Muscle Sarcoplasmic Reticulum Research Articles

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.

No voltage change at skeletal muscle SR membrane during Ca2+ release-just Mermaids on acid.

Calcium ions control multiple physiological functions by binding to extracellular and intracellular targets. One of the best-studied Ca2+-dependent functions is contraction of smooth and striated muscle tissue, which results from Ca2+ ligation to calmodulin and troponin C, respectively. Ca2+ signaling typically involves flux of the ion across membranes via specifically gated channel proteins. Because calcium ions are charged, they possess the ability to generate changes in the respective transmembrane voltage. Ca2+-dependent voltage alterations of the surface membrane are easily measured using microelectrodes. A well-known example is the characteristic plateau phase of the action potential in cardiac ventricular cells that results from the opening of voltage-gated L-type Ca2+ channels. Ca2+ ions are also released from intracellular storage compartments in many cells, but these membranes are not accessible to direct voltage recording with microelectrodes. In muscle, for example, release of Ca2+ from the sarcoplasmic reticulum (SR) to the myoplasm constitutes a flux that is considerably larger than the entry flux from the extracellular space. Whether this flux is accompanied by a voltage change across the SR membrane is an obvious question of mechanistic importance and has been the subject of many investigations. Because the tiny spaces enclosed by the SR membrane are inaccessible to microelectrodes, alternative methods have to be applied. In a study by Sanchez et al. (2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201812035) in this issue, modern confocal light microscopy and genetically encoded voltage probes targeted to the SR were applied in a new approach to search for changes in the membrane potential of the SR during Ca2+ release.

Cyclic Nucleotides Reduce Ryanodine Receptor Mediated Ca2+ Spark Activation Independent of Long Term Hypoxia in Ovine Fetal Pulmonary Arterial Myocytes

Ryanodine receptors (RyR) release Ca2+ from smooth muscle sarcoplasmic reticulum in rapid events known as Ca2+ sparks. These Ca2+ sparks activate voltage and Ca2+ gated potassium channels (BK channels) that cause cell repolarization. Ca2+ spark‐BK channel mediated cell repolarization dilates many types of vessels and is essential to pulmonary arterial (PA) vasodilation during the fetal transition to breathing air. Infants who do not successfully negotiate this transition are at risk of developing pulmonary hypertension, which has significant morbidity and mortality. Previous work shows that long term hypoxia (LTH) decreases Ca2+ spark events in fetal pulmonary arterial smooth muscle cells (PASMCs) even though the Ca2+ stores have functional RyRs. Additional studies illustrate that cyclic nucleotides can increase RyR activity, which led us to test the hypothesis that cyclic nucleotides could rescue RyR activity in myocytes of LTH fetuses. This was examined by treating PAs isolated from low (700m; LA) and high altitude (3801m; HA) near‐term fetal sheep with exogenous cyclic AMP (cAMP) and cyclic GMP (cGMP) or the methylxanthine IBMX, a non‐specific cAMP and cGMP phosphodiesterase inhibitor. RyR activity was measured by confocal imaging of Ca2+ sparks in fluo‐4 labeled PASMCs. The spatial and temporal aspects to the Ca2+ sparks were evaluated using SparkLab, a custom software package. Contrary to our hypothesis, the prevalence of Ca2+ sparks decreased in the presence of cAMP, cGMP, and IBMX for PASMC from both LA and HA fetal sheep. These data suggest that cyclic nucleotides reduce instead of increase vasodilatory capacity via RyR‐BK channel coupling mechanisms. Furthermore, increased cyclic nucleotides had no effect on the spatial‐temporal aspects to the Ca2+ sparks. Even though the signaling pathway remains unclear, the reduction of Ca2+ sparks by cyclic nucleotides is potentially a novel mechanism of RyR modulation in sheep fetal PASMC. This data suggests that increasing cyclic nucleotides may be contra‐indicated in newborn infants who are at risk of developing pulmonary hypertension.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. SH was a Loma Linda University Summer Undergraduate Research Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deciphering Functions of Intracellular Formaldehyde: Linking Cancer and Aldehyde Metabolism.

Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.

Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice.

Maintenance of muscle structure and function depends on the precise organization of contractile proteins into sarcomeres and coupling of the contractile apparatus to the sarcoplasmic reticulum (SR), which serves as the reservoir for calcium required for contraction. Several members of the Kelch superfamily of proteins, which modulate protein stability as substrate-specific adaptors for ubiquitination, have been implicated in sarcomere formation. The Kelch protein Klhl31 is expressed in a muscle-specific manner under control of the transcription factor MEF2. To explore its functions in vivo, we created a mouse model of Klhl31 loss of function using the CRISPR-Cas9 system. Mice lacking Klhl31 exhibited stunted postnatal skeletal muscle growth, centronuclear myopathy, central cores, Z-disc streaming, and SR dilation. We used proteomics to identify several candidate Klhl31 substrates, including Filamin-C (FlnC). In the Klhl31-knockout mice, FlnC protein levels were highly upregulated with no change in transcription, and we further demonstrated that Klhl31 targets FlnC for ubiquitination and degradation. These findings highlight a role for Klhl31 in the maintenance of skeletal muscle structure and provide insight into the mechanisms underlying congenital myopathies.

A pyridone derivative activates SERCA2a by attenuating the inhibitory effect of phospholamban

The cardiac sarco/endoplasmic reticulum Ca2+-dependent ATPase 2a (SERCA2a) plays a central role in Ca2+ handling within cardiomyocytes and is negatively regulated by phospholamban (PLN), a sarcoplasmic reticulum (SR) membrane protein. The activation of SERCA2a, which has been reported to improve cardiac dysfunction in heart failure, is a potential therapeutic approach for heart failure. Therefore, we developed a novel small molecule, compound A and characterized it both in vitro and in vivo. Compound A activated the Ca2+-dependent ATPase activity of cardiac SR vesicles but not that of skeletal muscle SR vesicles that lack PLN. The surface plasmon resonance assay revealed a direct interaction between compound A and PLN, suggesting that the binding of compound A to PLN attenuates its inhibition of SERCA2a, resulting in SERCA2a activation. This was substantiated by inhibition of the compound A-mediated increase in Ca2+ levels within the SR of HL-1 cells by thapsigargin, a SERCA inhibitor. Compound A also increased the Ca2+ transients and contraction and relaxation of isolated adult rat cardiomyocytes. In isolated perfused rat hearts, the compound A enhanced systolic and diastolic functions. Further, an infusion of compound A (30mg/kg, i.v. bolus followed by 2mg/kg/min, i.v. infusion) significantly enhanced the diastolic function in anesthetized normal rats. These results indicate that compound A is a novel SERCA2a activator, which attenuates PLN inhibition and enhances the systolic and diastolic functions of the heart in vitro and in vivo. Therefore, compound A might be a novel therapeutic lead for heart failure.

Sequencing of genes involved in the movement of calcium across human skeletal muscle sarcoplasmic reticulum: continuing the search for genes associated with malignant hyperthermia.

The genetic basis of malignant hyperthermia (MH) is not fully characterised and likely involves more than just the currently classified mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) and the gene encoding the α1 subunit of the dihydropyridine receptor (CACNA1S). In this paper we sequence other genes involved in calcium trafficking within skeletal muscle in patients with positive in vitro contracture tests, searching for alternative genes associated with MH. We identified four rare variants in four different genes (CACNB1, CASQ1, SERCA1 and CASQ2) encoding proteins involved in calcium handling in skeletal muscle in a cohort of 30 Australian MH susceptible probands in whom prior complete sequencing of RYR1 and CACNA1S had yielded no rare variants. These four variants have very low minor allele frequencies and while it is tempting to speculate that they have a role in MH, they remain at present variants of unknown significance. Nevertheless they provide the basis for a new set of functional studies, which may indeed identify novel players in MH.

Calsequestrins in skeletal and cardiac muscle from adult Danio rerio.

