Botulinum neurotoxin type A (BoNT/A) is used as a therapeutic tool to induce chemical denervation of spastically contracted muscles, yet the neurotoxin can also cause skeletal muscle atrophy. The underlying proteolytic mechanisms that induce this atrophy remain unclear. Our previous work has highlighted increased ubiquitin proteasome system (UPS) activity in soleus muscle of male Sprague Dawley rats following hind limb injection of BoNT/A, with the chymotrypsin-like activity of the 20s proteasome the most active. Thus, we chose to inhibit 20s proteasome activity in BoNT/A injected hind limb to determine the effect on soleus muscle atrophy. Epoxomicin is commonly used to inhibit the proteasome in vivo, binding specifically and irreversibly to the 20s proteasome catalytic subunits. Daily subcutaneous injections of epoxomicin abolished BoNT/A-induced elevations in 20s chymotrypsin-like activity both 3 days and 10 days post BoNT/A injection. Furthermore, BoNT/A-induced elevations in polyubiquitination remained elevated in BoNT/A + epoxomicin treated muscle, presumably due to epoxomicin's inhibition of the proteasome causing a back-up of polyubiquitinated proteins. Despite inhibition of the proteasome, epoxomicin was insufficient to significantly attenuate soleus muscle fiber atrophy 3 days following BoNT/A injection however, 10 days of daily epoxomicin injection was sufficient to spare ∼20% of muscle wasting. The mechanism of the remaining 80% of BoNT/A-induced atrophy presumably occurs via mechanisms outside of the 20s proteasome.

Botulinum neurotoxin type A (BoNT/A) is used clinically to induce therapeutic chemical denervation of spastically contracted skeletal muscles. However, BoNT/A administration can also cause atrophy. We sought to determine whether a major proteolytic pathway contributing to atrophy in multiple models of muscle wasting, the ubiquitin proteasome system (UPS), is involved in BoNT/A-induced atrophy. Three and ten days following BoNT/A injection of rat hindlimb, soleus muscle fiber cross-sectional area was reduced 25 and 65%, respectively. The transcriptional activity of NF-κB and Foxo was significantly elevated at 3 days (2- to 4-fold) and 10 days (5- to 6-fold). Muscle RING-finger protein-1 (MuRF1) activity was elevated (2-fold) after 3 days but not 10 days, while atrogin-1 activity was not elevated at any time point. BoNT/A-induced polyubiquitination occurred after 3 days (3-fold increase) but was totally absent after 10 days. Proteasome activity was elevated (1.5- to 2-fold) after 3 and 10 days. We employed the use of heat shock protein 70 (Hsp70) to inhibit NF-κB and Foxo transcriptional activity. Electrotransfer of Hsp70 into rat soleus, before BoNT/A administration, was insufficient to attenuate atrophy. It was also insufficient to decrease BoNT/A-induced Foxo activity at 3 days, although NF-κB activity was abolished. By 10 days both NF-κB and Foxo activation were abolished by Hsp70. Hsp70-overexpression was unable to alter the levels of BoNT/A-induced effects on MuRF1/atrogin-1, polyubiquitination, or proteasome activity. In conclusion, Hsp70 overexpression is insufficient to attenuate BoNT/A-induced atrophy. It remains unclear what proteolytic mechanism/s are contributing to BoNT/A-induced atrophy, although a Foxo-MuRF1-ubiquitin-proteasome contribution may exist, at least in early BoNT/A-induced atrophy. Further clarification of UPS involvement in BoNT/A-induced atrophy is warranted.

Heat shock protein 70 (Hsp70) is a ubiquitous, cytoprotective protein that is up‐regulated in response to cellular stress. Botulinum neurotoxin type A (BoNT/A) administration causes a chemical denervation which leads to skeletal muscle atrophy in rats. Previous work in skeletal muscle has shown that Hsp70 inhibits both NF‐κB and FOXO activation in response to certain stimuli. During periods of atrophy, Hsp70 levels are decreased, while NF‐κB and FOXO activity are increased and required for atrophy in some models. Therefore, the purpose of the current study was to determine if electrotransfer of Hsp70 into the soleus muscle of rats, prior to BoNT/A administration, is sufficient to inhibit atrophy and NF‐κB/FOXO activation. BoNT/A administration induced ~70% atrophy of the soleus. BoNT/A + Hsp70 administration elicited only ~50% atrophy. Hsp70 prevented the BoNT/A‐induced increase in total ubiquitin conjugated proteins. Furthermore, BoNT/A‐induced NF‐κB transactivation was abolished by Hsp70 and levels of FOXO3a‐phosphorylation were maintained. In conclusion, while Hsp70 decreases total ubiquitin conjugated proteins and attenuates a portion of the observed skeletal muscle atrophy, these data suggest that pathways other than NF‐κB/FOXO may predominate in control of protein degradation. Supported by Ipsen Pharmaceuticals.

