Effects of Resveratrol on the Recovery of Muscle Mass Following Disuse in the Plantaris Muscle of Aged Rats

Aging is associated with poor skeletal muscle regenerative ability following extended periods of hospitalization and other forms of muscular disuse. Resveratrol (3,5,4’-trihydroxystilbene) is a natural phytoalexin which has been shown in skeletal muscle to improve oxidative stress levels in muscles of aged rats. As muscle disuse and reloading after disuse increases oxidative stress, we hypothesized that resveratrol supplementation would improve muscle regeneration after disuse. A total of thirty-six male Fisher 344 × Brown Norway rats (32 mo.) were treated with either a water vehicle or resveratrol via oral gavage. The animals received hindlimb suspension for 14 days. Thereafter, they were either sacrificed or allowed an additional 14 day period of cage ambulation during reloading. A total of six rats from the vehicle and the resveratrol treated groups were used for the hindlimb suspension and recovery protocols. Furthermore, two groups of 6 vehicle treated animals maintained normal ambulation throughout the experiment, and were used as control animals for the hindlimb suspension and reloading groups. The data show that resveratrol supplementation was unable to attenuate the decreases in plantaris muscle wet weight during hindlimb suspension but it improved muscle mass during reloading after hindlimb suspension. Although resveratrol did not prevent fiber atrophy during the period of disuse, it increased the fiber cross sectional area of type IIA and IIB fibers in response to reloading after hindlimb suspension. There was a modest enhancement of myogenic precursor cell proliferation in resveratrol-treated muscles after reloading, but this failed to reach statistical significance. The resveratrol-associated improvement in type II fiber size and muscle mass recovery after disuse may have been due to decreases in the abundance of pro-apoptotic proteins Bax, cleaved caspase 3 and cleaved caspase 9 in reloaded muscles. Resveratrol appears to have modest therapeutic benefits for improving muscle mass after disuse in aging

Mitochondria Initiate and Regulate Sarcopenia

We present the hypothesis that an accumulation of dysfunctional mitochondria initiates a signaling cascade leading to motor neuron and muscle fiber death and culminating in sarcopenia. Interactions between neural and muscle cells that contain dysfunctional mitochondria exacerbate sarcopenia. Preventing sarcopenia will require identifying mitochondrial sources of dysfunction that are reversible

Regulation of Satellite Cell Function in Sarcopenia

The mechanisms contributing to sarcopenia include reduced satellite cell (myogenic stem cell) function that is impacted by the environment (niche) of these cells. Satellite cell function is affected by oxidative stress, which is elevated in aged muscles, and this along with changes in largely unknown systemic factors, likely contribute to the manner in which satellite cells respond to stressors such as exercise, disuse, or rehabilitation in sarcopenic muscles. Nutritional intervention provides one therapeutic strategy to improve the satellite cell niche and systemic factors, with the goal of improving satellite cell function in aging muscles. Although many elderly persons consume various nutraceuticals with the hope of improving health, most of these compounds have not been thoroughly tested, and the impacts that they might have on sarcopenia and satellite cell function are not clear. This review discusses data pertaining to the satellite cell responses and function in aging skeletal muscle, and the impact that three compounds: resveratrol, green tea catechins, and β-Hydroxy-β-methylbutyrate have on regulating satellite cell function and therefore contributing to reducing sarcopenia or improving muscle mass after disuse in aging. The data suggest that these nutraceutical compounds improve satellite cell function during rehabilitative loading in animal models of aging after disuse (i.e., muscle regeneration). While these compounds have not been rigorously tested in humans, the data from animal models of aging provide a strong basis for conducting additional focused work to determine if these or other nutraceuticals can offset the muscle losses, or improve regeneration in sarcopenic muscles of older humans via improving satellite cell function

A Role for Regulator of G Protein Signaling-12 (RGS12) in the Balance Between Myoblast Proliferation and Differentiation

Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking Rgs12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle Rgs12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of Rgs12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair

The Role of SIRT1 in Skeletal Muscle Function and Repair of Older Mice

Background Sirtuin 1 (SIRT1) is a NAD+ sensitive deacetylase that has been linked to longevity and has been suggested to confer beneficial effects that counter aging-associated deterioration. Muscle repair is dependent upon satellite cell function, which is reported to be reduced with aging; however, it is not known if this is linked to an aging-suppression of SIRT1. This study tested the hypothesis that Sirtuin 1 (SIRT1) overexpression would increase the extent of muscle repair and muscle function in older mice. Methods We examined satellite cell dependent repair in tibialis anterior, gastrocnemius, and soleus muscles of 13 young wild-type mice (20–30 weeks) and 49 older (80+ weeks) mice that were controls (n = 13), overexpressed SIRT1 in skeletal muscle (n = 14), and had a skeletal muscle SIRT1 knockout (n = 12) or a satellite cell SIRT1 knockout (n = 10). Acute muscle injury was induced by injection of cardiotoxin (CTX), and phosphate-buffered saline was used as a vector control. Plantarflexor muscle force and fatigue were evaluated before or 21 days after CTX injection. Satellite cell proliferation and mitochondrial function were also evaluated in undamaged muscles. Results Maximal muscle force was significantly lower in control muscles of older satellite cell knockout SIRT1 mice compared to young adult wild-type (YWT) mice (P \u3c 0.001). Mean contraction force at 40 Hz stimulation was significantly greater after recovery from CTX injury in older mice that overexpressed muscle SIRT1 than age-matched SIRT1 knockout mice (P \u3c 0.05). SIRT1 muscle knockout models (P \u3c 0.05) had greater levels of p53 (P \u3c 0.05 MKO, P \u3c 0.001 OE) in CTX-damaged tissues as compared to YWT CTX mice. SIRT1 overexpression with co-expression of p53 was associated with increased fatigue resistance and increased force potentiation during repeated contractions as compared to wild-type or SIRT1 knockout models (P \u3c 0.001). Muscle structure and mitochondrial function were not different between the groups, but proliferation of satellite cells was significantly greater in older mice with SIRT1 muscle knockout (P \u3c 0.05), but not older SIRT1 satellite cell knockout models, in vitro, although this effect was attenuated in vivo after 21 days of recovery. Conclusions The data suggest skeletal muscle structure, function, and recovery after CTX-induced injury are not significantly influenced by gain or loss of SIRT1 abundance alone in skeletal muscle; however, muscle function is impaired by ablation of SIRT1 in satellite cells. SIRT1 appears to interact with p53 to improve muscle fatigue resistance after repair from muscle injury

