Structure and composition of tubular aggregates of skeletal muscle fibres

Unusual regions of densely packed membranous tubules known as tubular aggregates (TAs) have been observed in skeletal muscle fibres of mammals under numerous pathological conditions but also in health. Their causality is unclear. It is neither known whether TAs are destructive and should be treated or whether they have a compensating function in an endangered muscle. In spite of many similarities, the histochemical, immunocytochemical and ultrastructural characteristics of tubular aggregates do vary. Histochemistry provided an overall characteristic of TAs as membranous inclusions with a variety of enzymatic activities. Immunocytochemical evidence revealed that tubular aggregates contain miscellaneous proteins and that derive from membranes of sarcoplasmic reticulum and mitochondria. No evidence for the presence of contractile and cytoskeletal proteins in TAs was found. Ultrastructurally, TAs are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments or inner tubules. Various reported types of tubular aggregates, namely, proliferating terminal cisterns, vesicular membrane collections, TAs with double-walled tubules, TAs with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, filamentous tubules, riesentubuli, and related membranous structures including cylindrical spirals are sumarized and analyzed here in detail.     

Expression of the sarco/endoplasmic Ca(2+)-ATPase, SERCA1a, in fibroblasts induces the formation of organelle membrane arrays

Members of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) family are transmembrane proteins that are essential for the function of intracellular Ca(2+) storage organelles. We found that overexpression of avian muscle SERCA1a in transfected mouse fibroblasts led to the appearance of tubular membrane bundles that we termed plaques. These structures were generated in transfected cells when SERCA1a protein expression approached the endogenous level measured in chicken skeletal muscle. Plaque membranes had associated ribosomes and contained endoplasmic reticulum (ER) proteins. Endogenous ER protein levels were not elevated in SERCA1a-expressing cells, indicating that plaques were not generalized proliferations of ER but rather a reorganization of existing organelle membrane. Plaque formation also was observed in cells expressing a green fluorescent protein-SERCA1a fusion protein (GFP-SERCA1a). GFP-SERCA1a molecules displayed extensive lateral mobility between plaques, suggesting the presence of membrane continuities between these structures. Plaques were induced in cells expressing cDNA encoding a catalytically silent SERCA1a mutant indicating that ER redistribution was driven by a structural feature of the enzyme. SERCA1a-induced plaque formation shares some characteristics of sarcoplasmic reticulum (SR) biogenesis during muscle differentiation, and high-level SERCA1a expression in vivo may contribute to the formation of SR from ER during embryonic myogenesis.     

Fatal mitochondrial cardiomyopathy in Kearns-Sayre syndrome with deficiency of cytochrome-c-oxidase in cardiac and skeletal muscle. An enzymehistochemical--ultra-immunocytochemical--fine structural study in longterm frozen autopsy tissue

Morphological studies in a 26-year-old man with long-standing Kearns-Sayre syndrome, with cardiac arrhythmias and a fatal congestive cardiomyopathy, revealed a mitochondrial myopathy of both skeletal and myocardial muscle (Hübner et al. 1986). Histochemical investigation of cytochrome-c-oxidase showed multiple enzyme defects of both cardiac and skeletal muscle present in myocytes with normal and abnormal numbers of mitochondria demonstrated by ultracytochemistry. Immunohistochemical studies with antibodies against the holoenzyme and various subunits revealed that in the heart the enzyme defect affected both contractile and conductive fibres and was characterized by a severe reduction but not a complete loss of nuclear and mitochondrially coded immunoreactive enzyme protein. In skeletal muscle, however, where up to 30% of the fibres lacked enzyme activity, immunoreactivity was reduced only very occasionally. These results are most consistent with a defective enzyme assembly in the inner mitochondrial membrane and probably indicate heterogeneity of mitochondria, i.e. organ-specific pathological reaction patterns.     