Skeletal muscles distal to the arterial blockage in Peripheral Arterial Disease (PAD) patients become ischemic during physical activity due to a mismatch between blood supply and the oxygen demands of the muscle. Thus, skeletal muscles of PAD patients undergo repeated cycles of ischemia-reperfusion daily as the patient is active, then rests. We hypothesize that this contributes to the muscle atrophy observed in PAD patients. The current work used a rodent model to determine whether repeated bouts of muscle contraction with restricted blood supply increases nuclear factor kappa B (NF-κB) signaling and causes skeletal muscle fiber atrophy. The femoral artery of rats was ligated (Lig) to restrict blood supply to muscles distal to the ligation and in vivo electrical stimulation (Stim) was used to cause repeated muscle contractions. Muscles were stimulated for 15 min, 4x/day, for 5 days. Lig & Stim caused an 18-fold increase in NF-κB activity, increased the expression of specific atrophy-related genes and caused a 35% decrease in skeletal muscle fiber cross sectional area, which were all abolished by in vivo gene transfer of a dominant negative inhibitor of κB α (IκBα). An NF-κB signaling pathway PCR array identified that several transcripts of the tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1) pathways were up-regulated in Lig & Stim muscles AND in the skeletal muscle of PAD patients. Treatment of C2C12 skeletal muscle cells with TNF-α, IL-1β or TNF-α plus IL-1β confirmed that these cytokines, alone and in combination, increase NF-κB-dependent transcription and atrophy gene expression. In conclusion, repeated muscle contractions (exercise) with restricted blood supply causes skeletal muscle atrophy which requires NF-κB activation. Supported by a James & Esther King Biomedical Research Program Grant, 08KN-07.

The arterial blockage in patients with peripheral arterial disease (PAD) restricts oxygen delivery to skeletal muscles distal to the blockage. In advanced-stage PAD patients, this creates a chronic ischemic condition in the affected muscles. However, in the majority of PAD patients, the muscles distal to the blockage only become ischemic during physical activity when the oxygen demands of these muscles are increased. Therefore, the skeletal muscle of most PAD patients undergoes repeated cycles of low-grade ischemia-reperfusion each time the patient is active and then rests. This has been speculated to contribute to the biochemical and morphological myopathies observed in PAD patients. The current study aimed to determine, using a rodent model, whether repeated hind limb muscle contractions during blood flow restriction to the hind limb muscles increases NF-κB activity. We, subsequently, determined whether an increase in NF-κB activity during this condition is required for the increased transcription of specific atrophy-related genes and muscle fiber atrophy. We found that hind limb muscle contractions during blood flow restriction to the limb increased NF-κB activity, the transcription of specific atrophy-related genes, and caused a 35% decrease in muscle fiber cross-sectional area. We further found that inhibition of NF-κB activity, via gene transfer of a dominant-negative inhibitor of κBα (d.n. IκBα), prevented the increase in atrophy gene expression and muscle fiber atrophy. These findings demonstrate that when blood flow to skeletal muscle is restricted, repeated cycles of muscle contraction can cause muscle fiber atrophy that requires NF-κB-IκBα signaling.

