HU elevated inducible nitric oxide synthase and returned to baseline during reloading, whereas 3-nitrotyrosine did not increase with HU and peaked at HU-R7. HU depressed levels of HSP25 phosphorylation at Ser82 and IGF-1. Although p-HSP25 and Akt phosphorylation (Ser473) recovered during early reloading, HSP70, heat shock factor 1, and IGF-1 remained depressed. HSP70, heat shock factor 1, and IGF-1 recovered, whereas p-Akt and 3-nitrotyrosine decreased to control levels at HU-R28. CONCLUSIONS Reloading elicited an early phase characterized by elevated nuclear factor κB activation, 3-nitrotyrosine, p-HSP25, and p-Akt levels and a delayed phase with recovery of HSP70, IGF-1, and muscle mass. We conclude that the reloading phenotype in skeletal muscle is expressed in two distinct phases related to (a) pro-inflammatory signaling and (b) muscle mass recovery.

Exercise training attenuates age-dependent elevation of angiotensin II type 1 receptor and Nox2 signaling in the rat heart

Fibrosis of the aging heart impedes cardiac function and increases the risk of arrhythmias and heart disease. Previously, we demonstrated that exercise-induced reduction of collagen I in the aging heart was linked to a suppression of oxidative stress and transforming growth factor-beta (TGF-ß). The renin-angiotensin II system (RAS) increases oxidative stress via NADPH oxidase-2 (Nox2) and thus elevates TGF-ß and collagen accumulation. Therefore, we tested the hypothesis that exercise training would alleviate age-related upregulation of the angiotensin II receptor I (AT1R) and NADPH oxidase-2 (Nox2), concomitant with suppression of TGF-β and fibrosis. Young (3 months, n=20) and old (31 months, n=20) Fischer 344 ×B rown Norway F1 (FBNF1) hybrid rats were assigned into sedentary and exercise groups, with exercise training rats training on a treadmill 45 min/day, 5 days/week for the next 12 weeks. Exercise training mitigated age-related upregulation of AT1R, Nox2 activity, and Nox2 subunits gp91phox and p47phox. Exercise training also attenuated TGF-ß positive staining and downstream effectors of fibrosis in the aging heart: connective tissue growth factor, phosphorylation of Smad2 at Ser423, myofibroblast proliferation, and collagen I-positive staining. Our results are consistent with the hypothesis that exercise training protects against age-dependent cardiac fibrosis by suppressing AT1R and Nox2 as part of a RAS-Nox2-TGF-β pathway.

Lifelong wheel running exercise and mild caloric restriction attenuate nuclear EndoG in the aging plantaris muscle

Apoptosis plays an important role in atrophy and sarcopenia in skeletal muscle. Recent evidence suggests that insufficient heat shock proteins (HSPs) may contribute to apoptosis and muscle wasting. In addition, long-term caloric restriction (CR) and lifelong wheel running exercise (WR) with CR provide significant protection against caspase-dependent apoptosis and sarcopenia. Caspase-independent mediators (endonuclease G: EndoG; apoptosis-inducing factor: AIF) of apoptosis are also linked to muscles wasting with disuse and aging. However, the efficacy of CR and WR with CR to attenuate caspase-independent apoptosis and preserve HSPs in aging skeletal muscle are unknown. Therefore, we tested the hypothesis that CR and WR with CR would ameliorate age-induced elevation of EndoG and AIF while protecting HSP27 and HSP70 levels in the plantaris. Male Fischer-344 rats were divided into 4 groups at 11weeks: ad libitum feeding until 6months (YAL); fed ad libitum until 24months old (OAL); 8%CR to 24months (OCR); WR+8%CR to 24months (OExCR). Nuclear EndoG levels were significantly higher in OAL (+153%) than in YAL, while CR (-38%) and WR with CR (-46%) significantly attenuated age-induced increment in nuclear EndoG. HSP27 (-63%) protein content and phosphorylation at Ser82 (-49%) were significantly lower in OAL than in YAL, while HSP27 protein content was significantly higher in OCR (+136%) and OExCR (+155%) and p-HSP27 (+254%) was significantly higher in OExCR compared with OAL, respectively. In contrast, AIF and HSP70 were unaltered by CR or WR with CR in aging muscle. These data indicate that CR and WR with CR attenuate age-associated upregulation of EndoG translocation in the nucleus, potentially involved with HSP27 signaling.

Effect of Eukarion‐134 on Akt–mTOR signalling in the rat soleus during 7 days of mechanical unloading

What is the central question of this study? Translocation of nNOSμ initiates catabolic signalling via FoxO3a and skeletal muscle atrophy during mechanical unloading. Recent evidence suggests that unloading‐induced muscle atrophy and FoxO3a activation are redox sensitive. Will a mimetic of superoxide dismutase and catalase (i.e. Eukarion‐134) also mitigate suppression of the Akt–mTOR pathway? What is the main finding and its importance? Eukarion‐134 rescued Akt–mTOR signalling and sarcolemmal nNOSμ, which were linked to protection against the unloading phenotype, muscle fibre atrophy and partial fibre‐type shift from slow to fast twitch. The loss of nNOSμ from the sarcolemma appears crucial to Akt phosphorylation and is redox sensitive, although the mechanisms remain unresolved.