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Earlier studies have shown that the progressive, unrelenting muscle loss associated with Duchenne muscular dystrophy (DMD) involves an imbalance between the rates of synthesis and degradation of muscle proteins. Although previous studies have suggested that oxandrolone may be beneficial in DMD, the mechanism of action of oxandrolone on muscle in DMD remains unclear. To address these issues, we combined stable isotope studies and gene expression analysis to measure the fractional synthesis rate of myosin heavy chain (MHC), the key muscle contractile protein, the transcript levels of the isoforms of MHC, and global gene expression profiles in four children with DMD before and after 3 mo of treatment with oxandrolone. Gastrocnemius muscle biopsies and blood samples were collected during the course of a primed 6-h continuous infusion of l-[U-(13)C]leucine on two separate occasions, before and after the 3-mo treatment with oxandrolone (0.1 mg.kg(-1).day(-1)). Gene expression analysis was done with microarrays and RT-qPCR. In response to the treatment, MHC synthesis rate increased 42%, and this rise was accounted for, at least in part, by an upregulation of the transcript for MHC8 (perinatal MHC). Gene expression data suggested a decrease in muscle regeneration as a consequence of oxandrolone therapy, presumably because of a decrease in muscle degeneration. These findings suggest that 1) oxandrolone has a powerful protein anabolic effect on a key contractile protein and 2) larger and longer-term studies are warranted to determine whether these changes translate into meaningful therapy for these patients.

The effects of the nitric oxide (NO) donor spermine NONOate (Sp-NO, 1.0 mM) on cross-bridge recruitment and cross-bridge cycling kinetics were studied in permeabilized rabbit psoas muscle fibers. Fibers were activated at various Ca2+ concentrations (pCa, negative logarithm of Ca2+ concentration), and the pCa at which force was maximal (pCa 4.0) and approximately 50% of maximal (pCa50 5.6) were determined. Fiber stiffness was determined using 1-kHz sinusoidal length perturbations, and the fraction of cross bridges in the force-generating state was estimated by the ratio of stiffness during maximal (pCa 4.0) and submaximal (pCa 5.6) Ca2+ activation to stiffness during rigor (at pCa 4.0). Cross-bridge cycling kinetics were evaluated by measuring the rate constant for force redevelopment after quick release (by 15% of optimal fiber length, L(o)) and restretch of the fiber to L(o). Exposing fibers to Sp-NO for 10 min reduced force and the fraction of cross bridges in the force-generating state at maximal and submaximal (pCa50) Ca2+ activation. However, the effects of Sp-NO were more pronounced during submaximal Ca2+ activation. Sp-NO also reduced the rate constant for force redevelopment but only during submaximal Ca2+ activation. We conclude that Sp-NO reduces Ca2+ sensitivity by decreasing the number of cross bridges in the strongly bound state and also impairs cross-bridge cycling kinetics during submaximal activation.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 microM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12 mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]i in activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]i level and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]i responses (25 +/- 7% reduction with 10 microM DEA-NO and 78 +/- 14% reduction with 100 microM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 microM) also reduced the amplitude of the caffeine-induced [Ca2+]i response. Similar data were obtained by using the chemically distinct NO donor S-nitroso-N-acetyl-penicillamine (100 microM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+ release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.

Numerous studies have explored the energetic properties of skeletal and cardiac muscle fibers. In this mini-review, we specifically explore the interactions between actin and myosin during cross-bridge cycling and provide a conceptual framework for the chemomechanical transduction that drives muscle fiber energetic demands. Because the myosin heavy chain (MHC) is the site of ATP hydrolysis and actin binding, we focus on the mechanical and energetic properties of different MHC isoforms. Based on the conceptual framework that is provided, we discuss possible sites where muscle remodeling may impact the energetic demands of contraction in skeletal and cardiac muscle.

Maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) and ATP consumption rate during maximum isometric activation (ATP(iso)) were determined in human vastus lateralis (VL) muscle fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP consumption [1 -- (ratio of ATP(iso) to V(max) ATPase)] varies across VL muscle fibers expressing different MHC isoforms. Biopsies were obtained from 12 subjects (10 men and 2 women; age 21--66 yr). A quantitative histochemical procedure was used to measure V(max) ATPase. In permeabilized fibers, ATP(iso) was measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption was lower for fibers coexpressing MHC(2X) and MHC(2A) compared with fibers singularly expressing MHC(2A) and MHC(slow) (39 vs. 52 and 56%, respectively). Tension cost (ratio of ATP(iso) to generated force) also varied with fiber type, being highest in fibers coexpressing MHC(2X) and MHC(2A). We conclude that fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such as susceptibility to fatigue.

In a previous study, we found that in house mice both genetic selection (10 generations of artificial selection for high voluntary activity on running wheels) and access to running wheels (7-8 weeks) elicited a modest increase in maximal oxygen consumption. Based on these results, we hypothesized that genetic selection would affect the changes in endurance and oxidative capacity of the medial gastrocnemius (MG) muscle induced by wheel access (training response). Wheel access increased the isotonic endurance of the MG in both genetically selected and random-bred (control) mice. However, this exercise-induced improvement in isotonic endurance of the MG was similar between genetically selected and control mice. Wheel access also increased the succinate dehydrogenase activity of MG muscle fibers in both selected and control lines. However, this exercise-induced increase in succinate dehydrogenase activity was comparable between genetically selected and control animals. Taken together, these results indicate that the modest increase in maximal oxygen consumption associated with genetic selection is not reflected by the training-induced changes in oxidative capacity and endurance of MG muscle fibers.

The present study used real-time confocal microscopy to examine the effects of the beta2-adrenoceptor agonist salbutamol on regulation of intracellular Ca2+ concentration ([Ca2+]i) in myotubes derived from neonatal mouse limb muscles. Immunocytochemical staining for ryanodine receptors and skeletal muscle myosin confirmed the presence of sarcomeres. The myotubes displayed both spontaneous and ACh-induced rapid (<2-ms rise time) [Ca2+]i transients. The [Ca2+]i transients were frequency modulated by both low and high concentrations of salbutamol. Exposure to alpha-bungarotoxin and tetrodotoxin inhibited ACh-induced [Ca2+]i transients and the response to low concentrations of salbutamol but not the response to higher concentrations. Preexposure to caffeine inhibited the subsequent [Ca2+]i response to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the [Ca2+]i response to caffeine. Inhibition of dihydropyridine-sensitive Ca2+ channels with nifedipine or PN-200-110 did not prevent [Ca2+]i elevations induced by higher concentrations of salbutamol. The effects of salbutamol were mimicked by the membrane-permeant analog dibutyryl adenosine 3', 5'-cyclic monophosphate. These data indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulum Ca2+ release.

The effects of the beta2-adrenoceptor agonist salbutamol (Slb) on isometric and isotonic contractile properties of the rat diaphragm muscle (Diamus) were examined. A loading dose of 25 microg/kg Slb was administered intracardially before Diamus excision to ensure adequate diffusion. Studies were then performed with 0.05 microM Slb in the in vitro tissue chamber. cAMP levels were determined by radioimmunoassay. Compared with controls (Ctl), cAMP levels were elevated after Slb treatment. In Slb-treated rats, isometric twitch and maximum tetanic force were increased by approximately 40 and approximately 20%, respectively. Maximum shortening velocity increased by approximately 15% after Slb treatment, and maximum power output increased by approximately 25%. During repeated isotonic activation, the rate of fatigue was faster in the Slb-treated Diamus, but both Slb-treated and Ctl Diamus fatigued to the same maximum power output. Still, endurance time during repetitive isotonic contractions was approximately 10% shorter in the Slb-treated Diamus. These results are consistent with the hypothesis that beta-adrenoceptor stimulation by Slb enhances Diamus contractility and that these effects of Slb are likely mediated, at least in part, by elevated cAMP.