Carbonic anhydrase in guinea pig skeletal muscle mitochondria

The presence of carbonic anhydrase activity was demonstrated in guinea pig skeletal muscle mitochondria purified by Percoll gradient centrifugation such that contamination by sarcoplasmic reticulum vesicles was less than 5%. Assay of purified heavy sarcoplasmic reticulum vesicles for carbonic anhydrase activity showed these to have somewhat less activity than the mitochondria, so that any contribution by sarcoplasmic reticulum vesicles to mitochondrial activity would be negligible. In agreement with this observation, rabbit skeletal muscle mitochondria prepared by the Percoll method had no detectable activity. Assay of the guinea pig muscle mitochondrial enzyme activity in the presence of Triton X-100 showed a sixfold greater activity than in its absence, indicating a matrix location for the carbonic anhydrase. The enzyme is highly sensitive to the sulfonamide inhibitor ethoxzolamide, with Ki = 8.7 nM. The activation energy obtained from the rate constant for CO2 hydration, kenz with units (mg/ml)-1 s-1, over the range 4 to 37 degrees C was 12.8 kcal/mol. These properties are those expected for a carbonic anhydrase of the CA II class of isozymes, rather than for CA I, CA III, and the liver mitochondrial enzyme CA V.     

骨骼肌中肌浆网/内质网钙ATP酶的功能及其作用机制

骨骼肌是机体生命活动和能量代谢的重要场所,其代谢紊乱会诱发一系列肌肉疾病。Ca²⁺作为肌肉收缩过程的重要调节器,在骨骼肌的功能行使中发挥重要作用。骨骼肌细胞中Ca²⁺浓度主要受肌浆网/内质网钙ATP酶(sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase, SERCA)的调节。SERCA利用ATP水解产生的能量介导胞质Ca²⁺进入肌浆网内腔,维持胞质Ca²⁺平衡。SERCA功能的失调会引发一系列骨骼肌疾病,而SERCA活性受部分肌浆网蛋白质的调控,跨膜蛋白质PLN、SLN、MRLN、DWORF和sAnk1以及胞质蛋白质THADA和SAR,其通过磷酸化,进而调控SERCA的功能。本文对骨骼肌中SERCA的功能、调控SERCA的相关功能蛋白质的结构及其作用机制进行了总结,以期为骨骼肌相关疾病的治疗提供最新的思路和方法。     

The sarcoplasmic reticulum Ca(2+)-ATPase, SERCA1a, contains endoplasmic reticulum targeting information.

The fast-twitch skeletal muscle Ca(2+)-ATPase isoenzyme, SERCA1a, is localized in chick skeletal myotubes to both the sarcoplasmic reticulum (SR) and to the nuclear envelope, an extension of the endoplasmic reticulum (ER). The ER labeling remained after cycloheximide treatment, indicating that it did not represent newly synthesized SERCA1a in transit to the SR. Expression of the cDNA encoding SERCA1a in cultured non-muscle cells led to the localization of the enzyme in the ER, as indicated by organelle morphology and the co-localization of SERCA1a with the endogenous ER luminal protein, BiP. Immunopurification analysis showed that SERCA1a was not bound to BiP, nor was any degradation apparent. Thus, the SR Ca(2+)-ATPase appears to contain ER targeting information.     

Specific absence of the alpha 1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis

Muscular dysgenesis is a lethal mutation in mice that results in a complete absence of skeletal muscle contraction due to the failure of depolarization of the transverse tubular membrane to trigger calcium release from the sarcoplasmic reticulum. In order to determine whether the defect in muscular dysgenesis leads to a specific loss of one of the components of excitation-contraction coupling or to a generalized loss of all components of excitation-contraction coupling, we have analyzed skeletal muscle from control and dysgenic mice for the sarcoplasmic reticulum and transverse tubular proteins which are believe to function in excitation-contraction coupling. We report that the proteins involved in sarcoplasmic reticulum calcium transport, storage, and release [Ca2+ + Mg2+)-ATPase, calsequestrin, and calcium release channel) are present in dysgenic muscle. Also present in dysgenic muscle is the 175/150-kDa glycoprotein subunit (alpha 2) of the dihydropyridine receptor. However, the 170-kDa dihydropyridine binding subunit (alpha 1) of the dihydropyridine receptor is absent in dysgenic muscle. These results suggest that the specific absence of the alpha 1 subunit of the dihydropyridine receptor is responsible for the defects in muscular dysgenesis and that the alpha 1 subunit of the dihydropyridine receptor is essential for excitation-contraction coupling in skeletal muscle.     

