scholarly journals Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle

2002 ◽  
Vol 92 (2) ◽  
pp. 435-445 ◽  
Author(s):  
Kevin Culligan ◽  
Niamh Banville ◽  
Paul Dowling ◽  
Kay Ohlendieck
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Muscular Dystrophy ◽  
Calcium Binding ◽  
Functional Decline ◽  
Two Dimensional ◽  
Drastic Reduction ◽  
Dystrophic Muscle ◽  
One Dimensional ◽  
Blotting Technique

Although the reduction in dystrophin-associated glycoproteins is the primary pathophysiological consequence of the deficiency in dystrophin, little is known about the secondary abnormalities leading to x-linked muscular dystrophy. As abnormal Ca2+ handling may be involved in myonecrosis, we investigated the fate of key Ca2+ regulatory membrane proteins in dystrophic mdx skeletal muscle membranes. Whereas the expression of the ryanodine receptor, the dihydropyridine receptor, the Ca2+-ATPase, and calsequestrin was not affected, a drastic decline in calsequestrin-like proteins of 150–220 kDa was observed in dystrophic microsomes using one-dimensional immunoblotting, two-dimensional immunoblotting with isoelectric focusing, diagonal two-dimensional blotting technique, and immunoprecipitation. In analogy, overall Ca2+ binding was reduced in the sarcoplasmic reticulum of dystrophic muscle. The reduction in Ca2+ binding proteins might be directly involved in triggering impaired Ca2+ sequestration within the lumen of the sarcoplasmic reticulum. Thus disturbed sarcolemmal Ca2+ fluxes seem to influence overall Ca2+homeostasis, resulting in distinct changes in the expression profile of a subset of Ca2+ handling proteins, which might be an important factor in the progressive functional decline of dystrophic muscle fibers.

Enzyme studies of skeletal muscle in mice with hereditary muscular dystrophy

1963 ◽  
Vol 205 (5) ◽  
pp. 897-901 ◽  
Author(s):  
Marilyn W. McCaman
Keyword(s):  
Skeletal Muscle ◽  
Muscular Dystrophy ◽  
Glutathione Reductase ◽  
Enzyme Activities ◽  
Lactic Dehydrogenase ◽  
Normal Muscle ◽  
Dystrophic Muscle ◽  
Nerve Section ◽  
Mouse Muscle ◽  
Dystrophic Mouse

The activities of 20 enzymes in normal, heterozygous, and dystrophic mouse muscle were studied by means of quantitative microchemical methods. Enzyme activities in normal and heterozygous muscle were essentially the same. In dystrophic muscle glucose-6-P dehydrogenase, 6-P-gluconic dehydrogenase, glutathione reductase, peptidase, ß-glucuronidase, and glucokinase activities were significantly higher than in normal muscle, while α-glycero-P dehydrogenase and lactic dehydrogenase activities were significantly lower. The pattern of enzyme activities found in normal gastrocnemius denervated by nerve section was strikingly similar to that in dystrophic muscle.

Diazepam reveals different rate-limiting processes in rat skeletal muscle contraction

1987 ◽  
Vol 65 (2) ◽  
pp. 272-273 ◽  
Author(s):  
Michael Chua ◽  
Angela F. Dulhunty
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Calcium Binding ◽  
Extensor Digitorum Longus ◽  
Calcium Uptake ◽  
Rat Skeletal Muscle ◽  
Skeletal Muscle Contraction ◽  
Slow Twitch ◽  
Fast Twitch ◽  
Rate Limiting

The action of the tranquilizer diazepam on rat skeletal muscle showed that relaxation of isometric twitches is controlled by different processes in extensor digitorum longus (fast-twitch) and soleus (slow-twitch) muscles. Diazepam caused an increase in the amplitude of twitches in fibres from both muscles but increased the twitch duration only in soleus. The amplitude of fused tetani were reduced in both muscles and the rate of relaxation after the tetanus slowed by as much as 34% when the amplitude of the tetanus was reduced by only 11%. The slower tetanic relaxation indicated that calcium uptake by the sarcoplasmic reticulum was slower than normal in slow- and fast-twitch fibres. We conclude therefore that calcium uptake by the sarcoplasmic reticulum is rate limiting for twitch relaxation in slow-twitch but not fast-twitch fibres and suggest that calcium binding to parvalbumin controls relaxation in the fast fibres.

Calcium binding to the sarcoplasmic reticulum of rabbit skeletal muscle

Biochemistry ◽  
1971 ◽  
Vol 10 (14) ◽  
pp. 2733-2737 ◽  
Author(s):  
Ronald A. Butow ◽  
Jean Chevallier
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Calcium Binding ◽  
Rabbit Skeletal Muscle

scholarly journals Iodination of Calsequestrin in the Sarcoplasmic Reticulum of Rabbit Skeletal Muscle: A Re-Examination

1979 ◽  
Vol 32 (2) ◽  
pp. 177 ◽  
Author(s):  
Ronald K Tume
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Calcium Binding ◽  
Local Environment ◽  
Maximal Inhibition ◽  
High Affinity ◽  
Tris Maleate ◽  
Sepharose 4B