Nitric oxide (NO) may exert direct effects on actin-myosin cross-bridge cycling by modulating critical thiols on the myosin head. In the present study, the effects of the NO donor sodium nitroprusside (SNP; 100 microM to 10 mM) on mechanical properties and actomyosin adenosinetriphosphatase (ATPase) activity of single permeabilized muscle fibers from the rabbit psoas muscle were determined. The effects of N-ethylmaleimide (NEM; 5-250 microM), a thiol-specific alkylating reagent, on mechanical properties of single fibers were also evaluated. Both NEM (>/=25 microM) and SNP (>/=1 mM) significantly inhibited isometric force and actomyosin ATPase activity. The unloaded shortening velocity of SNP-treated single fibers was decreased, but to a lesser extent, suggesting that SNP effects on isometric force and actomyosin ATPase were largely due to decreased cross-bridge recruitment. The calcium sensitivity of SNP-treated single fibers was also decreased. The effects of SNP, but not NEM, on force and actomyosin ATPase activity were reversed by treatment with 10 mM DL-dithiothreitol, a thiol-reducing agent. We conclude that the NO donor SNP inhibits contractile function caused by reversible oxidation of contractile protein thiols.

Diaphragm and latissimus dorsi muscle functions, histochemistries, and morphometries were studied in anesthetized male Yucatan minipigs with congestive heart failure (CHF) induced by supraventricular tachycardia (n = 5). Sham-operated animals served as a control group (n = 5). In CHF animals, transdiaphragmatic pressure measured during supramaximal phrenic stimulation was reduced by 40% at low frequencies (< or = 20 Hz) and by 60% at higher frequencies. Twitch amplitude and half-relaxation time were also decreased. The cross-sectional areas of type I, IIa, and IIb fibers were reduced in the diaphragm. The proportion of type I fibers increased, whereas type IIa fibers decreased. Succinate dehydrogenase activity was elevated in type IIa and IIb fibers, but diaphragmatic fatigability was not altered. CHF reduced latissimus dorsi isometric force by 40% for stimulation frequencies > or = 30 Hz. The cross-sectional area of latissimus dorsi type IIb fibers was decreased, but twitch characteristics, fiber type composition, succinate dehydrogenase activity, and fatigability were unchanged. Experimental CHF appears to cause greater intrinsic adaptive changes in the diaphragm compared with those in the latissimus dorsi in the minipig. For both muscles, reduced contractile function was associated with atrophy. Impaired performance of the diaphragm may also be attributed to an increase in the relative contribution of type I fibers to the total tension-generating capacity of the muscle and to the pathophysiological mechanisms underlying the shortened relaxation time of the twitch response.

Muscles of the male rat perineum attaching to the penis are the ischiocavernosus, the medial bulbocavernosus (BC), and the dorsal BC, also known as the levator ani (LA). The BC and the LA muscles are innervated by a morphologically, neurochemically, and physiologically homogeneous pool of motoneurons, the spinal nucleus of BC (SNB). The purpose of the present study was to determine whether BC and LA muscle fibers constitute histochemically and biochemically homogeneous populations, reflecting the homogeneity of the innervating motoneuron pool. Histochemical fiber type was based on the pH lability of myofibrillar adenosinetriphosphatase. Activity of the mitochondrial enzyme succinate dehydrogenase (SDH) was determined by using a quantitative histochemical procedure. A nonsexually dimorphic, androgen-insensitive muscle, the medial gastrocnemius (MG), was used as control. The superficial regions of the MG, BC, and LA muscles were composed exclusively of type IIb muscle fibers, whereas the fiber type composition of the deep region of the MG was mixed: 28.3% type I, 20.6% type IIa, 40.1% type IIx, and 11.0% type IIb. The SDH activities of type IIb fibers in the deep region of the MG ranged from 1.20 to 9.00 (mean 3.72 +/- 0.40) mmol fumarate.liter tissue-1.min-1. Fiber SDH activities in the superficial region of the MG ranged from 0.04 to 2.70 (mean 1.20 +/- 0.21) mmol fumarate.liter tissue-1.min-1. In the BC muscle, the SDH activities of the type IIb fibers ranged from undetectable to 1.80 with a mean of 0.62 +/- 0.05 mmol fumarate.liter tissue-1.min-1.(ABSTRACT TRUNCATED AT 250 WORDS)