Malignant hyperthermia: Effects of sarcoplasmic reticulum Ca2+ pump inhibition

In porcine malignant hyperthermia-susceptible (MHS) skeletal muscles, calcium release is abnormal and resting calcium may be elevated. Thus MHS muscles may have prolonged twitch relaxation and lower fusion frequencies, which would be augmented by inhibition of sarcoplasmic reticulum (SR) Ca²⁺ adenosine triphosphatase (ATPase) activity; bundles of intact muscle cells from MHS and normal pigs were used to investigate this possibility. Cooling and low-frequency stimulation, in combination, enhanced twitch fusion and prolonged twitch relaxation significantly more in MHS than in normal muscles (e.g., 34 ± 4% versus 16 ± 4% fusion, and 82.4 ± 9.4 ms versus 43.2 ± 7.8 ms half-relaxation time, for MHS and normal muscles, respectively). Similarly, inhibition of the SR Ca²⁺ ATPase by cyclopiazonic acid resulted in significantly greater twitch fusion in MHS muscles. These results were consistent with predicted effects of enhanced SR Ca²⁺ release and/or elevated resting calcium in MHS muscles and indicate that cooling during a malignant hyperthermia crisis could actually increase the force of muscle contractures. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:361–366, 1998.     

Effect of caffeine and high potassium on normal and dystrophic mouse EDL muscles at various developmental stages

EDL muscles from normal and dystrophic (dy^2j) mice of various ages were examined. Muscles were divided into three groups according to age: 7 to 14 days postnatal, 16 to 21 days postnatal, and 6 months old, to assess age and/or phenotype related differences in the muscle response to caffeine or high K⁺. The response of normal muscles to caffeine decreased with age and reached adult characteristics between the second and third week of postnatal life. Their response to high K⁺ also changed during post-natal development; specifically, the time taken to recover to 50% pretest twitch tension decreased with age, probably reflecting developmental changes in Cl^? conductance. Up to 21 days of age, the sensitivity of dystrophic muscles to both caffeine and high K⁺ was essentially similar to normal, while marked differences were observed in the adult. Taken altogether, our results suggest that while the maturation of a number of systems might be delayed in dystrophic muscles at preclinical stages of the disease, their e–c coupling and SR function (Ca²⁺ release and reuptake) appear to be quite normal. Our results further suggest that the “abnormal” responses of dystrophic muscles at more advanced stages of the disease, when challenged by drugs acting on either of these systems, may be explained in terms of changes in muscle fiber type proportions. © 1993 John Wiley & Sons, Inc.     

Myosin isoforms in hindlimb muscles of normal and dystrophic (ReJ129 dy/dy) mice.

Myosin isoform expression was studied in hindlimb muscles of control (Dy/Dy) and dystrophic (dy/dy) mice of the ReJ129 strain during postnatal development. Three myosin heavy chain isoforms (fast II-B MHC, neonatal MHC, and slow or I MHC) were identified using monoclonal antibodies. Only original fibers, i.e., fibers formed during fetal life, were studied. Necrotic and regenerating fibers were excluded. The disappearance of neonatal MHC was found to be delayed in all muscles of dystrophic mice, except the soleus. The fraction of fibers containing I MHC was similar in control and dystrophic animals at all ages, except during the third postnatal week. The developmental increase in the fraction of fibers expressing II-B MHC was interrupted in dystrophic mice by two marked declines. The first occurred during the second postnatal week at the beginning of the main wave of fiber necrosis, and the second occurred at between 30 and 90 postnatal days.     

Ca-, Sr-tension relationships and contraction velocities of human muscle fibers.

