90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we show that the isolated N-terminal kinase of RSK2 (amino acids 1-360) is phosphorylated at Ser(227) by PDK1, a constitutively active kinase, leading to 100-fold stimulation of kinase activity. In COS7 cells, ectopic PDK1 induced the phosphorylation of full-length RSK2 at Ser(227) and Ser(386), without involvement of ERK, leading to partial activation of RSK2. Similarly, two other members of the RSK family, RSK1 and RSK3, were partially activated by PDK1 in COS7 cells. Finally, our data indicate that full activation of RSK2 by growth factor requires the cooperation of ERK and PDK1 through phosphorylation of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.

Mutations in myostatin (GDF8) cause marked increases in muscle mass, suggesting that this transforming growth factor‐β (TGF‐β) superfamily member negatively regulates muscle growth. Myostatin blockade therefore offers a strategy for reversing muscle wasting in Duchenne's muscular dystrophy (DMD) without resorting to genetic manipulation. Here, we demonstrate that pharmacological blockade using a myostatin propeptide stabilized by fusion to IgG‐Fc improved pathophysiology of the mdx mouse model of DMD. Functional benefits evidenced by specific force improvement, exceeded those reported previously using myostatin antibody‐mediated blockade. More importantly, use of a propeptide blockade strategy obviates possibilities of anti‐idiotypic responses that could potentially limit the effectiveness of antibody‐mediated myostatin blockade strategies over time. This study provides a novel pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD and since it uses an endogenous inhibitor of myostatin should help circumvent technical hurdles and toxicity associated with conventional gene or cell based therapies. FASEB J. 19, 543–549 (2005)

Exercise intolerance is a prominent symptom in patients with mitochondrial myopathy (MM), but it is still unsettled whether exercise training is safe and beneficial for patients with MM. To address this, we studied the effect of 12 weeks cycle training on exercise capacity, quality of life and underlying molecular and cellular events in five patients with single large-scale deletions, one with a microdeletion and 14 with point mutations of mitochondrial DNA (mtDNA), and 13 healthy subjects. Each training session lasted 30 min, and was performed at an intensity of 70% of VO_2max (maximal oxygen uptake). Each subject performed 50 training sessions in 12 weeks. All subjects were evaluated before and after training, and 13 MM patients were studied after 8 weeks of deconditioning. Evaluation included VO_2max and mutation load and mtDNA quantity, mitochondrial enzymatic activity, and number of centrally nucleated, apoptotic, ragged red and cytochrome oxidase (COX)-negative fibres in muscle biopsies from the quadriceps muscle. After 12 weeks of training, VO_2max and muscle citrate synthase increased in MM (26 and 67%) and healthy (17 and 65%) subjects, while mtDNA quantity in muscle only increased in the MM patients (81%). In the MM patients, training did not change mtDNA mutation load in muscle, mitochondrial enzyme complex activities, muscle morphology and plasma creatine kinase. After deconditioning, VO_2max and citrate synthase activity returned to values before training, while muscle mtDNA mutation load decreased. These findings show that aerobic training efficiently improves oxidative capacity in MM patients. Based on unchanged levels of mutant load in muscle, morphological findings on muscle biopsy and plasma creatine kinase levels during training, the treatment appears to be safe. Regular, supervised aerobic exercise is therefore recommended in MM patients with the studied mutations.

Studies in a dystrophinopathy model (the mdx mouse) suggest that exercise training may be deleterious for muscle integrity, but exercise has never been studied in detail in humans with defects of dystrophin. We studied the effect of endurance training on conditioning in patients with the dystrophinopathy, Becker muscular dystrophy (BMD). Eleven patients with BMD and seven matched, healthy subjects cycled 50, 30 min sessions at 65% of their maximal oxygen uptake (VO_2max) over 12 weeks, and six patients continued cycling for 1 year. VO_2max, muscle biopsies, echocardiography, plasma creatine kinase (CK), lower extremity muscle strength and self-reported questionnaires were evaluated before, after 12 weeks and 1 year of training. Endurance training for 12 weeks, improved VO_2max by 47 ± 11% and maximal workload by 80 ± 19% in patients (P < 0.005). This was significantly higher than in healthy subjects (16 ± 2% and 17 ± 2%). CK levels did not increase with training, and number of central nuclei, necrotic fibres and fibres expressing neonatal myosin heavy chain did not change in muscle biopsies. Strength in muscles involved in cycle exercise (knee extension, and dorsi- and plantar-flexion) increased significantly by 13–40%. Cardiac pump function, measured by echocardiography, did not change with training. All improvements and safety markers were maintained after 1 year of training. Endurance training is a safe method to increase exercise performance and daily function in patients with BMD. The findings support an active approach to rehabilitation of patients with BMD.

Duchenne's muscular dystrophy (DMD) is a fatal neuromuscular disease caused by absence of dystrophin. Utrophin is a chromosome 6-encoded dystrophin-related protein (DRP), sharing functional motifs with dystrophin. Utrophin's ability to compensate for dystrophin during development and when transgenically overexpressed has provided an important impetus for identifying activators of utrophin expression. The utrophin promoter A is transcriptionally regulated in part by heregulin-mediated, extracellular signal-related kinase-dependent activation of the GABP _α/β transcription factor complex. Therefore, this pathway offers a potential mechanism to modulate utrophin expression in muscle. We tested the ability of heregulin to improve the dystrophic phenotype in the mdx mouse model of DMD. Intraperitoneal injections of a small peptide encoding the epidermal growth factor-like region of heregulin ectodomain for 3 months in vivo resulted in up-regulation of utrophin, a marked improvement in the mechanical properties of muscle as evidenced by resistance to eccentric contraction mediated damage, and a reduction of muscle pathology. The amelioration of dystrophic phenotype by heregulin-mediated utrophin up-regulation offers a pharmacological therapeutic modality and obviates many of the toxicity and delivery issues associated with viral vector-based gene therapy for DMD.