Loss of cIAP1 attenuates soleus muscle pathology and improves diaphragm function in mdx mice.

The cellular inhibitor of apoptosis 1 (cIAP1) protein is an essential regulator of canonical and noncanonical nuclear factor κB (NF-κB) signaling pathways. NF-κB signaling is known to play important roles in myogenesis and degenerative muscle disorders such as Duchenne muscular dystrophy (DMD), but the involvement of cIAP1 in muscle disease has not been studied directly. Here, we asked whether the loss of cIAP1 would influence the pathology of skeletal muscle in the mdx mouse model of DMD. Double-mutant cIAP1(-/-);mdx mice exhibited reduced muscle damage and decreased fiber centronucleation in the soleus, compared with single-mutant cIAP1(+/+);mdx mice. This improvement in pathology was associated with a reduction in muscle infiltration by macrophages and diminished expression of inflammatory cytokines such as IL-6 and tumor necrosis factor-α. Furthermore, the cIAP1(-/-);mdx mice exhibited reduced serum creatine kinase, and improved exercise endurance associated with improved exercise resilience by the diaphragm. Mechanistically, the loss of cIAP1 was sufficient to drive constitutive activation of the noncanonical NF-κB pathway, which led to increased myoblast fusion in vitro and in vivo. Collectively, these results show that the loss of cIAP1 protects skeletal muscle from the degenerative pathology resulting from systemic loss of dystrophin.

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle.

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated ß-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.

Fatigue preconditioning increases fatigue resistance in mouse flexor digitorum brevis muscles with non-functioning K(ATP) channels.

The objective of this study was to determine how an initial fatigue bout (FAT1 at 37°C) affects free myoplasmic Ca(2+) concentration and force ([Ca(2+)](i)/force) during a subsequent fatigue bout (FAT2) in mouse flexor digitorum brevis (FDB). During FAT1, both tetanic [Ca(2+)](i)/force decreased; however, they decreased to significantly lower levels when FAT1 was carried out in the presence of glibenclamide, a sarcolemmal K(ATP) (sK(ATP)) channel blocker. Glibenclamide also elicited greater increases in unstimulated [Ca(2+)](i)/force, which occurred when fibres failed to fully relax between contractions during FAT1. Finally, glibenclamide impaired force recovery after FAT1. The decreases in tetanic [Ca(2+)](i)/force and increases in unstimulated [Ca(2+)](i)/force were slower during FAT2 elicited 60 min after FAT1. Under control conditions, the effects were small with very few significant differences. In the presence of glibenclamide, on the other hand, the differences between FAT1 and FAT2 were very large. Unexpectedly, the differences in unstimulated and tetanic [Ca(2+)](i)/force between control and glibenclamide conditions observed during FAT1 were no longer observed during FAT2. The lack of differences was not related to a failure of glibenclamide to block K(ATP) channels during FAT2 because the effects of FAT1 on FAT2 were also observed using Kir6.2(-/-) mouse FDB, which lack sK(ATP) channel activity. The differences in [Ca(2+)](i)/force between FAT1 and FAT2 could be observed with FAT1 duration of just 30 s and a FAT1-FAT2 interval of at least 30 min. A modulation of factors involved in ischaemic pre-conditioning, i.e. A1-adenosine receptors, sK(ATP) and mitochondrial K(ATP) (mK(ATP)) channels, PKC and reactive oxygen species, during FAT1 had no effect on FAT2 fatigue kinetics. It is concluded that a preceding fatigue bout triggers an acute physiological process that prevents the contractile dysfunction induced by non-functioning K(ATP) channels.

Pharmacological activation of PPARbeta/delta stimulates utrophin A expression in skeletal muscle fibers and restores sarcolemmal integrity in mature mdx mice.

A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.

Contractile dysfunctions in ATP-dependent K+ channel-deficient mouse muscle during fatigue involve excessive depolarization and Ca2+ influx through L-type Ca2+ channels.

Muscles deficient in ATP-dependent potassium (KATP) channels develop contractile dysfunctions during fatigue that may explain their apparently faster rate of fatigue compared with wild-type muscles. The objectives of this study were to determine: (1) whether the contractile dysfunctions, namely unstimulated force and depressed force recovery, result from excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) whether reducing the magnitude of these two contractile dysfunctions reduces the rate of fatigue in KATP channel-deficient muscles. To reduce Ca2+ influx, we lowered the extracellular Ca2+ concentration ([Ca2+]o) from 2.4 to 0.6 mM or added 1 microM verapamil, an L-type Ca2+ channel blocker. Flexor digitorum brevis (FDB) muscles deficient in KATP channels were obtained by exposing wild-type muscles to 10 microM glibenclamide or by using FDB from Kir6.2-/- mice. Fatigue was elicited with one contraction per second for 3 min at 37 degrees C. In wild-type FDB, lowered [Ca2+]o or verapamil did not affect the decrease in peak tetanic force and unstimulated force during fatigue and force recovery following fatigue. In KATP channel-deficient FDB, lowered [Ca2+]o or verapamil slowed down the decrease in peak tetanic force recovery, reduced unstimulated force and improved force recovery. In Kir6.2-/- FDB, the rate of fatigue became slower than in wild-type FDB in the presence of verapamil. The cell membrane depolarized from -83 to -57 mV in normal wild-type FDB. The depolarizations in some glibenclamide-exposed fibres were similar to those of normal FDB, while in other fibres the cell membrane depolarized to -31 mV in 80 s, which was also the time when these fibres supercontracted. It is concluded that: (1) KATP channels are crucial in preventing excessive membrane depolarization and Ca2+ influx through L-type Ca2+ channels; and (2) they contribute to the decrease in force during fatigue.