Skeletal Muscle Sarcoplasmic Reticulum Ca2 Research Articles

Time course and fibre type-dependent nature of calcium-handling protein responses to sprint interval exercise in human skeletal muscle.

Sprint interval training (SIT) causes fragmentation of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor 1 (RyR1), 24h post-exercise, potentially signalling mitochondrial biogenesis by increasing cytosolic [Ca2+ ]. Yet, the time course and skeletal muscle fibre type-specific patterns of RyR1 fragmentation following a session of SIT remain unknown. Ten participants (n=4 females; n=6 males) performed a session of SIT (6×30s 'all-out' with 4.5min rest after each sprint) with vastus lateralis muscle biopsy samples collected before and 3, 6 and 24h after exercise. In whole muscle, full-length RyR1 protein content was significantly reduced 6h (mean (SD); -38 (38)%; P<0.05) and 24h post-SIT (-30 (48)%; P<0.05) compared to pre-exercise. Examining each participant's largest response in pooled samples, full-length RyR1 protein content was reduced in type II (-26 (30)%; P<0.05) but not type I fibres (-11 (40)%; P>0.05). Three hours post-SIT, there was also a decrease in sarco(endo)plasmic reticulum Ca2+ ATPase 1 in type II fibres (-23 (17)%; P<0.05) and sarco(endo)plasmic reticulum Ca2+ ATPase 2a in type I fibres (-19 (21)%; P<0.05), despite no time effect for either protein in whole muscle samples (P>0.05). PGC1A mRNA content was elevated 3 and 6h post-SIT (5.3- and 3.7-fold change from pre, respectively; P<0.05 for both), but peak PGC1A mRNA expression was not significantly correlated with peak RyR1 fragmentation (r2 =0.10; P>0.05). In summary, altered Ca2+ -handling protein expression, which occurs primarily in type II muscle fibres, may influence signals for mitochondrial biogenesis as early as 3-6h post-SIT in humans. KEY POINTS: Sprint interval training (SIT) has been shown to cause fragmentation of the sarcoplasmic reticulum calcium-release channel, ryanodine receptor 1 (RyR1), 24h post-exercise, which may act as a signal for mitochondrial biogenesis. In this study, the time course was examined of RyR1 fragmentation in human whole muscle and pooled type I and type II skeletal muscle fibres following a single session of SIT. Full-length RyR1 protein content was significantly lower than pre-exercise by 6h post-SIT in whole muscle, and fragmentation was detectable in type II but not type I fibres, though to a lesser extent than in whole muscle. The peak in PGC1A mRNA expression occurred earlier than RyR1 fragmentation. The increased temporal resolution and fibre type-specific responses for RyR1 fragmentation provide insights into its importance to mitochondrial biogenesis in humans.

Reduced sarcoplasmic reticulum Ca2+ ATPase activity underlies skeletal muscle wasting in asthma

AimsSkeletal muscle mass and strength are reduced in asthma and contribute to compromised functional capacity in asthmatic patients. However, an effective pharmacological intervention remains elusive, partly because molecular mechanisms dictating muscle decline in asthma are not known. MaterialsWe investigated the potential contribution(s) of skeletal muscle sarcoplasmic reticulum Ca2+ ATPase (SERCA) to muscle atrophy and weakness in asthmatic patients. Quadriceps muscle biopsies were taken from 58 to 72 years old male patients with mild and advanced asthma and the SERCA activity was analyzed in association with cellular redox environment and myonuclear domain (MND) size. Key findingsMaximal SERCA activity was reduced in skeletal muscles of mild and advanced asthmatics and was associated with reduced expression of SERCA2 protein and upregulation of sarcolipin, a SERCA inhibitory lipoprotein. We also found downregulation of Ca2+ release protein calstabin and upregulation of Ca2+ buffer, calsequestrin in skeletal muscles of asthmatic patients. The atrophic single muscle fibers had smaller cytoplasmic domains per myonucleus possibly indicating the reduced transcriptional reserves of individual myonuclei. Plasma periostin and CAF22 levels were significantly elevated in asthmatic patients and showed a strong correlation with hand-grip strength. These changes were accompanied by substantially elevated markers of global oxidative stress including lipid peroxidation and mitochondrial ROS production. ConclusionTaken together, our data suggest that muscle weakness and atrophy in asthma is in part driven by SERCA dysfunction and oxidative stress. The data propose SERCA dysfunction as a therapeutic intervention to address muscle decline in asthma.

