Francisco H. Andrade

EFFECT OF HYDROGEN PEROXIDE AND DITHIOTHREITOL ON CONTRACTILE FUNCTION OF SINGLE SKELETAL MUSCLE FIBRES FROM THE MOUSE

1 We used intact single fibres from a mouse foot muscle to study the role of oxidation‐reduction in the modulation of contractile function. 2 The oxidant hydrogen peroxide (H2O2, 100‐300 μM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. 3 The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. 4 The reductant dithiothreitol (DTT, 0.5‐1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. 5 These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation‐reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

Extraocular muscle is defined by a fundamentally distinct gene expression profile.

Skeletal muscle fibers are defined by patterned covariation of key traits that determine contractile and metabolic characteristics. Although the functional properties of most skeletal muscles result from their proportional content of a few conserved muscle fiber types, some, typically craniofacial, muscles exhibit fiber types that appear to lie outside the common phenotypic range. We analyzed gene expression profiles of three putative muscle classes, limb, masticatory, and extraocular muscle (EOM), in adult mice by high-density oligonucleotide arrays. Pairwise comparisons using conservative acceptance criteria identified expression differences in 287 genes between EOM and limb and/or masticatory muscles. Use of significance analysis of microarrays methodology identified up to 400 genes as having an EOM-specific expression pattern. Genes differentially expressed in EOM reflect key aspects of muscle biology, including transcriptional regulation, sarcomeric organization, excitation-contraction coupling, intermediary metabolism, and immune response. These patterned differences in gene expression define EOM as a distinct muscle class and may explain the unique response of these muscles in neuromuscular diseases.

Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation

Endogenous peroxides and related reactive oxygen species may influence various steps in the contractile process. Single mouse skeletal muscle fibers were used to study the effects of hydrogen peroxide (H2O2) and t‐butyl hydroperoxide (f‐BOOH) on force and myoplasmic Ca2+ concentration ([Ca2+]i). Both peroxides (10−1 to 10−5 M) decreased tetanic [Ca2+]i and increased force during submaximal tetani. Catalase (1 kU/ml) blocked the effect of H2O2, but not of t‐BOOH. The decrease in tetanic [Ca2+]i was constant, while the effect on force was biphasic: A transitory increase was followed by a steady decline to the initial level. Myofibrillar Ca2+ sensitivity remained increased during incubation with either peroxide. Only the highest peroxide concentration (10 μM) increased resting [Ca2+]i and slowed the return of [Ca2+]i to its resting level after a contraction, evidence of impaired sarcoplasmic reticulum Ca2+ re‐uptake. The peroxides increased maximal force production and the rate of force redevelopment, and decreased maximum shortening velocity. N‐ethylmaleimide (25 μM, thiol‐alkylating agent) prevented the response to 1 μM H2O2. These results show that myofibrillar Ca2+ sensitivity and cross‐bridge kinetics are influenced by H2O2 and t‐BOOH concentrations that approach those found physiologically, and these findings indicate a role for endogenous oxidants in the regulation of skeletal muscle function.

Effect of nitric oxide on single skeletal muscle fibres from the mouse

1 Single skeletal muscle fibres from a mouse foot muscle were used to investigate the effects of nitric oxide on contractile function. 2 We measured force production and myoplasmic free Ca2+ concentration ([Ca2+]i) in single fibres exposed to the nitric oxide donors S‐nitroso‐N‐acetylcysteine (SNAC) and nitroprusside. 3 The nitric oxide donors reduced myofibrillar Ca2+ sensitivity, whereas [Ca2+]i transients were increased during submaximal tetani. Force was largely unchanged. SNAC did not change maximum shortening velocity, the rate of force redevelopment, or force production at saturating [Ca2+]i. 4 The guanylyl cyclase inhibitor LY83583 increased tetanic [Ca2+]i but had no effect on Ca2+ sensitivity. LY83583 did not prevent the decrease in myofibrillar Ca2+ sensitivity in response to SNAC. The oxidizer sodium nitrite increased tetanic [Ca2+]i and decreased myofibrillar Ca2+ sensitivity. 5 We conclude that under our experimental conditions nitric oxide impairs Ca2+ activation of the actin filaments which results in decreased myofibrillar Ca2+ sensitivity.