The effect of caffeine on calcium efflux and calcium translocation in skeletal and visceral muscle.

1. KCl-induced depolarization resulted in a large stimulation of the 45Ca efflux from both cockroach skeletal muscle and rat ileal smooth muscle. 2. Caffeine (10 mM) induced a large stimulation of 45Ca efflux from skeletal muscle, but a fall in the efflux from ileal muscle, especially if the efflux was previously stimulated by KCl depolarization. 3. Caffeine inhibited calcium uptake by skeletal muscle mitochondria and sarcoplasmic reticulum, was without effect on ileal muscle mitochondria, but significantly increased caclium binding by ileal muscle membrane vesicular preparations. 4. The induction of contractures and stimulation of 45Ca efflux in skeletal muscle by caffeine are clearly related to inhibition of intracellular calcium binding by the sarcoplasmic reticulum and mitochondria. 5. The relaxation of ileal muscle by caffeine and the inhibition of fibre calcium efflux correlate well with caffeine enhancement of intracellular calcium binding. These experiments suggest that the membrane vesicular compartment may be the main agency centrally involved in fibre calcium regulation in this muscle during the contraction-relaxation cycle.     

A transmission delay and the effect of temperature at the triadic junction of skeletal muscle

The coupling process at the triadic junctions in skeletal muscle fibres is characterized by a significant latency between the depolarization of the transverse tubular membrane and the release of Ca from the sarcoplasmic reticulum. This time interval, the triadic delay, is sufficiently long to allow for the participation of a chemical process. The strong temperature dependence of the triadic delay (Q10 near 2.7) suggests that a sequence of chemical steps may link the electrical signal in the T-tubules to the opening of Ca channels in the terminal cisternae of the sarcoplasmic reticulum.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.     

Adenylate cyclase system of human skeletal muscle: Subcellular distribution and general properties

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

Solid-state NMR and functional measurements indicate that the conserved tyrosine residues of sarcolipin are involved directly in the inhibition of SERCA1

The transmembrane protein sarcolipin regulates calcium storage in the sarcoplasmic reticulum of skeletal and cardiac muscle cells by modulating the activity of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs). The highly conserved C-terminal region ((27)RSYQY-COOH) of sarcolipin helps to target the protein to the sarcoplasmic reticulum membrane and may also participate in the regulatory interaction between sarcolipin and SERCA. Here we used solid-state NMR measurements of local protein dynamics to illuminate the direct interaction between the Tyr(29) and Tyr(31) side groups of sarcolipin and skeletal muscle Ca(2+)-ATPase (SERCA1a) embedded in dioleoylphosphatidylcholine bilayers. Further solid-state NMR experiments together with functional measurements on SERCA1a in the presence of NAc-RSYQY, a peptide representing the conserved region of sarcolipin, suggest that the peptide binds to the same site as the parent protein at the luminal face of SERCA1a, where it reduces V(max) for calcium transport and inhibits ATP hydrolysis with an IC(50) of approximately 200 microM. The inhibitory effect of NAc-RSYQY is remarkably sequence-specific, with the native aromatic residues being essential for optimal inhibitory activity. This combination of physical and functional measurements highlights the importance of aromatic and polar residues in the C-terminal region of sarcolipin for regulating calcium cycling and muscle contractility.     

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.     

