Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.     

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin

Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.     

Morphology and molecular composition of sarcoplasmic reticulum surface junctions in the absence of DHPR and RyR in mouse skeletal muscle

Calcium release during excitation-contraction coupling of skeletal muscle cells is initiated by the functional interaction of the exterior membrane and the sarcoplasmic reticulum (SR), mediated by the "mechanical" coupling of ryanodine receptors (RyR) and dihydropyridine receptors (DHPR). RyR is the sarcoplasmic reticulum Ca(2+) release channel and DHPR is an L-type calcium channel of exterior membranes (surface membrane and T tubules), which acts as the voltage sensor of excitation-contraction coupling. The two proteins communicate with each other at junctions between SR and exterior membranes called calcium release units and are associated with several proteins of which triadin and calsequestrin are the best characterized. Calcium release units are present in diaphragm muscles and hind limb derived primary cultures of double knock out mice lacking both DHPR and RyR. The junctions show coupling between exterior membranes and SR, and an apparently normal content and disposition of triadin and calsequestrin. Therefore SR-surface docking, targeting of triadin and calsequestrin to the junctional SR domains and the structural organization of the two latter proteins are not affected by lack of DHPR and RyR. Interestingly, simultaneous lack of the two major excitation-contraction coupling proteins results in decrease of calcium release units frequency in the diaphragm, compared with either single knockout mutation.     

Increased susceptibility to isoproterenol-induced cardiac hypertrophy and impaired weight gain in mice lacking the histidine-rich calcium-binding protein

The sarcoplasmic reticulum (SR) plays a critical role in excitation-contraction coupling by regulating the cytoplasmic calcium concentration of striated muscle. The histidine-rich calcium-binding protein (HRCBP) is expressed in the junctional SR, the site of calcium release from the SR. HRCBP is expressed exclusively in muscle tissues and binds calcium with low affinity and high capacity. In addition, HRCBP interacts with triadin, a protein associated with the ryanodine receptor and thought to be involved in calcium release. Its calcium binding properties, localization to the SR, and interaction with triadin suggest that HRCBP is involved in calcium handling by the SR. To determine the function of HRCBP in vivo, we inactivated HRC, the gene encoding HRCBP, in mice. HRC knockout mice exhibited impaired weight gain beginning at 11 months of age, which was marked by reduced skeletal muscle and fat mass, and triadin protein expression was upregulated in the heart of HRC knockout mice. In addition, HRC null mice displayed a significantly exaggerated response to the induction of cardiac hypertrophy by isoproterenol compared to their wild-type littermates. The exaggerated response of HRC knockout mice to the induction of cardiac hypertrophy is consistent with a regulatory role for HRCBP in calcium handling in vivo and suggests that mutations in HRC, in combination with other genetic or environmental factors, might contribute to pathological hypertrophy and heart failure.     

Ca<Superscript>2+</Superscript> signaling in striated muscle: the elusive roles of triadin,junctin, and calsequestrin

This review focuses on molecular interactions between calsequestrin, triadin, junctin and the ryanodine receptor in the lumen of the sarcoplasmic reticulum. These interactions modulate changes in Ca²⁺ release in response to changes in the Ca²⁺ load within the sarcoplasmic reticulum store in striated muscle and are of fundamental importance to Ca²⁺ homeostasis, since massive adaptive changes occur when expression of the proteins is manipulated, while mutations in calsequestrin lead to functional changes which can be fatal. We find that calsequestrin plays a different role in the heart and skeletal muscle, enhancing Ca²⁺ release in the heart, but depressing Ca²⁺ release in skeletal muscle. We also find that triadin and junctin exert independent influences on the ryanodine receptor in skeletal muscle where triadin alone modifies excitation–contraction coupling, while junctin alone supports functional interactions between calsequestrin and the ryanodine receptor.     

Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with calsequestrin

Normal Ca²⁺ signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca²⁺ binding protein calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca²⁺ stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal calsequestrin and ryanodine receptors. Purified calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the calsequestrin re-associated channel complexes were further inhibited when luminal Ca²⁺ fell from 1 mM to ≤100 μM, as seen with native channels (containing endogenous calsequestrin/triadin/junctin). In contrast, skeletal calsequestrin had no effect on the triadin/ryanodine receptor complex and the channel activity of this complex increased when luminal Ca²⁺ fell, as seen with purified channels prior to triadin/calsequestrin re-association. Therefore in this cell free system, junctin alone mediates signals between luminal Ca²⁺, skeletal calsequestrin and skeletal ryanodine receptors and may curtail resting Ca²⁺ leak from the sarcoplasmic reticulum. We suggest that triadin serves a different function which may dominate during excitation–contraction coupling.     

6周有氧运动对高脂膳食的ApoE敲除小鼠骨骼肌钙调控的影响*

目的: 探讨6周有氧运动对高脂膳食的载脂蛋白E(ApoE)基因敲除小鼠骨骼肌肌浆网钙调控蛋白的影响。方法: 25只9周龄ApoE敲除小鼠(ApoE KO)随机选取5只ApoE KO小鼠进行最大跑速测试(以初始速度为4.8 m/min,坡度为0°,持续5 min后,每3 min速度增加1.2 m/min,直至力竭,最后速度为最大跑速,最大跑速的测试结果为(27.0±2.4)m/min,剩余20只ApoE KO小鼠随机分为ApoE KO小鼠高脂膳食组(KO)和ApoE KO小鼠高脂膳食+有氧运动组(KE),每组10只,同时以10只9周龄野生型C57BL/6J小鼠作为空白对照组(WT)。高脂饲料成分:脂肪含量为21%(w/w),胆固醇含量为1.5%(w/w)。KE组适应性训练1周后开始运动干预,运动方案为:40%最大跑速(10.8 m/min),运动时间40 min/d,频率每周3 d,共计6周。待末次运动后48 h,所有小鼠麻醉后经心脏穿刺处死后迅速分离双侧腓肠肌;可见光比色法检测骨骼肌Ca²⁺浓度;Western blot法检测小鼠骨骼肌肌浆网钙调控蛋白RyR、CaM、CaMKⅡ、SERCA1、SERCA2蛋白表达。结果: 与WT组相比,KO组小鼠骨骼肌Ca²⁺浓度显著降低(P＜0.01),骨骼肌肌浆网钙释放蛋白RyR、CaMKⅡ和钙回收蛋白SERCA1、SERCA2均显著降低(P＜ 0.05),但CaM蛋白无显著变化;与KO组相比,KE组小鼠骨骼肌Ca²⁺浓度和骨骼肌肌浆网钙回收蛋白SERCA1、SERCA2均显著升高(P＜0.05),但骨骼肌肌浆网钙释放蛋白RyR、CaM、CaMKⅡ蛋白表达均无显著性差异。结论: 高脂膳食可使ApoE敲除小鼠骨骼肌Ca²⁺浓度降低、肌浆网钙释放作用和钙回收作用减弱,6周有氧运动训练能够显著提高其Ca²⁺浓度、促进肌浆网钙回收作用。     

Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr^−/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.     

Phospholamban overexpression in transgenic rabbits

There has been considerable interest in pursuing phospholamban as a putative therapeutic target for overcoming depressed calcium handling in human heart failure. Studies predominantly done in mice have shown that phospholamban is a key regulator of sarcoplasmic reticulum calcium cycling and cardiac function. However, mice differ significantly from humans in how they regulate calcium, whereas rabbits better recapitulate human cardiac function and calcium handling. To investigate phospholamban’s role in the rabbit heart, transgenic rabbits that overexpressed wild-type phospholamban in the ventricular cardiomyocytes and slow-twitch skeletal muscles were generated. Rabbits expressing high levels of phospholamban were not viable due to severe skeletal muscle wasting, the onset of cardiac pathology and early death. A viable transgenic line exhibited a 30% increase in PLN protein levels in the heart. These animals showed isolated foci of cardiac pathology, but cardiac function as well as the response to β-adrenergic stimulation were normal. SR-calcium uptake measurements showed that the transgenic hearts had the expected reduced affinity for calcium. The data show that phospholamban-overexpressing transgenic rabbits differ markedly in phenotype from analogous transgenic mice in that rabbits are quite sensitive to alterations in phospholamban levels. Exceeding a relatively narrow window of phospholamban expression results in significant morbidity and early death.     