Negative chronotropism, positive inotropism and lusitropism of 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) on rat heart preparations occur through reduction of RyR2 Ca2+ leak

3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) is considered as an activator of the skeletal muscle sarcoplasmic reticulum (SR) Ca2+-uptake, endowed with antioxidant and L-type Ca2+ channel blocking activities.In this study we assessed the cardiac effects of DTBHA on Langendorff perfused rat hearts, isolated rat atria and rat cardiac SR membrane vesicles, as well as on several SERCA isoforms of membrane preparations. Moreover, in order to clarify its molecular mechanism of action Ca2+ imaging experiments were carried out on HEK293 cells transiently transfected with RyR2 channel. Docking of DTBHA at the rat RyR2 protein was investigated in silico.In Langendorff perfused rat hearts, DTBHA significantly increased, in a concentration-dependent manner, left ventricular pressure and diastole duration, while reducing heart rate and the time-constant of isovolumic relaxation, leaving unaltered coronary perfusion pressure. At the maximum concentration tested (30 µM), it significantly prolonged PQ interval, but left the corrected QT intervals unaffected. In spontaneously beating atria, DTBHA decreased sinus rate in a concentration-dependent manner.DTBHA, at concentrations higher than 10 µM, increased Ca2+ uptake in cardiac SR without affecting Ca2+-dependent ATPase activity assayed on several SERCA isoforms. Moreover, DTBHA antagonized thapsigargin-stimulated Ca2+ leak in cardiac SR and reduced caffeine-induced, RyR2-activated Ca2+ release in RyR2 expressing HEK293 cells. Using computational approaches, DTBHA showed a good affinity outline into binding sites of RyR2 protein.In conclusion, DTBHA behaved like a negative chronotropic, a positive inotropic and a lusitropic agent on rat heart preparations and improved cardiac SR Ca2+ uptake by lowering SR Ca2+ leak.

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle.

K201 (JTV-519) may prevent abnormal Ca(2+) leak from the sarcoplasmic reticulum (SR) in the ischemic heart and skeletal muscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1 and RyR2, respectively). We tested direct modulation of the SR Ca(2+)-stimulated ATPase (SERCA) and RyRs by K201. In isolated cardiac and SkM SR microsomes, K201 slowed the rate of SR Ca(2+) loading, suggesting potential SERCA block and/or RyR agonism. K201 displayed Ca(2+)-dependent inhibition of SERCA-dependent ATPase activity, which was measured in microsomes incubated with 200, 2, and 0.25 µM Ca(2+) and with the half-maximal K201 inhibitory doses (IC50) estimated at 130, 19, and 9 µM (cardiac muscle) and 104, 13, and 5 µM (SkM SR). K201 (≥5 µM) increased RyR1-mediated Ca(2+) release from SkM microsomes. Maximal K201 doses at 80 µM produced ∼37% of the increase in SkM SR Ca(2+) release observed with the RyR agonist caffeine. K201 (≥5 µM) increased the open probability (Po) of very active ("high-activity") RyR1 of SkM reconstituted into bilayers, but it had no effect on "low-activity" channels. Likewise, K201 activated cardiac RyR2 under systolic Ca(2+) conditions (∼5 µM; channels at Po ∼0.3) but not under diastolic Ca(2+) conditions (∼100 nM; Po < 0.01). Thus, K201-induced the inhibition of SR Ca(2+) leak found in cell-system studies may relate to potentially potent SERCA block under resting Ca(2+) conditions. SERCA block likely produces mild SR depletion in normal conditions but could prevent SR Ca(2+) overload under pathologic conditions, thus precluding abnormal RyR-mediated Ca(2+) release.

