SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca2? released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.     

Calumenin, a multiple EF-hands Ca2+-binding protein, interacts with ryanodine receptor-1 in rabbit skeletal sarcoplasmic reticulum

Calumenin is a multiple EF-hand Ca2+-binding protein located in endo/sarcoplasmic reticulum of mammalian tissues. In the present study, we cloned two rabbit calumenin isoforms (rabbit calumenin-1 and -2, GenBank Accession Nos. SY225335 and AY225336, respectively) by RT-PCR. Both isoforms contain a 19 aa N-terminal signal sequence, 6 EF-hand domains, and a C-terminal ER/SR retrieval signal, HDEF. Both calumenin isoforms exist in rabbit cardiac and skeletal muscles, but calumenin-2 is the main isoform in skeletal muscle. Presence of calumenin in rabbit sarcoplasmic reticulum (SR) was identified by Western blot analysis. GST-pull down and co-immunoprecipitation experiments showed that ryanodine receptor 1 (RyR1) interacted with calumenin-2 in millimolar Ca2+ concentration range. Experiments of gradual EF-hand deletions suggest that the second EF-hand domain is essential for calumenin binding to RyR1. Adenovirus-mediated overexpression of calumenin-2 in C2C12 myotubes led to increased caffeine-induced Ca2+ release, but decreased depolarization-induced Ca2+ release. Taken together, we propose that calumenin-2 in the SR lumen can directly regulate the RyR1 activity in Ca2+-dependent manner.     

Subcellular distribution of ryanodine receptors in the cardiac muscle of carp (Cyprinus carpio)

We examined the subcellular localization of ryanodine receptors (RyR) in the cardiac muscle of carp using biochemical, immunohistochemical, and electron microscopic methods and compared it with those of rats and guinea pigs. To achieve this goal, an anti-RyR antibody was newly raised against a synthetic peptide corresponding to an amino acid sequence that was conserved among all sequenced RyRs. Western blot analysis using this antibody detected a single RyR band following the SDS-PAGE of sarcoplasmic reticulum (SR) membranes from carp atrium and ventricle as well as from mammalian hearts and skeletal muscles. The carp heart band had slightly greater mobility than those of mammalian hearts. Although immunohistochemical staining showed evident striations corresponding to the Z lines in longitudinal sections of mammalian hearts, clusters of punctate staining, in contrast, were distributed ubiquitously throughout carp atrium and ventricle. Electron microscopic images of the carp myocardium showed that the SR was observed largely as the subsarcolemmal cisternae and the reticular SR, suggesting that the RyR is localized in the junctional and corbular SR.     

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.     

Properties of ryanodine receptor in rat muscles submitted to unloaded conditions

Unloading of skeletal muscles by hindlimb unweighting is known to induce muscle atrophy and a shift toward faster contractile properties associated with an increase in the expression of fast contractile proteins, particularly in slow soleus muscles. Contractile properties suggest that slow soleus muscles acquire SR properties close to those of a faster one. We studied the expression and properties of the sarcoplasmic reticulum calcium release (RyR) channels in soleus and gastrocnemius muscles of rats submitted to hindlimb unloading (HU). An increase in RyR1 and a slight decrease in RyR3 expression was detected in atrophied soleus muscles only after 4 weeks of HU. No variation appeared in fast muscles. [(3)H]Ryanodine binding experiments showed that HU neither increased the affinity of the receptors for [(3)H]ryanodine nor changed the caffeine sensitivity of [(3)H]ryanodine binding. Our results suggested that not only RyR1 but also RyR3 expression can be regulated by muscle activity and innervation in soleus muscle. The changes in the RyR expression in slow fibers suggested a transformation of the SR from a slow to a fast phenotype.     

The novel skeletal muscle sarcoplasmic reticulum JP-45 protein. Molecular cloning,tissue distribution,developmental expression,and interaction with alpha 1.1 subunit of the voltage-gated calcium channel

JP-45 is a novel integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal muscle cDNA library. Mouse skeletal muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases approximately 3-fold during aging. Double immunofluorescence of adult skeletal muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the alpha1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal muscle excitation-contraction coupling.     

Morphological and biochemical correlates of skeletal muscle contractility in the cat. II. Physiological and biochemical studies.

Isometric twitch characteristics and biochemical parameters of isolated myosin and sarcoplasmic reticulum have been compared in three cat hind limb muscles. The fast twitch caudofemoralis and the slow twitch soleus are almost pure muscles as judged from histochemical studies. Isolated myosin from the caudofemoralis is not only 2- to 3-fold higher in its ATPase activities than that of the soleus, but also in non-dissociated forms has greater electrophoretic mobility than the soleus myosin. Purified myosins from fast muscles as well as soleus exhibited three light chains upon electrophoresis. However, the intact non-solubilized myosins differed in electrophoretic mobilities. The sarcoplasmic reticulum fraction isolated from caudfemoralis exhibits faster rates of Ca++ binding and uptake than soleus, and when fit to a two component model, the caudofemoralis SR exhibits a higher amount of a fast binding site than does soleus SR, features reflected in differences in the relaxation time of the two muscles. In contrast, the fast twitch tibialis anterior has been shown to be a gradient of fiber types and its isometric twitch may be separated by selective nerve stimulation, into a fast and a slow twitch component. Our findings that myosin fractions, as well as sarcoplasmic reticulum fractions isolated from these two components differ with respect to their biochemical characteristics add support to the possibility of a dual function in this muscle.     