This study compared the efficacy of a 7% glucose polymer beverage containing electrolytes (GP) versus a nonnutrient, nonelectrolyte placebo (P) in maintaining blood homeostasis during recovery from football and determined whether consumption of the GP beverage improved anaerobic performance immediately after football competition when compared with the placebo. Forty-four high school football players participated in a 50-play scrimmage designed to simulate game conditions. At each of six periods before and during the scrimmage, players consumed 170 ml of the GP or P beverage. Eight maximal-effort 40-yd sprints (40-sec rest intervals) were performed before and after the scrimmage to assess the decrement in anaerobic performance from the scrimmage. Venous blood samples were drawn before and after the scrimmage and analyzed. The pre- to postscrimmage differences in mean and peak sprint velocities did not differ between treatments, nor did body weight and plasma. In contrast, the percent decrease in plasma volume was significantly greater in the P group. Postscrimmage increases in glucose and insulin were greater in the GP group. These data suggest that CHO-electrolyte drinks do not prevent a decline in anaerobic performance when compared to water, but a CHO-electrolyte drink is more effective in maintaining PV than water during recovery from anaerobic exercise.

We examined reactive oxygen species as upstream activators of nuclear factor kappa B; (NF-kappa B) and forkhead box O (Foxo) in skeletal muscle during disuse atrophy. Call an enzyme that degrades H2O2, was overexpressed in soleus muscles via plasmid injection prior to 7 days of hindlimb immobilization. The increased catalase activity abolished immobilization-induced transactivation of both NF-kappa B and Foxo and attenuated the loss of muscle mass. Thus, H2O2 may be an important initiator of these signaling pathways that lead to muscle atrophy. Muscle Nerve 41: 110-113, 2010

Botulinum neurotoxin type A (BoNT/A) is commonly used as a therapeutic treatment for a number of conditions associated with muscle spasticity in humans. BoNT/A creates a partial paralysis in skeletal muscle by inhibiting release of acetylcholine at the neuromuscular junction; effectively creating a chemical denervation. Like mechanical denervation, this results in significant skeletal muscle atrophy. Our prior studies have shown that heat shock protein 70 (Hsp70) is significantly reduced during various models of skeletal muscle atrophy and, if overexpressed, provides protection against atrophy. PURPOSE: To determine the effect of BoNT/A on Hsp70 expression and CSA in skeletal muscle. METHODS: Male, Sprague-Dawley rats were injected with either a control buffer or three units of Botulinum type A in the triceps surae muscle group. Hindlimbs were then stimulated to confirm force deficit in the triceps surae group and animals were allowed to ambulate normally for either 7 or 14 days. Soleus, plantaris, and gastrocnemius muscles were removed, weighed, and rapidly frozen for cross sectional analysis and Hsp70 protein expression. RESULTS: Force deficits of ∼80% confirmed chemical denervation of the triceps surae muscle group. Gastrocnemius, soleus, and plantaris muscle weights were all significantly decreased (∼36%; ∼31%; ∼54%, respectively) 14 days after injection. Similarly, soleus muscle fiber CSA was significantly decreased (∼25%) in injected limbs. Hsp70 protein expression remained unchanged in the plantaris and soleus muscles 7 and 14 days after BoNT/A injection. CONCLUSION: These data suggest that small doses of BoNT/A cause significant muscle atrophy in a short time period. Unlike other reduced-use models of atrophy, BoNT/A does not cause a reduction in Hsp70. Supported by Ipsen Pharmaceuticals, Ltd.

The purpose of the current study was to determine whether heat shock protein 70 (Hsp70) directly regulates forkhead box O (FOXO) signaling in skeletal muscle. This aim stems from previous work demonstrating that Hsp70 overexpression inhibits disuse-induced FOXO transactivation and prevents muscle fiber atrophy. However, although FOXO is sufficient to cause muscle wasting, no data currently exist on the requirement of FOXO signaling in the progression of physiological muscle wasting, in vivo. In the current study we show that specific inhibition of FOXO, via expression of a dominant-negative FOXO3a, in rat soleus muscle during disuse prevented >40% of muscle fiber atrophy, demonstrating that FOXO signaling is required for disuse muscle atrophy. Subsequent experiments determined whether Hsp70 directly regulates FOXO3a signaling when independently activated in skeletal muscle, via transfection of FOXO3a. We show that Hsp70 inhibits FOXO3a-dependent transcription in a gene-specific manner. Specifically, Hsp70 inhibited FOXO3a-induced promoter activation of atrogin-1, but not MuRF1. Further studies showed that a FOXO3a DNA-binding mutant can activate MuRF1, but not atrogin-1, suggesting that FOXO3a activates these two genes through differential mechanisms. In summary, FOXO signaling is required for physiological muscle atrophy and is directly inhibited by Hsp70.