Roles of Long-range Electrostatic Domain Interactions and K+ in Phosphoenzyme Transition of Ca2+-ATPase

Sarcoplasmic reticulum Ca(2+)-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca(2+) transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 m inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K(+) is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.

Sarcolemmal Calcium Influx in Malignant Hyperthermia Susceptible Muscle

The most common cause of malignant hyperthermia (MH) susceptibility is a mutation in RyR1 (ryanodine receptor type 1), the skeletal muscle sarcoplasmic reticulum Ca2+ release channel. Muscles expressing MH-RyR1s have an increased cytosolic free Ca2+ concentration ([Ca2+]i) at rest which is exacerbated by volatile anesthetics. The Ca2+ overload induced by anesthetics is fatal unless treated with dantrolene. Here we show that the sarcolemmal Ca2+ influx and [Ca2+]i are greater at rest in MH-RyR1R163C myotubes than in WT cells. Gd+3 and GsMTx-4 are more effective than BTP2 or over-expression of a dominant negative Orai1 (E190Q) in decreasing [Ca2+]i in MH-RyR1R163C myotubes suggesting that it is mediated by an alternative non-Stim1/Orai1 entry pathway. Exposure to a DAG analog induced a larger increase in Ca2+ in MH-RyR1R163C myotubes than in WT cells and was completely blocked by Gd3+ and GsMTx-4 suggesting that TRPC3/6 could be involved. Further support is that these non-selective cationic channels are over-expressed and that [Na]i is elevated in MHRyR1R163C cells. In vivo in MH-RyR1R163C/Wt muscle intracellular Ca2+ and Na+ overload can be blocked by dantrolene, and attenuated by Gd3+ and GsMTx-4. Exposure of MH-RyR1R163C/Wt mice to halothane markedly increased both [Ca2+]i and [Na+]i in their muscle fibers. These effects of halothane were markedly attenuated by Gd+3 or GsMTx-4 and completely suppressed by dantrolene. Taken together these results strongly suggest that sarcolemmal Ca2+ and Na+ entry via TRPC3/6 are important contributors to the altered Ca2+ and Na+ homeostasis observed in MH muscle both at rest and during an MH crisis.

RyR1-mediated Ca2+ Leak and Ca2+ Entry Determine Resting Intracellular Ca2+ in Skeletal Myotubes

The control of resting free Ca(2+) in skeletal muscle is thought to be a balance of channels, pumps, and exchangers in both the sarcolemma and sarcoplasmic reticulum. We explored these mechanisms using pharmacologic and molecular perturbations of genetically engineered (dyspedic) muscle cells that constitutively lack expression of the skeletal muscle sarcoplasmic reticulum Ca(2+) release channels, RyR1 and RyR3. We demonstrate here that expression of RyR1 is responsible for more than half of total resting Ca(2+) concentration ([Ca(2+)](rest)) measured in wild type cells. The elevated [Ca(2+)](rest) in RyR1-expressing cells is not a result of active gating of the RyR1 channel but instead is accounted for by the RyR1 ryanodine-insensitive Ca(2+) leak conformation. In addition, we demonstrate that basal sarcolemmal Ca(2+) influx is also governed by RyR1 expression and contributes in the regulation of [Ca(2+)](rest) in skeletal myotubes.