Muscle fibers from the lateral gastrocnemius or intercostal muscles of 7 normal adult males were chemically skinned (sarcolemma disrupted) and isolated fibers were divided into two parts for histochemical determination of fiber type and physiologic studies. The Ca- and Sr-induced tension relationships and maximum contraction velocities were measured. Slow twitch fibers developed tension at lower concentrations of Ca or Sr than fast twitch fibers. The difference between fast and slow twitch fibers was greatest when Sr was the activating cation. Fast and slow twitch fibers was greatest when Sr was the activating cation. Fast and slow twitch fibers generated similar maximum tensions. The contraction velocities of fast twitch fibers were more than two-fold greater than slow twitch fibers. Fast-oxidative-glycolytic (FOG, type IIA) and fast-glycolytic (FG, type IIB) fibers had similar Ca- and Sr-tension relationships and contraction velocities.     

Response of a fast muscle from normal and dystrophic (dy2j) mice to a local decrease in extracellular Ca2+ induced at different stages of postnatal life

It has previously been reported that reducing Ca2+ entry into muscle fibres was beneficial to dystrophic muscles. In this study, we examined the effect, on the force output and contractile properties of the tibialis anterior muscle, of a local decrease in extracellular Ca2+, produced in normal and dystrophic mice at various stages of postnatal life by applying a small strip of silicon rubber containing a calcium chelator (BAPTA). Lowering extracellular Ca2+ in this way at an early stage of postnatal life (11-16 days) interferes with normal development in that, 3-5 weeks after the initial operation, the treated TA muscles from normal mice are weaker and their contractile speed is slower than that of their untreated counterparts. In contrast, the same procedure has a beneficial effect on dystrophic muscles in that they produce more force than untreated controls. Our results show that these changes are not related to changes in the total number of muscle fibres or fibre type proportions. These changes are temporary and by 8-12 weeks after the operation, the treated muscles are indistinguishable from controls. Finally, our results also indicate that skeletal muscles from older animals, both normal and dystrophic, become insensitive to this manipulation. These results provide the first evidence for a difference in the sensitivity of normal immature and normal adult skeletal muscles to their extracellular Ca2+ environment. They also suggest that in this context, dystrophic muscles might already differ from normal at a stage prior to the clinical expression of the symptoms of the disease.     

Identification of inactive ecto-5'-nucleotidase in normal mouse muscle and its increased activity in dystrophic Lama2(dy) mice.

Ecto-5'-nucleotidase (eNT) activity and protein in normal (NM) and merosin-deficient dystrophic (DM) Lama2(dy) mice muscle were studied. eNT activity in DM was three- to four-fold that in NM. eNT in NM and DM displayed the same kinetic properties. Slot and Western blotting revealed that the immunoreactive protein was two to three times more abundant in control muscle, when NM and DM samples with the same eNT activity were compared, indicating that mouse muscle contains catalytically inactive eNT components. eNT activity and protein peaks coincided in sedimentation analyses, revealing that inactive eNT occurs as dimers. Most eNT activity and protein of NM bound to Lens culinaris (LCA) or Ricinus communis (RCA) agglutinins, but half of the activity and one-third of the protein bound to wheat germ agglutinin (WGA). Although WGA interaction did not permit full separation of inactive eNT, the results suggest that similar proportions of active species with and without WGA reactivity occur in mouse muscle, whereas a great fraction of the inactive eNT variants lack WGA reactivity. Because the level of eNT protein was little modified in DM, the higher eNT activity in dystrophic than in control muscle may result from misregulation in the synthesis of active and inactive eNT species or from conversion of inactive into active components.     