Reactive oxygen species and fatigue-induced prolonged low-frequency force depression in skeletal muscle fibres of rats, mice and SOD2 overexpressing mice

Skeletal muscle often shows a delayed force recovery after fatiguing stimulation, especially at low stimulation frequencies. In this study we focus on the role of reactive oxygen species (ROS) in this fatigue‐induced prolonged low‐frequency force depression. Intact, single muscle fibres were dissected from flexor digitorum brevis (FDB) muscles of rats and wild‐type and superoxide dismutase 2 (SOD2) overexpressing mice. Force and myoplasmic free [Ca2+] ([Ca2+]i) were measured. Fibres were stimulated at different frequencies before and 30 min after fatigue induced by repeated tetani. The results show a marked force decrease at low stimulation frequencies 30 min after fatiguing stimulation in all fibres. This decrease was associated with reduced tetanic [Ca2+]i in wild‐type mouse fibres, whereas rat fibres and mouse SOD2 overexpressing fibres instead displayed a decreased myofibrillar Ca2+ sensitivity. The SOD activity was ∼50% lower in wild‐type mouse than in rat FDB muscles. Myoplasmic ROS increased during repeated tetanic stimulation in rat fibres but not in wild‐type mouse fibres. The decreased Ca2+ sensitivity in rat fibres could be partially reversed by application of the reducing agent dithiothreitol, whereas the decrease in tetanic [Ca2+]i in wild‐type mouse fibres was not affected by dithiothreitol or the antioxidant N‐acetylcysteine. In conclusion, we describe two different causes of fatigue‐induced prolonged low‐frequency force depression, which correlate to differences in SOD activity and ROS metabolism. These findings may have clinical implications since ROS‐mediated impairments in myofibrillar function can be counteracted by reductants and antioxidants, whereas changes in SR Ca2+ handling appear more resistant to interventions.

Persistent over-expression of specific CC class chemokines correlates with macrophage and T-cell recruitment in mdx skeletal muscle

Prior studies and the efficacy of immunotherapies provide evidence that inflammation is mechanistic in pathogenesis of Duchenne muscular dystrophy. To identify putative pro-inflammatory mechanisms, we evaluated chemokine gene/protein expression patterns in skeletal muscle of mdx mice. By DNA microarray, reverse transcription-polymerase chain reaction, quantitative polymerase chain reaction, and immunoblotting, convergent evidence established the induction of six distinct CC class chemokine ligands in adult MDX: CCL2/MCP-1, CCL5/RANTES, CCL6/mu C10, CCL7/MCP-3, CCL8/MCP-2, and CCL9/MIP-1gamma. CCL receptors, CCR2, CCR1, and CCR5, also showed increased expression in mdx muscle. CCL2 and CCL6 were localized to both monocular cells and muscle fibers, suggesting that dystrophic muscle may contribute toward chemotaxis. Temporal patterns of CCL2 and CCL6 showed early induction and maintained expression in mdx limb muscle. These data raise the possibility that chemokine signaling pathways coordinate a spatially and temporally discrete immune response that may contribute toward muscular dystrophy. The chemokine pro-inflammatory pathways described here in mdx may represent new targets for treatment of Duchenne muscular dystrophy.

Constitutive properties, not molecular adaptations, mediate extraocular muscle sparing in dystrophic mdx mice