Glutathione transport in the endo/sarcoplasmic reticulum

Glutathione transport through the endo/sarcoplasmic reticulum (ER/SR) membrane might play a role in the maintenance of the thiol redox potential difference between the lumen and the cytosol. The transport of glutathione (both GSH and glutathione disulfide, GSSG) is entirely different in the ER and SR membranes. The transport measurements based on either rapid filtration or light scattering techniques revealed that the SR membrane transports glutathione much faster than the hepatic ER membrane or microsomal membranes prepared from heart or brain. The fastest transport has been measured in the membrane of muscle terminal cisternae, which is enriched in ryanodine receptor type 1 (RyR1). All the studied membranes have been found to be equally impermeable to various hydrophilic substances of similar size to glutathione, thus the glutathione transport in muscle microsomes and terminal cysternae as well as the correlation between the rate of glutathione transport and the abundance of RyR1 are specific. In both muscle microsomes and terminal cysternae, glutathione influx can be either inhibited or activated by antagonists and agonists of the ryanodine receptor, respectively, while these agents do not influence the transport of other small permeant molecules. These findings strongly suggest that the ryanodine receptor channel activity is directly associated with glutathione transport activity in the skeletal muscle sarcoplasmic reticulum membrane.     

Deduced amino acid sequence and E1-E2 equilibrium of the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle. Comparison with the Ca(2+)-ATPase of rabbit fast twitch muscle.

The cDNA encoding a Ca(2+)-transport ATPase of frog (Rana esculenta) skeletal muscle was isolated and characterized. The deduced amino acid sequence, consisting of 994 residues, showed 89% identity to the fast twitch muscle sarcoplasmic reticulum Ca(2+)-ATPases of chicken and rabbit. Northern blot analysis using a fragment of this cDNA as probe detected a 5.0 kb message in frog skeletal muscle but did not detect any mRNA encoding sarcoplasmic reticulum Ca(2+)-ATPase in frog cardiac muscle. The enzymatic properties of the amphibian skeletal muscle Ca(2+)-ATPase were compared with those of the rabbit fast twitch muscle Ca(2+)-ATPase by functional expression of the cDNAs in COS-1 cells. The amphibian Ca(2+)-ATPase displayed a reduced apparent affinity for Ca2+ and an increased apparent affinity for the inhibitors, vanadate and thapsigargin, relative to the mammalian enzyme. This may be explained by a mechanism in which relatively more of the E2 conformation accumulated in the frog Ca(2+)-ATPase than in the mammalian enzyme.     

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.     

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).     

Redox modulation of diaphragm contractility: Interaction between DHPR and RyR channels

Previous reports indicate that reactive oxygen species (ROS) may modulate contractility in skeletal muscle. Although Ca(2+)-sensitivity of the contractile apparatus appears to be a primary site of regulation, dihydropyridine receptor (DHPR or L-type Ca(2+) channels) and calcium efflux in isolated sarcoplasmic reticulum (SR) vesicles appear to be redox sensitive as well. However, DHPR as a target is poorly understood in intact muscles at body temperature, particularly in the diaphragm, a muscle more dependent on external Ca(2+) than locomotor muscles. Previously, we reported that oxidant challenge via xanthine oxidase (XO) alters the K(+) contractures in diaphragm fiber bundles, suggestive of a role of L-type Ca(2+) channels. Contractility of isolated rat diaphragm fiber bundles revealed a biphasic response to ROS challenge that was dose and time dependent. Potentiation of twitch and low-frequency diaphragm fiber bundle contractility with 0.02 U?ml(-1) XO was reversible or partially preventable with washout, dithiothreitol, and the SOD/catalase mimetic EUK-134. The RyR antagonist ruthenium red inhibited xanthine oxidase-induced potentiation, while the RyR agonist caffeine elevated diaphragm twitch and low-frequency tension in a non-additive manner by 55% when introduced simultaneously with ROS challenge. The DHPR antagonist nitrendipine (15 μM) inhibited elevation in low-frequency diaphragm tension produced by ROS challenge. Caffeine threshold tension curves were shifted to the left with 0.02 U?ml(-1) XO, but this effect was partially reversed with 15 μM nitrendipine. These results are consistent with the hypothesis that DHPR redox state and RyR function are modulated in an interactive manner, affecting contractility in intact diaphragm fiber bundles.     