Calsequestrin (Casq) is a high capacity, low affinity Ca(2+)-binding protein, critical for Ca(2+)-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located.

Skeletal Muscle Phospholipid Metabolism Regulates Insulin Sensitivity and Contractile Function.

Skeletal muscle insulin resistance is an early defect in the development of type 2 diabetes. Lipid overload induces insulin resistance in muscle and alters the composition of the sarcoplasmic reticulum (SR). To test the hypothesis that skeletal muscle phospholipid metabolism regulates systemic glucose metabolism, we perturbed choline/ethanolamine phosphotransferase 1 (CEPT1), the terminal enzyme in the Kennedy pathway of phospholipid synthesis. In C2C12 cells, CEPT1 knockdown altered SR phospholipid composition and calcium flux. In mice, diet-induced obesity, which decreases insulin sensitivity, increased muscle CEPT1 expression. In high-fat diet-fed mice with skeletal muscle-specific knockout of CEPT1, systemic and muscle-based approaches demonstrated increased muscle insulin sensitivity. In CEPT1-deficient muscles, an altered SR phospholipid milieu decreased sarco/endoplasmic reticulum Ca(2+) ATPase-dependent calcium uptake, activating calcium-signaling pathways known to improve insulin sensitivity. Altered muscle SR calcium handling also rendered these mice exercise intolerant. In obese humans, surgery-induced weight loss increased insulin sensitivity and decreased skeletal muscle CEPT1 protein. In obese humans spanning a spectrum of metabolic health, muscle CEPT1 mRNA was inversely correlated with insulin sensitivity. These results suggest that high-fat feeding and obesity induce CEPT1, which remodels the SR to preserve contractile function at the expense of insulin sensitivity.

Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca(2+)-ATPase Inhibitors and Structure-Activity Relationships.

Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 μM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented.

Oxidative stress, mitochondrial damage, and cores in muscle from calsequestrin-1 knockout mice

BackgroundMutations in the gene encoding ryanodine receptor type-1 (RYR1), the calcium ion (Ca2+) release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, are linked to central core disease (CCD) and malignant hyperthermia (MH) susceptibility. We recently reported that mice lacking the skeletal isoform of calsequestrin (CASQ1-null), the primary Ca2+ buffer in the SR of skeletal muscle and a modulator of RYR1 activity, exhibit lethal heat- and anesthetic-induced hypermetabolic episodes that resemble MH events in humans.MethodsWe compared ultrastructure, oxidative status, and contractile function in skeletal fibers of extensor digitorum longus (EDL) muscles in wild type (WT) and CASQ1-null mice at different ages (from 4 to 27 months) using structural, biochemical, and functional assays.ResultsAbout 25% of fibers in EDL muscles from CASQ1-null mice of 14 to 27 months of age exhibited large areas of structural disarray (named core-like regions), which were rarely observed in muscle from age-matched WT mice. To determine early events that may lead to the formation of cores, we analyzed EDL muscles from adult mice: at 4 to 6 months of age, CASQ1-null mice (compared to WT) displayed significantly reduced grip strength (40 ± 1 vs. 86 ± 1 mN/gr) and exhibited an increase in the percentage of damaged mitochondria (15.1% vs. 2.6%) and a decrease in average cross-sectional fiber area (approximately 37%) in EDL fibers. Finally, oxidative stress was also significantly increased (25% reduction in ratio between reduced and oxidized glutathione, or GSH/GSSG, and 35% increase in production of mitochondrial superoxide flashes). Providing ad libitum access to N-acetylcysteine in the drinking water for 2 months normalized GSH/GSSG ratio, reduced mitochondrial damage (down to 8.9%), and improved grip strength (from 46 ± 3 to 59 ± 2 mN/gr) in CASQ1-null mice.ConclusionsOur findings: 1) demonstrate that ablation of CASQ1 leads to enhanced oxidative stress, mitochondrial damage, and the formation of structural cores in skeletal muscle; 2) provide new insights in the pathogenic mechanisms that lead to damage/disappearance of mitochondria in cores; and 3) suggest that antioxidants may provide some therapeutic benefit in reducing mitochondrial damage, limiting the development of cores, and improving muscle function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0035-9) contains supplementary material, which is available to authorized users.