Type 3 and type 1 ryanodine receptors are localized in triads of the same mammalian skeletal muscle fibers.

The type 3 ryanodine receptor (RyR3) is a ubiquitous calcium release channel that has recently been found in mammalian skeletal muscles. However, in contrast to the skeletal muscle isoform (RyR1), neither the subcellular distribution nor the physiological role of RyR3 are known. Here, we used isoform-specific antibodies to localize RyR3 in muscles of normal and RyR knockout mice. In normal hind limb and diaphragm muscles of young mice, RyR3 was expressed in all fibers where it was codistributed with RyR1 and with the skeletal muscle dihydropyridine receptor. This distribution pattern indicates that RyR3 is localized in the triadic junctions between the transverse tubules and the sarcoplasmic reticulum. During development, RyR3 expression declined rapidly in some fibers whereas other fibers maintained expression of RyR3 into adulthood. Comparing the distribution of RyR3-containing fibers with that of known fiber types did not show a direct correlation. Targeted deletion of the RyR1 or RyR3 gene resulted in the expected loss of the targeted isoform, but had no adverse effects on the expression and localization of the respective other RyR isoform. The localization of RyR3 in skeletal muscle triads, together with RyR1, is consistent with an accessory function of RyR3 in skeletal muscle excitation-contraction coupling.     

Deletion of hexose-6-phosphate dehydrogenase activates the unfolded protein response pathway and induces skeletal myopathy

Hexose-6-phosphate dehydrogenase (H6PD) is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver H6PD is required for the 11-oxoreductase activity of 11beta-hydroxysteroid dehydrogenase type 1, which converts inactive 11-oxo-glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in type II (fast) fibers, which have increased glycogen content. Here, we show that H6PD null mice develop a severe skeletal myopathy characterized by switching of type II to type I (slow) fibers. Running wheel activity and electrically stimulated force generation in isolated skeletal muscle are both markedly reduced. Affected muscles have normal sarcomeric structure at the electron microscopy level but contain large intrafibrillar membranous vacuoles and abnormal triads indicative of defects in structure and function of the sarcoplasmic reticulum (SR). SR proteins involved in calcium metabolism, including the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), calreticulin, and calsequestrin, show dysregulated expression. Microarray analysis and real-time PCR demonstrate overexpression of genes encoding proteins in the unfolded protein response pathway. We propose that the absence of H6PD induces a progressive myopathy by altering the SR redox state, thereby impairing protein folding and activating the unfolded protein response pathway. These studies thus define a novel metabolic pathway that links ER stress to skeletal muscle integrity and function.     

Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.     

Cloning and characterization of a new isoform of skeletal muscle triadin

We have shown that several isoforms of triadin, a protein involved in calcium release process through the ryanodine receptor, are expressed in rat skeletal muscle, and we have cloned two of these isoforms. One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, and the second one, shorter, is a truncated form of the previous one, but with a new unique COOH-terminal end. We propose to name the two proteins identified here Trisk 95 and Trisk 51. We have produced antibodies specific to each isoform. Using these antibodies, we have shown that the newly identified protein, Trisk 51, is actually expressed in adult rat skeletal muscle and also in rat embryo skeletal muscle. Immunofluorescent labeling of rat skeletal muscle with anti-Trisk 95, anti-Trisk 51, or anti-ryanodine receptor antibodies shows a similar localization of these proteins, in the tissue. Transfection of L6 cells with cDNA of Trisk 51 or Trisk 95 leads to the expression of proteins with the expected molecular weight, identical to those detected in rat skeletal muscle. Both proteins appear during differentiation of satellite cells in myotubes which may indicate the involvement of these two isoforms in the building of a functional calcium release machinery.     