The exposed proteins of sarcoplasmic reticulum (SR) vesicles from skeletal muscle were iodinated with the use of Sepharose 4B-bound lactoperoxidase, so that the location of the proteins in the membrane could be determined. It was found that the pattern of protein labelling could be modified simply by changing the constituents of the incubation media. This implies that the position or configuration of a particular protein in the membrane can be altered by the local environment. When the reaction was performed in the presence of 25 mM tris-maleate, pH 7 �0, alone, the Ca2+ pump ATPase (molecular weight 105000) and calsequestrin (47000) were both heavily labelled, suggesting they are at least partially exposed on the outer surface of the membrane. By contrast the high affinity calcium-binding protein (55000) was not labelled. However, when the vesicles were iodinated under conditions that were suitable for ATPase activity and Ca2+ accumulation, namely in the presence of 25 mM tris-maleate, pH 7 �0, 5 mM ATP, 5 mM Mg2+ and 0�05 mM Ca2+, a different pattern of labelling was obtained. No labelling of calsequestrin was observed whereas the extent of labelling of the Ca2+ pump ATPase remained about the same. The inclusion of anyone of the additives mentioned was effective in inhibiting the iodination of calsequestrin in the SR vesicle. When added alone, Ca2+ was more effective than Mg2+ in preventing labelling of calsequestrin. Half-maximal inhibition was observed at concentrations of approximately 0�05 mM Ca 2+ and 0�2-0�3 mM Mg2+ . Although much reduced, significant labelling of calsequestrin was observed even in the presence of 5 mM ATP. Investigations with partially purified calsequestrin revealed that the iodination of calsequestrin was the same in both the presence and absence of 1 mM Ca2 +. Therefore the reduction in label observed in intact SR vesicles probably represents a change in the location of calsequestrin within the membrane, rather than inhibition by Ca2+ of the iodination sites of the protein itself.

scholarly journals BETs inhibition attenuates oxidative stress and preserves muscle integrity in Duchenne muscular dystrophy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Marco Segatto ◽  
Roberta Szokoll ◽  
Raffaella Fittipaldi ◽  
Cinzia Bottino ◽  
Lorenzo Nevi ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mdx Mouse ◽  
Early Event ◽  
Dystrophic Muscle ◽  
Protein Levels ◽  
Bet Inhibitors ◽  
Ros Metabolism

AbstractDuchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD.

scholarly journals The Influence of Hydrogen Ion Concentration on Calcium Binding and Release by Skeletal Muscle Sarcoplasmic Reticulum

1972 ◽  
Vol 59 (1) ◽  
pp. 22-32 ◽  
Author(s):  
Yoshiaki Nakamaru ◽  
Arnold Schwartz
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Calcium Binding ◽  
Calcium Release ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Ph Change ◽  
Release Process ◽  
Hydrogen Ion Concentration ◽  
Calcium Loading

Calcium release and binding produced by alterations in pH were investigated in isolated sarcoplasmic reticulum (SR) from skeletal muscle. When the pH was abruptly increased from 6.46 to 7.82, after calcium loading for 30 sec, 80–90 nanomoles (nmole) of calcium/mg protein were released. When the pH was abruptly decreased from 7.56 to 6.46, after calcium loading for 30 sec, 25–30 nmole of calcium/mg protein were rebound. The calcium release process was shown to be a function of pH change: 57 nmole of calcium were released per 1 pH unit change per mg protein. The amount of adenosine triphosphate (ATP) bound to the SR was not altered by the pH changes. The release phenomenon was not due to alteration of ATP concentration by the increased pH. Native actomyosin was combined with SR in order to study the effectiveness of calcium release from the SR by pH change in inducing super-precipitation of actomyosin. It was found that SR, in an amount high enough to inhibit superprecipitation at pH 6.5, did not prevent the process when the pH was suddenly increased to 7.3, indicating that the affinity of SR for calcium depends specifically on pH. These data suggest the possible participation of hydrogen ion concentration in excitation-contraction coupling.

A novel technique for the isolation of transverse tubular membrane from dystrophic skeletal muscle

1978 ◽  
Vol 36 (2) ◽  
pp. 640-641
Author(s):  
Roger Sabbadini ◽  
Donald Scales ◽  
Cecelia Nixon
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Buoyant Density ◽  
Dystrophic Muscle ◽  
Tubular Membrane ◽  
Novel Technique ◽  
High Fraction ◽  
T Tubules ◽  
Dystrophic Chicken

Introduction A particularly high fraction of T tubular membrane is apparent in standard isolations of microsomes derived from dystrophic muscle. We describe here a method for separating vesicles of T tubules from vesicles of sarcoplasmic reticulum (SR). The method assumes that only SR membranes are capable of ATP-dependent Ca2+ transport, and in the presence of the precipitating anion oxalate, the buoyant density of oxalate- filled SR exceeds that of non-calcium transporting membranes. These latter microsomes are tentatively identified as T tubular membrane based on both morphological and biochemical observations.Methods As described previously microsomes were isolated from dystrophic chicken pectoral is muscle. The crude microsomes were incubated for 10 min. at 37°C in a medium containing 20 mM MOPS (pH 6. 8), 2 mM CaCl2, 2 mM EGTA, 5 mM Mg-ATP, 80 mM K-oxalate. This was cooled to 5°C and a 30 ml aliquot was layered on a 4 ml cushion of 52% sucrose, 20 mM MOPS. The microsomes were centrifuged for 45 min. at 82, 500 g in a Beckman L5-50 ultracentrifuge.

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

2006 ◽  
Vol 291 (2) ◽  
pp. C245-C253 ◽  
Author(s):  
Alessandra Nori ◽  
Giorgia Valle ◽  
Elena Bortoloso ◽  
Federica Turcato ◽  
Pompeo Volpe
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Calcium Binding ◽  
Calcium Release ◽  
Structural Information ◽  
Muscle Fibers ◽  
Physiological Role ◽  
Homogeneous Distribution ◽  
Skeletal Muscle Fibers ◽  
Domain Iii

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C2C12 myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HAΔ24NH2, CS-HAΔ2D, CS-HAΔ3D, and CS-HAΔHT, a double mutant of the NH2 terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAΔHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C2C12 myotubes, as judged by immunofluorescence, not only CS-HAΔHT but also CS-HAΔ3D and CS-HAΔ2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C2C12 myoblasts, and only CS-HAΔ3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH2 terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation.

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