Effect of Azumolene on RyR1-Dependent Store Operated Calcium Entry in RyR1-Expressing, Non Excitable Cells

Store-Operated Calcium Entry (SOCE) restores Ca2+ to depleted endoplasmic reticulum (ER) from the extracellular space via a multiprotein complex involving plasma membrane Orai1 and TRPC1, and ER membrane resident STIM1. Dantrolene and azumolene suppress the rise in intracellular Ca2+ seen during skeletal muscle during excitation-contraction coupling and in malignant hyperthermia, a hypermetabolic pharmacogenetic sensitivity to volatile anesthetics. Azumolene inhibits a component of SOCE coupled to activation of RyR1, the skeletal muscle sarcoplasmic reticulum Ca2+ release channel, but not Ca2+ release itself. Classical SOCE, activated by SR Ca2+-ATPase inhibitors, is unaffected. Thus, azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1. We used CHO cells stably transfected with RyR1 (C1148) and wild type (CHO-wt) to determine whether these distinguishable mechanisms of Ca2+entry are present universally, or only in excitable cells. SOCE was measured using Mn2+ quenching of Fura-2 fluorescence. C1148 cells expressing RyR1, but not CHO-wt, had high intrinsic Mn2+-quenching that is inhibited by azumolene and by the specific SOCE inhibitor BTP2, while low intrinsic Ca2+ entry of CHO-wt was unaffected by these drugs. On contrast, SOCE stimulated by the SR Ca2+-ATPase inhibitor, CPA (10μM), was inhibited by BTP2, but not by azumolene. Knockdown of STIM1 levels using shRNA demonstrates inhibition of RyR1-coupled SOCE. Immunocytochemistry of C1148 cells shows colocalization of RyR1 and STIM1 proteins in the presence of the RyR1 agonists, caffeine and ryanodine, and these proteins co-immunoprecipitate, suggesting they are interacting proteins. Thus, in RyR1-expressing, non-excitable cells, azumolene inhibits RyR1-dependent SOCE, but not Ca2+-ATPase-dependent SOCE, and suggests that STIM1, as one of the components of the SOCE machinery, may need to interact with RyR1 in this pathway.

Regulation of the Cardiac Muscle Ryanodine Receptor by O2 Tension and S-Nitrosoglutathione

The cardiac and skeletal muscle sarcoplasmic reticulum ryanodine receptor Ca(2+) release channels contain thiols that are potential targets of endogenously produced reactive oxygen and nitrogen intermediates. Previously, we showed that the skeletal muscle ryanodine receptor (RyR1) has O(2)-sensitive thiols; only when these thiols are in the reduced state (pO(2) approximately 10 mmHg) can physiological concentrations of NO (nanomolar) activate RyR1. Here, we report that cardiac muscle ryanodine receptor (RyR2) activity also depends on pO(2), but unlike RyR1, RyR2 was not activated or S-nitrosylated directly by NO. Rather, activation and S-nitrosylation of RyR2 required S-nitrosoglutathione. The effects of peroxynitrite were indiscriminate on RyR1 and RyR2. Our results indicate that both RyR1 and RyR2 are pO(2)-responsive yet point to different mechanisms by which NO and S-nitrosoglutathione influence cardiac and skeletal muscle sarcoplasmic reticulum Ca(2+) release.

Intermolecular Interactions in the Mechanism of Skeletal Muscle Sarcoplasmic Reticulum Ca2+-ATPase (SERCA1): Evidence for a Triprotomer