Comparative biochemical study of sarcolemma and sarcoplasmic reticulum fractions isolated from mouse skeletal and cardiac muscles

1. Ca-ATPase activity, calcium-binding proteins and Concanavalin-A-bound glycoproteins of sarcolemma and sarcoplasmic reticulum were compared in mouse cardiac and skeletal muscles. 2. Ca-ATPase activity and calsequestrin were quite reduced in cardiac muscle, and the quantity of calcium bound to these two proteins was practically negligible, contrary to what was observed with skeletal muscle. In addition, the quantity of lipid bound calcium was not greater in cardiac muscle than in skeletal muscle. 3. Certain proteins seemed exclusively specific for skeletal muscle, including a 30,000 mol. wt glycoprotein which was totally absent in cardiac muscle sarcolemma.     

Altered function in atrium of transgenic mice overexpressing triadin 1

Triadin 1 is a protein in the cardiac junctional sarcoplasmic reticulum (SR) that interacts with the ryanodine receptor, junctin, and calsequestrin, proteins that are important for Ca(2+) release. To better understand the role of triadin 1 in SR-Ca(2+) release, we studied the time-dependent expression of SR proteins and contractility in atria of 3-, 6-, and 18-wk-old transgenic mice overexpressing canine cardiac triadin 1 under control of the alpha-myosin heavy chain (MHC) promoter. Three-week-old transgenic atria exhibited mild hypertrophy. Finally, atrial weight was increased by 110% in 18-wk-old transgenic mice. Triadin 1 overexpression was accompanied by time-dependent changes in the protein expression of the ryanodine receptor, junctin, and cardiac/slow-twitch muscle SR Ca(2+)-ATPase isoform. Force of contraction was already decreased in 3-wk-old transgenic atria. The application of caffeine led to a positive inotropic effect in transgenic atria of 3-wk-old mice. Rest pauses resulted in an increased potentiation of force of contraction after restimulation in 3- and 6-wk-old mice and a reduced potentiation of force of contraction in 18-wk-old transgenic mice. Hence, triadin 1 overexpression triggered time-dependent alterations in SR protein expression, Ca(2+) homeostasis, and contractility, indicating for the first time an inhibitory function of triadin 1 on SR-Ca(2+) release in vivo.     

Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins

This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice as well as dramatic alterations in intracellular localization of key components of the Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.     

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.     

Calsequestrin (CASQ1) rescues function and structure of calcium release units in skeletal muscles of CASQ1-null mice

Amplitude of Ca(2+) transients, ultrastructure of Ca(2+) release units, and molecular composition of sarcoplasmic reticulum (SR) are altered in fast-twitch skeletal muscles of calsequestrin-1 (CASQ1)-null mice. To determine whether such changes are directly caused by CASQ1 ablation or are instead the result of adaptive mechanisms, here we assessed ability of CASQ1 in rescuing the null phenotype. In vivo reintroduction of CASQ1 was carried out by cDNA electro transfer in flexor digitorum brevis muscle of the mouse. Exogenous CASQ1 was found to be correctly targeted to the junctional SR (jSR), as judged by immunofluorescence and confocal microscopy; terminal cisternae (TC) lumen was filled with electron dense material and its width was significantly increased, as judged by electron microscopy; peak amplitude of Ca(2+) transients was significantly increased compared with null muscle fibers transfected only with green fluorescent protein (control); and finally, transfected fibers were able to sustain cytosolic Ca(2+) concentration during prolonged tetanic stimulation. Only the expression of TC proteins, such as calsequestrin 2, sarcalumenin, and triadin, was not rescued as judged by Western blot. Thus our results support the view that CASQ1 plays a key role in both Ca(2+) homeostasis and TC structure.     

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.