Immunolocalization of triadin,DHP receptors,and ryanodine receptors in adult and developing skeletal muscle of rats

The dihydropyridine receptors (DHPR) and the ryanodine receptors (RyR) are well-characterized proteins of the triad junctions of skeletal muscle fibers. Recently, a newly discovered 95-kDa protein, triadin, has been purified from rabbit skeletal muscle heavy sarcoplasmic reticulum (SR) vesicles. WE have used indirect immunogold EM to localize triadin to the junctional face of the SR in isolated triads. In addition, we have used indirect immunofluorescence to localize triadin in relation to the DHPR and the RyR in adult and developing rat skeletal muscle. In double immunolabeling experiments of longitudinally oriented adult rat skeletal muscle tissue, triadin-specific and RyR-specific antibodies resulted in a characteristic striated staining pattern. The staining arising from these antibodies completely overlapped when examined by computer analysis of digitized laser scanning confocal microscopy images. A similar result was obtained in double staining experiments using antibodies raised against the DHPR and the RyR suggesting that all three proteins are located in the triads in situ. The developmental expression of the three triad proteins was examined using double labeling of skeletal muscle tissue from several fetal and early postnatal ages. The staining patterns of triadin, RyR, and DHPR antibodies were overlapping throughout development, suggesting that from their earliest appearance the three proteins are components of the triads. © John Wiley & Sons, Inc.     

The effects of altered metabolism (hypothyroidism) on muscle repair in the mdx dystrophic mouse

After dystrophic damage, the limb muscles of the mdx mouse recover very effectively compared to muscles in Duchenne muscular dystrophy (DMD) patients. Since thyroid hormone is required for muscle development and integrity, we examined whether a deficiency of the hormone, induced by 0.05% propylthiouracil (PTU) in drinking water over 8 weeks, would be deleterious to the myogenesis and muscle repair in control and mdx mice. Measured metabolic and growth parameters confirmed hypothyroidism in PTU-treated mice. Histological and morphometric techniques were used to study myogenesis and the repair of the tibialis anterior muscle (TA) after crush injury in mdx mice and their nondystrophic controls (C57B1/10ScSn). After 8 weeks, PTU-treated TA from mdx mice had larger crush sites and lower myotube density than TA in untreated mdx mice. In unoperated mdx TA, there was a larger proportionate area of active dystrophy and smaller fiber diameter in PTU-treated than in untreated mdx TA, which suggested that PTU increased the activity of dystrophy as well. In contrast, in control TA neither the regeneration of myotubes or fiber diameter were affected significantly by PTU. Therefore, these results suggest that mdx muscle regeneration is more affected by hypothyroidism than normal muscle repair. This may be due to the larger pool of muscle precursors in mdx than control muscle, and a possible impairment of precursor cell proliferation or fusion during myotube formation. © 1994 John Wiley & Sons, Inc.     

Effect of creatine supplementation on skeletal muscle of mdx mice

Dystrophic mice (mdx) and their controls (C57/Bl10) were fed for 1 month with a diet with or without creatine (Cr) enrichment. Cr supplementation reduced mass (by 19%, P < 0.01) and mean fiber surface (by 25%, P < 0.05) of fast-twitch mdx muscles. In both strains, tetanic tension increased slightly (9.2%) without reaching statistical significance (P = 0.08), and relaxation time increased by 16% (P < 0.001). However, Cr had no protective effect on the other hallmarks of dystrophy such as susceptibility to eccentric contractions; large numbers of centrally nucleated fibers in tibialis anterior; and elevated total calcium content, which increased by 85% (P = 0.008) in gastrocnemius mdx muscles. In conclusion, Cr may be a positive intervention for improving function of dystrophic muscle.     

Sarcoplasmic reticulum: the dynamic calcium governor of muscle

The sarcoplasmic reticulum (SR) provides feedback control required to balance the processes of calcium storage, release, and reuptake in skeletal muscle. This balance is achieved through the concerted action of three major classes of SR calcium-regulatory proteins: (1) luminal calcium-binding proteins (calsequestrin, histidine-rich calcium-binding protein, junctate, and sarcalumenin) for calcium storage; (2) SR calcium release channels (type 1 ryanodine receptor or RyR1 and IP3 receptors) for calcium release; and (3) sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pumps for calcium reuptake. Proper calcium storage, release, and reuptake are essential for normal skeletal muscle function. We review SR structure and function during normal skeletal muscle activity, the proteins that orchestrate calcium storage, release, and reuptake, and how phenotypically distinct muscle diseases (e.g., malignant hyperthermia, central core disease, and Brody disease) can result from subtle alterations in the activity of several key components of the SR calcium-regulatory machinery.     