Extraocular muscle (EOM) is spared in Duchenne muscular dystrophy. Here, we tested putative EOM sparing mechanisms predicted from existing dystrophinopathy models. Data show that mdx mouse EOM contains dystrophin‐glycoprotein complex (DGC)‐competent and DGC‐deficient myofibers distributed in a fiber type‐specific pattern. Up‐regulation of a dystrophin homologue, utrophin, mediates selective DGC retention. Counter to the DGC mechanical hypothesis, an intact DGC is not a precondition for EOM sarcolemmal integrity, and active adaptation at the level of calcium homeostasis is not mechanistic in protection. A partial, fiber type‐specific retention of antiischemic nitric oxide to vascular smooth muscle signaling is not a factor in EOM sparing, because mice deficient in dystrophin and α‐syntrophin, which localizes neuronal nitric oxide synthase to the sarcolemma, have normal EOMs. Moreover, an alternative transmembrane protein, α7β1 integrin, does not appear to substitute for the DGC in EOM. Finally, genomewide expression profiling showed that EOM does not actively adapt to dystrophinopathy but identified candidate genes for the constitutive protection of mdx EOM. Taken together, data emphasize the conditional nature of dystrophinopathy and the potential importance of nonmechanical DGC roles and support the hypothesis that broad, constitutive structural cell signaling, and/or biochemical differences between EOM and other skeletal muscles are determinants of differential disease responsiveness.

Nitric oxide: biologic effects on muscle and role in muscle diseases

Nitric oxide is a ubiquitous cell-signaling molecule involved in regulation of numerous homeostatic mechanisms and in mediation of tissue injury. Nitric oxide influences contraction, blood flow, and metabolism, as well as myogenesis. Nitric oxide exerts its influence by activation of guanylate cyclase and nitrosylation of proteins, which include glyceraldehyde-3-phosphate dehydrogenase, the ryanodine receptor and actomyosin ATPase. Skeletal muscle expresses all three isoforms of the nitric oxide synthase, including a muscle-specific splice variant; expression of the isoforms is fiber-type specific and influenced by age and disease. Nitric oxide produced with certain systemic conditions and local inflammation is likely toxic to skeletal muscle, either directly or in reactions with oxygen-derived radicals. Although nitric oxide impacts on many functions in muscle, its effects are subtle, and much work remains to be done to determine its importance in the pathogenesis of muscle diseases.

Perinatal exercise improves glucose homeostasis in adult offspring

Emerging research has shown that subtle factors during pregnancy and gestation can influence long-term health in offspring. In an attempt to be proactive, we set out to explore whether a nonpharmacological intervention, perinatal exercise, might improve offspring health. Female mice were separated into sedentary or exercise cohorts, with the exercise cohort having voluntary access to a running wheel prior to mating and during pregnancy and nursing. Offspring were weaned, and analyses were performed on the mature offspring that did not have access to running wheels during any portion of their lives. Perinatal exercise caused improved glucose disposal following an oral glucose challenge in both female and male adult offspring (P < 0.05 for both). Blood glucose concentrations were reduced to lower values in response to an intraperitoneal insulin tolerance test for both female and male adult offspring of parents with access to running wheels (P < 0.05 and P < 0.01, respectively). Male offspring from exercised dams showed increased percent lean mass and decreased fat mass percent compared with male offspring from sedentary dams (P < 0.01 for both), but these parameters were unchanged in female offspring. These data suggest that short-term maternal voluntary exercise prior to and during healthy pregnancy and nursing can enhance long-term glucose homeostasis in offspring.

Eye muscle sparing by the muscular dystrophies: Lessons to be learned?

The devastating consequences of the various muscular dystrophies are even more obvious when a muscle or muscle group is spared. The study of the exceptional cell or tissue responses may prove to be of considerable value in the analysis of disease mechanisms. The small muscles responsible for eye movements, the extraocular muscles, have functional and morphological characteristics that set them aside from other skeletal muscles. Notably, these muscles are clinically unaffected in Duchenne/Becker, limb‐girdle, and congenital muscular dystrophies, pathologies due to a broken mechanical or signaling linkage between the cytoskeleton and the extracellular matrix. Uncovering the strategies used by the extraocular muscles to “naturally” protect themselves in these diseases should contribute to knowledge of both pathogenesis and treatment. We propose that careful investigation of the cellular determinants of extraocular muscle‐specific properties may provide insights into how these muscles avoid or adapt to the cascade of events leading to myofiber degeneration in the muscular dystrophies. Microsc. Res. Tech. 48:192–203, 2000.