A Pilot Study of Age-Related Changes in the Sphingolipid Composition of the Rat Sarcoplasmic Reticulum.

Muscle strength declines more rapidly than muscle size, manifesting as a loss of muscle quality. One putative contributor to this impairment of muscle quality is impaired sarcoplasmic reticulum (SR) function. The principal objective of this study was to characterize the sphingolipid composition of the SR in adult and aging rat muscles. A secondary, exploratory objective was to test for associations between SR sphingolipids and SR function (i.e., Ca2+ release). Using an animal model, the objectives were evaluated in a pre-clinical, cross-sectional study. Data were collected in an academic research laboratory. Medial gastrocnemius muscles of adult (n=8; 7-8 months) and aged (n=8; 24-25 months), male F344/BN hybrid rats were processed to extract SR. Sphingolipids in the SR were measured using tandem mass spectrometry. Fatty acid concentrations within the major sphingolipid classes were evaluated via Principal Component Analysis (PCA). In a subset of samples, SR Ca2+ release rates were determined using fluorometric methods, and associations with specific (based on results of PCA) fatty acid concentrations were evaluated. Aging SR showed an overall decline in the ratio of unsaturated to saturated fatty acids. Age-specific differences were observed for hexosylceramide and ceramide-1-phosphate. Within subset of samples with SR Ca2+ release data, a significant negative association between Ca2+ release and C1P18:0 and trends for positive associations with hexCER24:0 and 24:1 were observed. These preliminary, pre-clinical data suggest that changes in SR sphingolipids may play a role in age-related impairment of muscle function. Further work is needed to explore this hypothesis, as SR sphingolipids may prove a fruitful target for interventions, be they physical (i.e., exercise), nutritional or pharmacological.

DHBP Block of Ryanodine Receptor Channels

1,1′-diheptyl-4,4′-bipyridinium dibromide (DHBP), a divalent organic cation, was reported to inhibit muscle contraction, which was attributed to inhibition of ryanodine receptor (RyR) mediated Ca2+ release from the sarcoplasmic reticulum (SR). Still, single RyR channel studies are lacking and the mechanism by which DHBP blocks RyRs is unknown. We reconstituted RyRs from skeletal muscle and heart SR microsomes into planar bilayers. We observed with DHBP addition to the cytosolic surface of RyRs (clamped at Vm = 0 mV) decreased channel conductance (IC50 ∼ 1 mM). The percentage block in channel conductance elicited by DHBP was invariable in the range −30 to +30 mV. DHBP did not affect RyR gating characteristics, which was measured under Ca2+ conditions that produced low, moderate or high Po and in the presence/absence of physiological modulators (ATP, Mg2+). DHBP added to luminal and cytosolic sides of the RyRs, resulted in additive effects on channel conductance. DHBP also induced inhibition of RyR-mediated SR Ca2+ release from skeletal SR microsomes. A similar block of RyRs conductance was observed with other bipyridinum derivatives, none of which affected SERCA ATPase activity. Previous reports in the literature indicated block of RyRs by DHBP in cells required lower drug concentrations, suggesting a possible cellular mechanism of DHBP accumulation. In summary, DHBP blocks RyRs Ca2+ conductance resembling the action of polyamines and organic cations. DHBP properties make it an attractive experimental probe for understanding the role of RyR conductance in Ca2+ release from intracellular Ca2+ stores (Supported by American Heart Association and Eskridge Foundation).