Carbonic anhydrases IV and IX: subcellular localization and functional role in mouse skeletal muscle

The subcellular localization of carbonic anhydrase (CA) IV and CA IX in mouse skeletal muscle fibers has been studied immunohistochemically by confocal laser scanning microscopy. CA IV has been found to be located on the plasma membrane as well as on the sarcoplasmic reticulum (SR) membrane. CA IX is not localized in the plasma membrane but in the region of the t-tubular (TT)/terminal SR membrane. CA IV contributes 20% and CA IX 60% to the total CA activity of SR membrane vesicles isolated from mouse skeletal muscles. Our aim was to examine whether SR CA IV and TT/SR CA IX affect muscle contraction. Isolated fiber bundles of fast-twitch extensor digitorum longus and slow-twitch soleus muscle from mouse were investigated for isometric twitch and tetanic contractions and by a fatigue test. The muscle functions of CA IV knockout (KO) fibers and of CA IX KO fibers do not differ from the function of wild-type (WT) fibers. Muscle function of CA IV/XIV double KO mice unexpectedly shows a decrease in rise and relaxation time and in force of single twitches. In contrast, the CA inhibitor dorzolamide, whether applied to WT or to double KO muscle fibers, leads to a significant increase in rise time and force of twitches. It is concluded that the function of mouse skeletal muscle fibers expressing three membrane-associated CAs, IV, IX, and XIV, is not affected by the lack of one isoform but is possibly affected by the lack of all three CAs, as indicated by the inhibition studies.     

Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle.

Skeletal muscle Ca(2+) release units (CRUs) are junctions of the surface membrane/T-tubule system and the sarcoplasmic reticulum (SR) that function in excitation-contraction coupling. They contain high concentrations of dihydropyridine receptors (DHPRs) in the T-tubules and of ryanodine receptors (RyR) in the SR and they are positioned at specific locations in the sarcomere. In order to characterize the sequence of developmental steps leading to the specific molecular and structural organization of CRUs, we applied a range of imaging techniques that allowed us to follow the differentiation of the membrane compartments and the expression of junctional proteins in developing mouse diaphragm muscle. We find that docking of the two membrane systems precedes the incorporation of the RyRs into the junctions, and that T-tubule/SR junctions are formed and positioned at the I-A interface at a stage when the orientation of T-tubule is predominantly longitudinal. Thus, the sequence of developmental events is first the docking of T-tubules and SR, secondly the incorporation of RyR in the junctions, thirdly the positioning of the junctions in the sarcomere, and only much later the transverse orientation of the T-tubules. These sequential stages suggests an order of inductive processes for the molecular differentiation and structural organization of the CRUs in skeletal muscle development.     

TRPC3-interacting triadic proteins in skeletal muscle

The expression of TRPC3 (canonical-type transient receptor potential cation channel type 3) is tightly regulated during skeletal muscle cell differentiation, and a functional interaction between TRPC3 and RyR1 [(ryanodine receptor type 1), an SR (sarcoplasmic reticulum) Ca2+-release channel] regulates the gain of SR Ca2+ release during EC (excitation-contraction) coupling. However, it has not been possible to demonstrate direct protein-protein interactions between TRPC3 and RyR1. To identify possible candidate(s) for a linker protein(s) between TRPC3 and RyR1 in skeletal muscle, in the present study we performed MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of a cross-linked triadic protein complex from rabbit skeletal triad vesicles and co-immunoprecipitation assays using primary mouse skeletal myotubes. From these studies, we found that six triadic proteins, that are known to regulate RyR1 function and/or EC coupling [TRPC1, JP2 (junctophilin 2), homer, mitsugumin 29, calreticulin and calmodulin], interacted directly with TRPC3 in a Ca2+-independent manner. However we again found no direct interaction between TRPC3 and RyR1. TRPC1 was identified as a potential physical link between TRPC3 and RyR1, as it interacted with both TRPC3 and RyR1, and JPs showed subtype-specific interactions with both RyR1 and TRPC3 (JP1-RyR1 and JP2-TRPC3). These results support the hypothesis that TRPC3 and RyR1 are functionally engaged via linker proteins in skeletal muscle.     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling

Much recent progress has been made in our understanding of the mechanism of sarcoplasmic reticulum Ca²⁺ release in skeletal muscle. Vertebrate skeletal muscle excitation-contraction (E-C) coupling is thought to occur by a mechanical coupling mechanism involving protein-protein interactions that lead to activation of the sarcoplasmic reticulum (SR) ryanodine receptor (RyR)/Ca²⁺ release channel by the voltage-sensing transverse (T–) tubule dihydropyridine receptor (DHPR)/Ca²⁺ channel. In a subsequent step, the released Ca²⁺ amplify SR Ca²⁺ release by activating release channels that are not linked to the DHPR. Experiments with mutant muscle cells have indicated that skeletal muscle specific DHPR and RyR isoforms are required for skeletal muscle E-C coupling. A direct functional and structural interaction between a DHPR-derived peptide and the RyR has been described. The interaction between the DHPR and RyR may be stabilized by other proteins such as triadin (a SR junctional protein) and modulated by phosphorylation of the DHPR.     

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation-contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are "leaky." RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.     

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.