Native membrane sarcoplasmic reticulum (SR) Ca(2+)-ATPase isolated from skeletal muscle (SERCA1) exhibits oligomeric kinetic behavior [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. In the present study we used quenched-flow mixing, electron paramagnetic resonance (EPR), and chemical cross-linking to probe for intermolecular interactions at physiological (0.1 M) and high (0.4 M) KCl. Exposure of SR membranes to water- and lipid-soluble cross-linking reagents revealed a mixture of SERCA1 oligomeric species consisting mainly of dimers and trimers. Titration of iodoacetamide spin-labeled SERCA1 with AMPPCP in the presence of 10 microM Ca(2+) and 0.1 M KCl revealed high- (K(D) = 45 microM) and low-affinity (K(D) = 315 microM) nucleotide binding sites in a 2:1 ratio, respectively. Raising the [KCl] to 0.4 M increased the fraction of weak binding sites and lowered the K(D) of the high-affinity component (20 microM). Phosphorylation by 10 microM ATP at 21 degrees C and 0.1 M KCl produced an early burst of P(i) production without a corresponding decline in the steady-state phosphoenzyme (EP) level. The steady-state EP level was twice as large as the P(i) burst and received equal contributions from E1P and E2P. Chasing the phosphoenzyme at 0.4 M KCl and 2 degrees C with ADP revealed a biphasic time course of E1P formation with a slow phase that matched the kinetics of the transient EPR signal from the spin-labeled Ca(2+)-ATPase. The absence of a fast component in the EPR signal excludes E1P as its source. Instead, it arises from a slow, KCl-dependent transformation at the start of the cycle which controls the formation of downstream intermediates with an increased mole fraction of rotationally restricted probes. We modeled this behavior with a SERCA1 trimer in which the formation of E1P/E2/E2P from E1ATP/E2P/E1P results from concerted transformations in the subunits coupling phosphorylation (E1ATP --> E1P + ADP) to dephosphorylation (E2P --> E2 + P(i)) and the conversion of E1P to E2P.

FKBP12 deficiency reduces strength deficits after eccentric contraction-induced muscle injury

Strength deficits associated with eccentric contraction-induced muscle injury stem, in part, from excitation-contraction uncoupling. FKBP12 is a 12-kDa binding protein known to bind to the skeletal muscle sarcoplasmic reticulum Ca2+ release channel [ryanodine receptor (RyR1)] and plays an important role in excitation-contraction coupling. To assess the effects of FKBP12 deficiency on muscle injury and recovery, we measured anterior crural muscle (tibialis anterior and extensor digitorum longus muscles) strength in skeletal muscle-specific FKBP12-deficient and wild-type (WT) mice before and after a single bout of 150 eccentric contractions, as well as before and after the performance of six injury bouts. Histological damage of the tibialis anterior muscle was assessed after injury. Body weight and peak isometric and eccentric torques were lower in FKBP12-deficient mice compared with WT mice. There were no differences between FKBP12-deficient and WT mice in preinjury peak isometric and eccentric torques when normalized to body weight, and no differences in the relative decreases in eccentric torque with a single or multiple injury bouts. After a single injury bout, FKBP12-deficient mice had less initial strength deficits and recovered faster (especially females) than WT mice, despite no differences in the degree of histological damage. After multiple injury bouts, FKBP12-deficient mice recovered muscle strength faster than WT mice and exhibited significantly less histological muscle damage than WT mice. In summary, FKBP12 deficiency results in less initial strength deficits and enhanced recovery from single (especially females) and repeated bouts of injury than WT mice.

Effects of Azumolene on Ca2+Sparks in Skeletal Muscle Fibers

Azumolene is an analog of dantrolene, the only approved medicine for treatment of malignant hyperthermia (MH). The pharmacological mechanism of these drugs is to inhibit skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release by modulating the activity of the SR ryanodine receptor (RyR) Ca2+ release channel. To investigate the effects of azumolene on SR Ca2+ channel gating within skeletal muscle fibers, we monitored Ca2+ sparks in permeabilized frog skeletal muscle fibers. Application of 0.0001 to 10 microM azumolene suppressed the frequency of spontaneous Ca2+ sparks in a dose-dependent manner (EC50 = 0.25 microM; Hill coefficient = 1.44), but it did not cause systematic dose-dependent effects on the properties of the Ca2+ sparks. These results suggest that azumolene decreases the likelihood of Ca2+ release channel openings that initiate Ca2+ sparks, thereby decreasing spark frequency, but it has little effect on aggregate Ca2+ channel open times during a spark. To assess azumolene inhibition of RyRs activated in a manner analogous to those activated during an MH episode, we applied DP4, a synthetic peptide corresponding to a central region of RyR1 (Leu2442 to Pro2477), which mimics an MH modification. Azumolene also decreased Ca2+ spark frequency in a dose-dependent manner without altering spark properties in the DP4 MH model. We conclude that azumolene suppresses the opening rate but not the open time of RyR Ca2+ release channels within skeletal fibers.

