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.     

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.     

The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium

The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.     

Calsequestrin Is an Inhibitor of Skeletal Muscle Ryanodine Receptor Calcium Release Channels

We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca²⁺] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (∼35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.     

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.     

Supramolecular calsequestrin complex.

As recently demonstrated by overlay assays using calsequestrin-peroxidase conjugates, the major 63 kDa Ca(2+)-binding protein of the sarcoplasmic reticulum forms complexes with itself, and with junctin (26 kDa), triadin (94 kDa) and the ryanodine receptor (560 kDa) [Glover, L., Culligan, K., Cala, S., Mulvey, C. & Ohlendieck, K. (2001) Biochim. Biophys. Acta1515, 120-132]. Here, we show that variations in the relative abundance of these four central elements of excitation-contraction coupling in different fiber types, and during chronic electrostimulation-induced fiber type transitions, are reflected by distinct alterations in the calsequestrin overlay binding patterns. Comparative immunoblotting with antibodies to markers of the junctional sarcoplasmic reticulum, in combination with the calsequestrin overlay binding patterns, confirmed a lower ryanodine receptor expression in slow soleus muscle compared to fast fibers, and revealed a drastic reduction of the RyR1 isoform in chronic low-frequency stimulated tibialis anterior muscle. The fast-to-slow transition process included a distinct reduction in fast calsequestrin and triadin and a concomitant reduction in calsequestrin binding to these sarcoplasmic reticulum elements. The calsequestrin-binding protein junctin was not affected by the muscle transformation process. The increase in calsequestrin and decrease in junctin expression during postnatal development resulted in similar changes in the intensity of binding of the calsequestrin conjugate to these sarcoplasmic reticulum components. Aged skeletal muscle fibers tended towards reduced protein interactions within the calsequestrin complex. This agrees with the physiological concept that the key regulators of Ca(2+) homeostasis exist in a supramolecular membrane assembly and that protein-protein interactions are affected by isoform shifting underlying the finely tuned adaptation of muscle fibers to changed functional demands.     

Calsequestrin and the calcium release channel of skeletal and cardiac muscle

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.     

Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin

In mammalian striated muscles, ryanodine receptor (RyR), triadin, junctin, and calsequestrin form a quaternary complex in the lumen of sarcoplasmic reticulum. Such intermolecular interactions contribute not only to the passive buffering of sarcoplasmic reticulum luminal Ca2+, but also to the active Ca2+ release process during excitation-contraction coupling. Here we tested the hypothesis that specific charged amino acids within the luminal portion of RyR mediate its direct interaction with triadin. Using in vitro binding assay and site-directed mutagenesis, we found that the second intraluminal loop of the skeletal muscle RyR1 (amino acids 4860-4917), but not the first intraluminal loop of RyR1 (amino acids 4581-4640) could bind triadin. Specifically, three negatively charged residues Asp4878, Asp4907, and Glu4908 appear to be critical for the association with triadin. Using deletional approaches, we showed that a KEKE motif of triadin (amino acids 200-232) is essential for the binding to RyR1. Because the second intraluminal loop of RyR has been previously shown to contain the ion-conducting pore as well as the selectivity filter of the Ca2+ release channel, and Asp4878, Asp4907, and Glu4908 residues are predicted to locate at the periphery of the pore assembly of the channel, our data suggest that a physical interaction between RyR1 and triadin could play an active role in the overall Ca2+ release process of excitation-contraction coupling in muscle cells.     

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle.

Ca(2+)-handling proteins are important regulators of the excitation-contraction-relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca(2+)-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca(2+) reservoir and thereby occupies a central position in Ca(2+) homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca(2+) concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca(2+)-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca(2+) homeostasis is regulated by direct protein-protein interactions.     

Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing triadin 1

Triadin 1 is a major transmembrane protein in cardiac junctional sarcoplasmic reticulum (SR), which forms a quaternary complex with the ryanodine receptor (Ca(2+) release channel), junctin, and calsequestrin. To better understand the role of triadin 1 in excitation-contraction coupling in the heart, we generated transgenic mice with targeted overexpression of triadin 1 to mouse atrium and ventricle, employing the alpha-myosin heavy chain promoter to drive protein expression. The protein was overexpressed 5-fold in mouse ventricles, and overexpression was accompanied by cardiac hypertrophy. The levels of two other junctional SR proteins, the ryanodine receptor and junctin, were reduced by 55% and 73%, respectively, in association with triadin 1 overexpression, whereas the levels of calsequestrin, the Ca(2+)-binding protein of junctional SR, and of phospholamban and SERCA2a, Ca(2+)-handling proteins of the free SR, were unchanged. Cardiac myocytes from triadin 1-overexpressing mice exhibited depressed contractility; Ca(2+) transients decayed at a slower rate, and cell shortening and relengthening were diminished. The extent of depression of cell shortening of triadin 1-overexpressing cardiomyocytes was rate-dependent, being more depressed under low stimulation frequencies (0.5 Hz), but reaching comparable levels at higher frequencies of stimulation (5 Hz). Spontaneously beating, isolated work-performing heart preparations overexpressing triadin 1 also relaxed at a slower rate than control hearts, and failed to adapt to increased afterload appropriately. The fast time inactivation constant, tau(1), of the l-type Ca(2+) channel was prolonged in transgenic cardiomyocytes. Our results provide evidence for the coordinated regulation of junctional SR protein expression in heart independent of free SR protein expression, and furthermore suggest an important role for triadin 1 in regulating the contractile properties of the heart during excitation-contraction coupling.     

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.     

Luminal calcium regulation of calcium release from sarcoplasmic reticulum

This article discusses how changes in luminal calcium concentration affect calcium release rates from triad-enriched sarcoplasmic reticulum vesicles, as well as single channel opening probability of the ryanodine receptor/calcium release channels incorporated in bilayers. The possible participation of calsequestrin, or of other luminal proteins of sarcoplasmic reticulum in this regulation is addressed. A comparison with the regulation by luminal calcium of calcium release mediated by the inositol 1,4,5-trisphosphate receptor/calcium channel is presented as well.     

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.     

Identification of triadin 1 as the predominant triadin isoform expressed in mammalian myocardium.

Triadin is an integral membrane protein of sarcoplasmic reticulum shown to interact with the ryanodine receptor/Ca(2+) release channel, junctin, and calsequestrin. Several triadin isoforms have been postulated to exist in cardiac muscle, but to date none has been conclusively identified. Here, we show that only triadin 1 is significantly expressed. We cloned and sequenced cDNAs encoding canine cardiac triadin 1 and 3 but found no evidence for triadin 2. From deduced primary structures, antibodies against domains common to all triadins and an antibody against the unique C terminus of triadin 1 were raised. All antibodies detected two prominent proteins of molecular masses 35 and 40 kDa on immunoblots from cardiac microsomes, including the antibody that recognizes only triadin 1. The 40-kDa mobility form was shown to correspond to the glycosylated form of triadin 1, not a distinct triadin 2 isoform as previously hypothesized. Confirming this, overexpression of triadin 1 in transgenic mouse hearts produced both the 35-kDa deglycosylated and the 40-kDa glycosylated mobility forms. The glycosylation site of triadin 1 was localized to asparagine residue 75, and its bitopic arrangement in the membrane was confirmed. Although a 92-kDa immunoreactive protein could be tentatively identified in myocardium as triadin 3, its expression level was insignificant (相似文献   

Localization and characterization of the calsequestrin-binding domain of triadin 1. Evidence for a charged beta-strand in mediating the protein-protein interaction