Heat shock protein 25/27 (Hsp25/27) is a cytoprotective protein that is ubiquitously expressed in most cells, and is up-regulated in response to cellular stress. Previous work, in nonmuscle cells, has shown that Hsp27 inhibits TNF-alpha-induced NF-kappaB activation. During skeletal muscle disuse, Hsp25/27 levels are decreased and NF-kappaB activity increased, and this increase in NF-kappaB activity is required for disuse muscle atrophy. Therefore, the purpose of the current study was to determine whether electrotransfer of Hsp27 into the soleus muscle of rats, prior to skeletal muscle disuse, is sufficient to inhibit skeletal muscle disuse atrophy and NF-kappaB activation. The 35% disuse muscle-fiber atrophy observed in nontransfected fibers was attenuated by 50% in fibers transfected with Hsp27. Hsp27 also inhibited the disuse-induced increase in MuRF1 and atrogin-1 transcription by 82 and 40%, respectively. Furthermore, disuse- and IKKbeta-induced NF-kappaB transactivation were abolished by Hsp27. In contrast, Hsp27 had no effect on Foxo transactivation. In conclusion, Hsp27 is a negative regulator of NF-kappaB in skeletal muscle, in vivo, and is sufficient to inhibit MuRF1 and atrogin-1 and attenuate skeletal muscle disuse atrophy.

This study determined the effects of heat shock protein 70 (Hsp70) overexpression on disuse muscle atrophy in senescent rats. Solei of young and senescent rats were co-injected with Hsp70 plus a nuclear factor kappa B (NF-κΒ) reporter plasmid. After 4 days, the hind limbs of half the young and senescent rats were immobilized for 6 days with the remainder serving as weight bearing controls. Hsp70 protein levels and cross-sectional area decreased in both groups (~20%) after immobilization. Atrophy was prevented in those fibers overexpressing Hsp70. NF-κΒ activity increased in the soleus of both young (three-fold) and senescent (five-fold) animals after immobilization and was prevented by Hsp70 overexpression. Inhibitor of κΒ decreased in young (~30%) and senescent (~10%) animals with immobilization and returned to normal with Hsp70. Heat shock protein 70 overexpression prevents disuse atrophy in senescent rats, possibly through suppression of the NF-κB pathway.

To ensure safety and predictable clinical efficacy, the biological activity of type A botulinum toxin (BoNT-A) preparations must remain consistent. Several methods have been employed to assess consistency but lack clinical applicability and/or are associated with animal welfare concerns. Here, we describe a novel in vivo rat muscle force model for evaluating the biological activity of formulated BoNT-A product (Dysport) prepared from bulk toxin batches manufactured at different facilities. Toxin activity was assessed by measuring muscle force generation over time in the triceps surae muscles in the rat hind leg. Animals received 0.1 ml gelatine phosphate buffer (negative vehicle control) or 0.1 or 1.0 LD50 units of BoNT-A in phosphate buffer. Batch equivalence and consistency were confirmed by the lack of significant differences in muscle force generation and duration of effect between each test batch and the reference preparation tested in the same series of experiments. The reduction in muscle force generation was dose-related and reproducible for all active treatment groups. At appropriate dose levels, the rat muscle force model is a reliable tool for measuring biological activity in bulk toxin batches used to formulate clinical product and demonstrates the consistency of batches manufactured over many years.

We have previously shown oxidative stress and oedema, caused by both xanthine oxidase‐derived oxidants and infiltrating neutrophils, within skeletal muscle after contractile‐induced claudication. The purpose of this study was to determine whether supplementation with antioxidant vitamins attenuates the oxidative stress, neutrophil infiltration and oedema associated with an acute bout of contractile‐induced claudication. Rats received vehicle, vitamin C, vitamin E or vitamin C + E for 5 days prior to contractile‐induced claudication. Force production was significantly reduced in the claudicant limbs of all groups compared with the control (sham) limb of control animals. Contractile‐induced claudication caused a significant increase in protein oxidation, lipid peroxidation, neutrophil infiltration and oedema compared with sham muscles. Supplementation with vitamin C, E or C + E prevented the increases in each of these, and there were no differences between groups. These findings suggest that, in an animal model of exercise‐induced claudication, neutrophil chemotaxis is caused by oxidizing species and that antioxidant supplementation can prevent oxidative damage, neutrophil infiltration and oedema following an acute bout of contractile‐induced claudication.