The Effects of Calcium-β-Hydroxy-β-Methylbutyrate on Aging-Associated Apoptotic Signaling and Muscle Mass and Function in Unloaded but Nonatrophied Extensor Digitorum Longus Muscles of Aged Rats

Beta-hydroxy-beta-methylbutyrate (HMB), a naturally occurring leucine metabolite, has been shown to attenuate plantar flexor muscle loss and increase myogenic stem cell activation during reloading after a period of significant muscle wasting by disuse in old rodents. However, it was less clear if HMB would alter dorsiflexor muscle response to unloading or reloading when there was no significant atrophy that was induced by unloading. In this study, we tested if calcium HMB (Ca-HMB) would improve muscle function and alter apoptotic signaling in the extensor digitorum longus (EDL) of aged animals that were unloaded but did not undergo atrophy. The EDL muscle was unloaded for 14 days by hindlimb suspension (HS) in aged (34-36 mo.) male Fisher 344×Brown Norway rats. The rats were removed from HS and allowed normal cage ambulation for 14 days of reloading (R). Throughout the study, the rats were gavaged daily with 170 mg of Ca-HMB or water 7 days prior to HS, then throughout 14 days of HS and 14 days of recovery after removing HS. The animals’ body weights were significantly reduced by ~18% after 14 days of HS and continued to decline by ~22% during R as compared to control conditions; however, despite unloading, EDL did not atrophy by HS, nor did it increase in mass after R. No changes were observed in EDL twitch contraction time, force production, fatigue resistance, fiber cross-sectional area, or markers of nuclear apoptosis (myonuclei + satellite cells) after HS or R. While HS and R increased the proapoptotic Bax protein abundance, BCL-2 abundance was also increased as was the frequency of TUNEL-positive myonuclei and satellite cells, yet muscle mass and fiber cross-sectional area did not change and Ca-HMB treatment had no effect reducing apoptotic signaling. These data indicate that (i) increased apoptotic signaling preceded muscle atrophy or occurred without significant EDL atrophy and (ii) that Ca-HMB treatment did not improve EDL signaling, muscle mass, or muscle function in aged rats, when HS and R did not impact mass or function

Ankyrin repeat domain protein 2 and inhibitor of DNA binding 3 cooperatively inhibit myoblast differentiation by physical interaction.

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB(mdm)) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program. J Biol Chem 2013 Aug 23; 288(34):24560-8

Fatigue index.

<p>Plantaris muscle fatigability was expressed as a fatigue index, and calculated as the percent of the initial force ( average of the first three contractions, divided by the force at the end of the fatigue protocol (an average of the force generated in the final three contractions) in a series of 128 consecutive contractions. * <i>P</i><0.05 <i>vs</i>. cage control; † <i>P</i><0.05 vehicle <i>vs</i>. resveratrol.</p

Body and muscle wet weights.

<p><i>A</i>) Bodyweight measurements of all animals were taken immediately prior to the start of the one-week pretreatment period (Start), prior to suspension (0), following hindlimb suspension (HS), and again following the reloading period recovery in cage control (n=12), vehicle-treated (n=12) or resveratrol-treated (n=12) animals. <i>B</i>. Plantaris muscle wet weights were obtained following HS or R. * <i>P</i><0.05 <i>vs</i>. cage control.</p

Skeletal Muscle Gene Expression Profile in Response to Caloric Restriction and Aging: A Role for SirT1

SirT1 plays a crucial role in the regulation of some of the caloric restriction (CR) responsive biological pathways. Aging suppresses SirT1 gene expression in skeletal muscle, suggesting that aging may affect the role of CR in muscle. To determine the role of SirT1 in the regulation of CR regulated pathways in skeletal muscle, we performed high-throughput RNA sequencing using total RNA isolated from the skeletal muscles of young and aged wild-type (WT), SirT1 knockout (SirT1-KO), and SirT1 overexpression (SirT1-OE) mice fed to 20 wk ad libitum (AL) or 40% CR diet. Our data show that aging repressed the global gene expression profile, which was restored by CR via upregulating transcriptional and translational process-related pathways. CR inhibits pathways linked to the extracellular matrix and cytoskeletal proteins regardless of aging. Mitochondrial function and muscle contraction-related pathways are upregulated in aged SirT1 KO mice following CR. SirT1 OE did not affect whole-body energy expenditure or augment skeletal muscle insulin sensitivity associated pathways, regardless of aging or diet. Overall, our RNA-seq data showed that SirT1 and CR have different functions and activation of SirT1 by its activator or exercise may enhance SirT1 activity that, along with CR, likely have a better functional role in aging muscle