Electro-Chemical Modeling Challenges of Biological Ion Pumps

We outline the basic operational, structural and functional features of ion motive ATPases: transmembrane proteins central to biological functions of all animal cells. As an example we discuss the modeling problems associated with the operation of the surface membrane Na(+),K(+)-ATPase and skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase and focus on the frameworks required for their solution. There are three basic problems: identification of the pathway for ion permeation, prediction of ion binding rate coefficients and affinities based on the structure of the protein, and prediction of conformational changes of protein structure and the associated movement of charges within the membrane dielectric. A solution strategy useful in approaching the first two problems and preliminary results obtained using molecular dynamics simulations are also presented.

Autosomal dominant Brody disease cosegregates with a chromosomal (2;7)(p11.2;p12.1) translocation in an Italian family.

Brody disease is a rare muscle disorder characterized by exercise-induced impairment in muscle relaxation, due to a markedly reduced influx of calcium ions in the sarcoplasmic reticulum. A subset of autosomal recessive families harbour mutations in the ATP2A1 gene, encoding the fast-twitch skeletal muscle sarcoplasmic reticulum Ca(2+) ATPase (SERCA1). Rare autosomal dominant families have been described, in which ATP2A1 was excluded as the causative gene, further supporting genetic heterogeneity. We report four individuals from a three-generation Italian family with a clinical phenotype of Brody disease, in which linkage analysis excluded ATP2A1 as the responsible gene. The disease cosegregates in an autosomal dominant fashion with an apparently balanced constitutional chromosome translocation (2;7)(p11.2;p12.1), suggesting a causal relationship between the rearrangement and the phenotype. FISH analysis using YAC and PAC clones as probes refined the breakpoint regions to genomic segments of about 164 and 120 kb, respectively, providing a possible clue to pinpoint the location of a novel gene responsible for this rare muscle disorder.

The C Terminus (Amino Acids 75–94) and the Linker Region (Amino Acids 42–54) of the Ca2+-binding Protein S100A1 Differentially Enhance Sarcoplasmic Ca2+ Release in Murine Skinned Skeletal Muscle Fibers

S100A1, a Ca2+-binding protein of the EF-hand type, is most highly expressed in striated muscle and has previously been shown to interact with the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) isoform. However, it was unclear whether S100A1/RyR1 interaction could modulate SR Ca2+ handling and contractile properties in skeletal muscle fibers. Since S100A1 protein is differentially expressed in fast- and slow-twitch skeletal muscle, we used saponin-skinned murine Musculus extensor digitorum longus (EDL) and Musculus soleus (Soleus) fibers to assess the impact of S100A1 protein on SR Ca2+ release and isometric twitch force in functionally intact permeabilized muscle fibers. S100A1 equally enhanced caffeine-induced SR Ca2+ release and Ca2+-induced isometric force transients in both muscle preparations in a dose-dependent manner. Introducing a synthetic S100A1 peptide model (devoid of EF-hand Ca2+-binding sites) allowed identification of the S100A1 C terminus (amino acids 75-94) and hinge region (amino acids 42-54) to differentially enhance SR Ca2+ release with a nearly 3-fold higher activity of the C terminus. These effects were exclusively based on enhanced SR Ca2+ release as S100A1 influenced neither SR Ca2+ uptake nor myofilament Ca2+ sensitivity/cooperativity in our experimental setting. In conclusion, our study shows for the first time that S100A1 augments contractile performance both of fast- and slow-twitch skeletal muscle fibers based on enhanced SR Ca2+ efflux at least mediated by the C terminus of S100A1 protein. Thus, our data suggest that S100A1 may serve as an endogenous enhancer of SR Ca2+ release and might therefore be of physiological relevance in the process of excitation-contraction coupling in skeletal muscle.