Sirs,The recent review by Drs de Almeida and de Boulle [1], describing probable diffusion characteristics of the botulinum neurotoxin products when used in aesthetic and other indications, has soug...

Heat shock protein 70 (Hsp70) is a highly conserved and ubiquitous protein that is reported to provide cytoprotection in various cell types and tissues. However, the importance of Hsp70 expression during skeletal muscle atrophy, when Hsp70 levels are significantly decreased, is not known. The current study aimed to determine whether plasmid-mediated overexpression of Hsp70, in the soleus muscle of rats, was sufficient to regulate specific atrophy signaling pathways and attenuate skeletal muscle disuse atrophy. We found that Hsp70 overexpression prevented disuse muscle fiber atrophy and inhibited the increased promoter activities of atrogin-1 and MuRF1. Importantly, the transcriptional activities of Foxo3a and NF-kappaB, which are implicated in the regulation of atrogin-1 and MuRF1, were abolished by Hsp70. These data suggest that Hsp70 may regulate key atrophy genes through inhibiting Foxo3a and NF-kappaB activities during disuse. Indeed, we show that specific inhibition of Foxo3a prevented the increases in both atrogin-1 and MuRF1 promoter activities during disuse. However, inhibition of NF-kappaB did not affect the activation of either promoter, suggesting its requirement for disuse atrophy is through its regulation of other atrophy genes. We conclude that overexpression of Hsp70 is sufficient to inhibit key atrophy signaling pathways and prevent skeletal muscle atrophy.

In recent years, an increasing number Of Studies have demonstrated that a myopathy is present, contributes, and, to a certain extent, determines the pathogenesis of peripheral arterial occlusive disease. These works provide evidence that a state of repetitive cycles of exercise-induced ischemia followed by reperfusion at rest operates in patients with peripheral arterial occlusive disease and mediates a large number of structural and metabolic changes in the muscle, resulting in reduced strength and function. The key players in this process appear to be defective mitochondria that, through multilevel failure in their roles as energy, oxygen radical species, and apoptosis regulators, produce and sustain a progressive decline in muscle performance. In this 2-part review, the currently available evidence that characterizes the nature and mechanisms responsible for this myopathy is highlighted. In part 1, the functional and histomorphological characteristics of the myopathy were reviewed, and the main focus was on the biochemistry and bioenergetics of its mitochondriopathy. In part 2, accumulating evidence that oxidative stress related to ischemia reperfusion is probably the major operating mechanism of peripheral arterial occlusive disease myopathy is reviewed. Important new findings of a possible neuropathy and a shift in muscle fiber type are also reviewed. Learning more about these mechanisms will enhance our understanding. of the degree to which they are preventable and treatable.

Abnormal mitochondrial function is present in patients with peripheral arterial disease and may contribute to its clinical manifestations. However, the specific biochemical mitochondrial defects and their association with increased oxidative stress have not been fully characterized. Gastrocnemius muscle was obtained from peripheral arterial disease patients (n = 25) and age-matched controls (n = 16) and mitochondrial parameters were measured. Complexes I through IV of the electron transport chain were individually evaluated to assess for isolated defects. Muscle was also evaluated for protein and lipid oxidative changes by measuring the levels of protein carbonyls, lipid hydroperoxides, and total 4-hydroxy-2-nonenal binding and for the activities of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase. Mitochondrial electron transport chain complexes I, III, and IV in arterial disease patients demonstrated significant reductions in enzymatic activities and mitochondrial respiration compared to controls. Oxidative stress biomarker analysis demonstrated significantly increased levels of protein carbonyls, lipid hydroperoxides, and 4-hydroxy-2-nonenal compared to control muscle. Antioxidant enzyme activities were altered, with a significant decrease in superoxide dismutase activity and significant increases in catalase and glutathione peroxidase. Peripheral arterial disease is associated with abnormal mitochondrial function and evidence of significant oxidative stress.