Triadin is an integral membrane protein of the junctional sarcoplasmic reticulum that binds to the high capacity Ca(2+)-binding protein calsequestrin and anchors it to the ryanodine receptor. The lumenal domain of triadin contains multiple repeats of alternating lysine and glutamic acid residues, which have been defined as KEKE motifs and have been proposed to promote protein associations. Here we identified the specific residues of triadin responsible for binding to calsequestrin by mutational analysis of triadin 1, the major cardiac isoform. A series of deletional fusion proteins of triadin 1 was generated, and by using metabolically labeled calsequestrin in filter-overlay assays, the calsequestrin-binding domain of triadin 1 was localized to a single KEKE motif comprised of 25 amino acids. Alanine mutagenesis within this motif demonstrated that the critical amino acids of triadin binding to calsequestrin are the even-numbered residues Lys(210), Lys(212), Glu(214), Lys(216), Gly(218), Gln(220), Lys(222), and Lys(224). Replacement of the odd-numbered residues within this motif by alanine had no effect on calsequestrin binding to triadin. The results suggest a model in which residues 210-224 of triadin form a beta-strand, with the even-numbered residues in the strand interacting with charged residues of calsequestrin, stabilizing a "polar zipper" that links the two proteins together. This small, highly charged beta-strand of triadin may tether calsequestrin to the junctional face membrane, allowing calsequestrin to sequester Ca(2+) in the vicinity of the ryanodine receptor during Ca(2+) uptake and Ca(2+) release.     

Triadin binding to the C-terminal luminal loop of the ryanodine receptor is important for skeletal muscle excitation contraction coupling

Ca(2+) release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca(2+) to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation-contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca(2+) release during excitation-contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca(2+) release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca(2+) release during excitation-contraction coupling in skeletal muscle.     

The conformation of calsequestrin determines its ability to regulate skeletal ryanodine receptors

Ca2+ efflux from the sarcoplasmic reticulum decreases when store Ca2+ concentration falls, particularly in skinned fibers and isolated vesicles where luminal Ca2+ can be reduced to very low levels. However ryanodine receptor activity in many single channel studies is higher when the luminal free Ca2+ concentration is reduced. We investigated the hypothesis that prolonged exposure to low luminal Ca2+ causes conformational changes in calsequestrin and deregulation of ryanodine receptors, allowing channel activity to increase. Lowering of luminal Ca2+ from 1 mM to 100 microM for several minutes resulted in conformational changes with dissociation of 65-75% of calsequestrin from the junctional face membrane. The calsequestrin remaining associated no longer regulated channels. In the absence of this regulation, ryanodine receptors were more active when luminal Ca2+ was lowered from 1 mM to 100 microM. In contrast, when ryanodine receptors were calsequestrin regulated, lowering luminal Ca2+ either did not alter or decreased activity. Ryanodine receptors are regulated by calsequestrin under physiological conditions where calsequestrin is polymerized. Since depolymerization occurs slowly, calsequestrin can regulate the ryanodine receptor and prevent excess Ca2+ release when the store is transiently depleted, for example, during high frequency activity or early stages of muscle fatigue.     

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.     

Increased sensitivity of the ryanodine receptor to halothane-induced oligomerization in malignant hyperthermia-susceptible human skeletal muscle.

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the alpha2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014-0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+ uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.     

Regulation of myocardial function by histidine-rich, calcium-binding protein

Impaired sarcoplasmic reticulum (SR) Ca release has been suggested to contribute to the depressed cardiac function in heart failure. The release of Ca from the SR may be regulated by the ryanodine receptor, triadin, junctin, calsequestrin, and a histidine-rich, Ca-binding protein (HRC). We observed that the levels of HRC were reduced in animal models and human heart failure. To gain insight into the physiological function of HRC, we infected adult rat cardiac myocytes with a recombinant adenovirus that contains the full-length mouse HRC cDNA. Overexpression (1.7-fold) of HRC in adult rat cardiomyocytes was associated with increased SR Ca load (28%) but decreased SR Ca-induced Ca release (37%), resulting in impaired Ca cycling and depressed fractional shortening (36%) as well as depressed rates of shortening (38%) and relengthening (33%). Furthermore, the depressed basal contractile and Ca kinetic parameters in the HRC-infected myocytes remained significantly depressed even after maximal isoproterenol stimulation. Interestingly, HRC overexpresssion was accompanied by increased protein levels of junctin (1.4-fold) and triadin (1.8-fold), whereas the protein levels of ryanodine receptor, calsequestrin, phospholamban, and sarco(endo)plasmic reticulum Ca-ATPase remained unaltered. Collectively, these data indicate that alterations in expression levels of HRC are associated with impaired cardiac SR Ca homeostasis and contractile function.