FKBP12 Binding to RyR1 Modulates Excitation-Contraction Coupling in Mouse Skeletal Myotubes

The skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel or ryanodine receptor (RyR1) binds four molecules of FKBP12, and the interaction of FKBP12 with RyR1 regulates both unitary and coupled gating of the channel. We have characterized the physiologic effects of previously identified mutations in RyR1 that disrupt FKBP12 binding (V2461G and V2461I) on excitation-contraction (EC) coupling and intracellular Ca2+ homeostasis following their expression in skeletal myotubes derived from RyR1-knockout (dyspedic) mice. Wild-type RyR1-, V246I-, and V2461G-expressing myotubes exhibited similar resting Ca2+ levels and maximal responses to caffeine (10 mm) and cyclopiazonic acid (30 microm). However, maximal voltage-gated Ca2+ release in V2461G-expressing myotubes was reduced by approximately 50% compared with that attributable to wild-type RyR1 (deltaF/Fmax = 1.6 +/- 0.2 and 3.1 +/- 0.4, respectively). Dyspedic myotubes expressing the V2461I mutant protein, that binds FKBP12.6 but not FKBP12, exhibited a comparable reduction in voltage-gated SR Ca2+ release (deltaF/Fmax = 1.0 +/- 0.1). However, voltage-gated Ca2+ release in V2461I-expressing myotubes was restored to a normal level (deltaF/Fmax = 2.9 +/- 0.6) following co-expression of FKBP12.6. None of the mutations that disrupted FKBP binding to RyR1 significantly affected RyR1-mediated enhancement of L-type Ca2+ channel activity (retrograde coupling). These data demonstrate that FKBP12 binding to RyR1 enhances the gain of skeletal muscle EC coupling.

The inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by macrocyclic lactones and cyclosporin A.

The pharmacology of macrocyclic lactones is varied, with many beneficial effects in treating disease processes. FK-506, rapamycin and ascomycin have been utilized as immunosuppressant agents. Ivermectin is typically used to treat parasitic worm infections in mammals. Another immunosuppressant, cyclosporin A, is a cyclic oligotide that has similar immunosuppressant properties to those exerted by macrocyclic lactones. Here we report on the inhibition by these compounds of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pumps. Ivermectin, cyclosporin A and rapamycin all inhibited the skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1). In addition, although ivermectin inhibited brain microsomal endoplasmic reticulum (type 2b) Ca(2+)-ATPase, cyclosporin A and rapamycin did not. As cyclosporin A also did not inhibit cardiac Ca(2+)-ATPase activity, this would suggest that it could be an isoform-specific inhibitor. Ivermectin was shown to be the most potent Ca(2+)-ATPase inhibitor of the macrocyclic lactones (IC(50)=7 microM). It appears to show a 'competitive' inhibition with respect to high concentrations of ATP by increasing the regulatory binding site K(m) but without affecting the catalytic site K(m). In addition, ivermectin stabilizes the ATPase in an E1 conformational state, and inhibits Ca(2+) release from the enzyme during turnover. This would suggest that ivermectin inhibits Ca(2+) release from the luminal binding sites of the phosphoenzyme intermediate, a step that is known to be accelerated by high [ATP].