A differential pattern of gene expression in skeletal muscle of tumor‐bearing rats reveals dysregulation of excitation–contraction coupling together with additional muscle alterations

Introduction: Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass. Methods: By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia‐inducing tumor, the AH‐130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor‐bearing animals, as compared with their healthy counterparts. Results: The results show important differences in gene expression in EDL skeletal muscle between tumor‐bearing animals with cachexia and control animals. Conclusions: The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome. Muscle Nerve 49 : 233–248, 2014     

A comparative freeze‐fracture study of plasma membrane of dystrophic skeletal muscles in dy/dy mice with merosin (laminin 2) deficiency and mdx mice with dystrophin deficiency

The intramembranous particle (IMP), orthogonal array (OA) and orthogonal array subunit particle (OASP) densities of skeletal muscle plasma membranes of merosin deficient dy/dy mice and their control mice at 7, 14 and 28 days after birth were analysed by freeze‐fracture electron microscopy. Similar studies were performed on dystrophin‐deficient mdx mice with mild muscle weakness at 28 days after birth for the comparison with those of dy/dy mice with severe muscle weakness at the same age. In the pre‐clinical stage of dy/dy mice at 14 days after birth, the membranes showed a significantly decreased density of OAs ( P <0.01 by Wilcoxon rank‐sum test) as compared with control mice, while those in the clinical stage of dy/dy mice at 28 days after birth showed normal IMP density but a marked depletion of OA density ( P <0.01). Moreover, at 28 days after birth, the reduction of OAs in the plasma membranes of dy/dy mice was more marked than that of mdx mice ( P <0.05 by Wilcoxon rank‐sum test). These results provided us with the information that the OA density was affected more severely with merosin deficiency than with dystrophin deficiency, and again supported our previously proposed concept that the clinical severity in muscular dystrophies correlated with the OA density.     

The Ca(V) 1.2 Ca(2+) channel is expressed in sarcolemma of type I and IIa myofibers of adult skeletal muscle

Although Ca(2+)-dependent signaling pathways are important for skeletal muscle plasticity, the sources of Ca(2+) that activate these signaling pathways are not completely understood. Influx of Ca(2+) through surface membrane Ca(2+) channels may activate these pathways. We examined expression of two L-type Ca(2+) channels in adult skeletal muscle, the Ca(V) 1.1 and Ca(V) 1.2, with isoform-specific antibodies in Western blots and immunocytochemistry assays. Consistent with a large body of work, expression of the Ca(V) 1.1 was restricted to skeletal muscle where it was expressed in T-tubules. Ca(V) 1.2 was also expressed in skeletal muscle, in the sarcolemma of type I and IIa myofibers. Exercise-induced alterations in muscle fiber types cause a concomitant increase in the number of both Ca(V) 1.2 and type IIa-positive fibers. Taken together, these data suggest that the Ca(V) 1.2 Ca(2+) channel is expressed in adult skeletal muscle in a fiber type-specific manner, which may help to maintain oxidative muscle phenotype.     

Subacute necrotizing encephalomyelopathy (Leigh syndrome) associated with disturbed oxidation of pyruvate, malate and 2-oxoglutarate in muscle and liver

We studied a 17-year-old girl with subacute necrotizing encephalomyelopathy (Leigh syndrome). Lactate and pyruvate levels were increased in serum and cerebrospinal fluid. The oxidation rates of all substrates tested, i.e. pyruvate in liver, and pyruvate, malate and 2-oxoglutarate in muscle, were decreased, as was the production of adenosine triphosphate plus creatine phosphate. Cytochrome content was normal. The data imply a defect in oxidative phosphorylation, outside the cytochrome region.