Adenylate cyclase system of human skeletal muscle: Characteristics of catecholamine stimulation and nucleotide regulation

The subcellular localization of adenylate cyclase was examined in human skeletal muscle. Three major subcellular membrane fractions, plasmalemma, sarcoplasmic reticulum and mitochondria, were characterized by membrane-marker biochemical studies, by dodecyl sulfate polycrylamide gel electrophoresis and by electron microscopy. About 60% of the adenylate cyclase of the homogenate was found in the plasmalemmal fraction and 10–14% in the sarcoplasmic reticulum and mitochondria. When the plasmalemmal preparation was subjected to discontinuous sucrose gradients, the distribution of adenylate cyclase in different subfractions closely paralleled that of (Na⁺ + K⁺)-ATPase. The highest specific activity was found in a fraction which setteled at the 0.6–0.8 M sucrose interface. The electron microscopic study of this fraction revealed the presence of flattened sacs of variable sizes and was devoid of mitochondrial and myofibrillar material. The electron microscopy of each fraction supported the biochemical studies with enzyme markers. The three major membrane fractions also contained a low K_m phosphodiesterase activity, the highest specific activity being associated with sarcoplasmic reticulum.The plasmalemmal adenylate cyclase was more sensitive to catecholamine stimulation than that associated with sarcoplasmic reticulum or mitochondria. The catecholamine-sensitive, but not the basal, enzyme was further stimulated by GTP. The plasmalemmal adenylate cyclase had typical Michaelis-Menten kinetics with respect to ATP and the apparent K_m for ATP was approx. 0.3. mM. The pH optimum for that enzyme was 7.5. The enzyme required Mg²⁺, and the concentration to achieve half-maximal stimulation was approx. 3 mM. Higher concentrations of Mg²⁺ (about 10 mM) were inhibitory. Solubilization of the plasmalemmal membrane fraction with Lubrol-PX resulted in preferential extraction of 106 000- and 40 000-dalton protein components. The solubilized adenylate cyclase lost its sensitivity for catecholamine stimulation, and the extent of fluoride stimulation was reduced to one-sixth of that of the intact membranes. It is concluded that the catalytically active and hormone-sensitive adenylate cyclase is predominantly localized in the surface membranes of the cells within skeletal muscle. (That “plasmalemmal” fraction is considered likely to contain, in addition to plasmalemma of muscle cells, plasmalemma of bloodvessel cells (endothelium, and perhaps smooth muscle) which may be responsible for a certain amount of the adenylate cyclase activity and other propertiesobserved in that fraction.)The method of preparation used in this study provides a convenient material for evaluating the catecholamine-adenylate cyclase interactions in human skeletal muscle.     

Impaired mitochondrial oxidative phosphorylation in skeletal muscle of the dystrophin-deficient mdx mouse

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca²⁺ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

The ultrahistochemical picture of the so-called reversed ATPase in the gastrocnemius muscle of the rat.

The ultrahistochemical localization of the "reversed" ATPase activity was investigated. Red muscle fibres showed permanent sarcomere contraction, enzymatic activity in the inner membrane and matrix of mitochondria, and large, osmiophilic, probably calcium-containing structures within mitochondria and on their outside. White muscle fibre sarcomeres were relaxed, and activity within their sarcoplasmic reticulum was marked, but slight in the mitochondria. The relaxed state of the sarcomere in the white muscle fibres is supposed to be connected with inactivation of myofibrillar ATPase by acid preincubation, whereas red muscle contraction indicates that acid preincubation does not inactivate their myofibrillar ATPase. That the product of its activity failed to become visible in the sarcomeres is probably due to imperfection of the method. Two sub-types of red muscle fibres were distinguished: those showing only enzymatic activity in mitochondria, and those containing large intra- and extramitochondrial osmiophilic structures. The origin and composition of these structures is difficult to explain. A relation seems to exist between their presence within mitochondria and outside.     

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.     

A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.     

Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.     

Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca²⁺-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca²⁺ homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca²⁺ concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.