3,5-di- t-butyl-4-hydroxyanisole (DTBHA) activation of rat skeletal muscle sarcoplasmic reticulum Ca 2+-ATPase

3,5-Di- t-butyl-4-hydroxyanisole (DTBHA) increased in a concentration-dependent manner (calculated p ec 50 = 4.55 ± 0.18 M) the oxalate-stimulated Ca 2+-pumping rate of rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Kinetic analysis of this effect suggested that the activation of SR Ca 2+-ATPase operated by (DTBHA) was of both mixed and non-competitive type with respect to ATP in the range of concentrations 0.1–0.5 mM and above 1 mM, respectively; furthermore, it was independent of the free Ca 2+ concentrations. This indicated that the enzyme activation took place through the acceleration of the enzyme-substrate complex breakdown. Moreover, it appeared that its target site was cyclopiazonic acid sensitive. The uncommon ability of (DTBHA) to upregulate SR Ca 2+ uptake is of interest in view of its possible use for treating pathological conditions characterised by cell Ca 2+ overload as well as genetic disorders where SR Ca 2+ homeostasis is altered.

Protein Kinase A Transgenes

The β-adrenergic receptor/adenylyl cyclase/protein kinase A (PKA) axis is the central signaling pathway that serves to stimulate cardiac function. It is classically perceived as a linear signaling cascade (Figure), and cAMP is thought to be the second messenger responsible for the positive inotropic, chronotropic, and lusitropic effects of catecholamines. New data gained from a series of transgenic animals overexpressing various elements of this axis show that this system has a far more complex makeup than previously thought. Transgenic models of the cardiac β-adrenergic receptor/PKA system. The signaling cascade is represented as a simple linear chain. Proteins that are detrimental when overexpressed are shown in black; those causing beneficial effects upon overexpression are depicted in white. PLB indicates phospholamban. It has been known for about two decades that the β-adrenergic receptor system is dysfunctional in heart failure.1 The loss of receptor responsiveness has been attributed to a reduced β-receptor number, particularly of the β1-subtype, plus a functional desensitization of the remaining receptors—presumably mediated via increased activity of G protein–coupled receptor kinases.2 The extent of the receptor downregulation and the functional loss correlate with the severity of the disease.3,4 This phenomenon has long been regarded as detrimental to the compromised function of the failing heart, and it has been proposed that restoration of β-adrenergic receptor responsiveness might be a strategy to improve cardiac function. However, recent clinical studies and observations on a panel of transgenic animals suggest that the opposite is the case. Sustained stimulation or overexpression of cardiac β1-adrenergic receptors leads to hypertrophy, fibrosis, and heart failure,5–7 and blockade of β-adrenergic receptors with several antagonists—bisoprolol, carvedilol, and metoprolol—improves survival in heart failure patients.8 Thus, the loss of β-adrenergic receptor responsiveness in heart failure is more likely to represent a protective mechanism than …

The effects of phenothiazines and other calmodulin antagonists on the sarcoplasmic and endoplasmic reticulum Ca 2+ pumps

The effects of a number of phenothiazines and other calmodulin antagonists on the Ca 2+-ATPase activity of sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) were investigated. The drugs used in this study were trifluoperazine, calmidazolium, fluphenazine, chlorpromazine, W-7, and calmodulin-binding peptide. Our results showed that calmidazolium and calmodulin-binding peptide were the most potent inhibitors of skeletal muscle SR Ca 2+-ATPase activity (isoform SERCA 1) ( ic 50 values of 0.5 and 7 μM, respectively), while W-7 was the least potent inhibitor ( ic 50, 125 μM). All of the antagonists had little effect on the cerebellar ER Ca 2+-ATPase activity (isoform SERCA 2b), except for trifluoperazine, which had a biphasic effect, causing stimulation at low concentrations and inhibition at higher concentrations. Our results suggest that the effects of these calmodulin antagonists are independent of calmodulin and that they inhibit the Ca 2+-ATPase in an isoform-specific manner. It was found that these antagonists inhibit the skeletal muscle isoform of the Ca 2+ pump by altering the Ca 2+ affinity and the associated Ca 2+-binding steps, as well as possibly stabilising the E1 conformational state of the enzyme.