G.P.159: Cylindrical spirals originate from sarcoplasmic reticulum of muscles in two Chinese siblings

Cylindrical spirals are morphological abnormalities, reported in fewer than 15 cases so far and presenting with a wide variety of clinical phenotypes. To clarify the nature of cylindrical spirals, we characterized the proteins in muscle specimens from two Chinese siblings presented clinically with muscle weakness and ultrastructurally with cylindrical spirals using a series of antibodies to sarcoplasmic reticulum and its related structures. On electron microscope, the cylindrical spirals are consisted of 10–20 lamellae wrapped around a central core containing glycogen granules. The immunohistochemical stains showed that all of these key calcium-regulatory proteins of the skeletal muscle sarcoplasmic reticulum, including SERCA1 (longitudinal sarcoplasmic reticulum), calsequestrin (terminal cisternae) and RyR1 (junctional sarcoplasmic reticulum) were expressed in cylindrical spirals. The T-tubule special resident DHPR and nuclear membrane protein emerin were also present in cylindrical spirals, while the Golgi apparatus marker GM130 and the myofilament protein myosin as well as the cellular skeleton protein desmin were not found in cylindrical spirals. Moreover, antibodies to LC3 and Lamp2 relevant to autophagosome and lysosome were negative. Taken together, these results suggested that cylindrical spirals could originate from the whole sarcoplasmic reticulum containing the junctional, longitudinal and cisternae components of the SR and consequently affected the calcium homeostasis.

Activation and propagation of Ca2+ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle.

Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation-contraction coupling, requires virtually simultaneous release of large amounts of Ca(2+) from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca(2+) movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca(2+) within its lumen to promote the propagation of Ca(2+) release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg(2+)] ([Mg(2+)]cyto) induced a leak of Ca(2+) through RyRs, causing a reduction in SR Ca(2+) buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca(2+)]SR. The local rise in [Ca(2+)]SR, an intra-SR Ca(2+) transient, induced a local diffusely rising [Ca(2+)]cyto. A prolonged Ca(2+) wave lasting tens of seconds or more was generated from these events. Ca(2+) waves were dependent on the diffusion of Ca(2+) within the lumen of the SR and ended as [Ca(2+)]SR dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca(2+)]SR and the activation of secondary Ca(2+) waves in the persistent presence of low [Mg(2+)]cyto if the threshold [Ca(2+)]SR for RyR opening could be reached. Secondary Ca(2+) waves occurred without an abrupt reduction in SR Ca(2+) buffering power. Ca(2+) release and wave propagation occurred in the absence of Ca(2+)-induced Ca(2+) release. These observations are consistent with the activation of Ca(2+) release through RyRs of lowered cytoplasmic inhibition by [Ca(2+)]SR or store overload-induced Ca(2+) release. Restitution of SR Ca(2+) buffering power to its initially high value required imposing normal resting ionic conditions in the cytoplasm, which re-imposed the normal resting inhibition on the RyRs, allowing [Ca(2+)]SR to return to endogenous levels without activation of store overload-induced Ca(2+) release. These results are discussed in the context of how pathophysiological Ca(2+) release such as that occurring in malignant hyperthermia can be generated.

A Mouse Model for Dominant Collagen VI Disorders: HETEROZYGOUS DELETION OF Col6a3 EXON 16

Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the Col6a3 gene. The resulting heterozygous mouse, Col6a3(+/d16), produced comparable amounts of normal Col6a3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The Col6a3(+/d16) mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The Col6a3(+/d16) mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.

Muscle Glycogen Content Modifies SR Ca2+ Release Rate in Elite Endurance Athletes

The aim of the present study was to investigate the influence of muscle glycogen content on sarcoplasmic reticulum (SR) function and peak power output (Wpeak) in elite endurance athletes. Fourteen highly trained male triathletes (VO2max = 66.5 ± 1.3 mL O2·kg·min), performed 4 h of glycogen-depleting cycling exercise (HRmean = 73% ± 1% of maximum). During the first 4 h of recovery, athletes received either water (H2O) or carbohydrate (CHO), separating alterations in muscle glycogen content from acute changes affecting SR function and performance. Thereafter, all subjects received CHO-enriched food for the remaining 20-h recovery period. Immediately after exercise, muscle glycogen content and SR Ca release rate was reduced to 32% ± 4% (225 ± 28 mmol·kg dw) and 86% ± 2% of initial levels, respectively (P < 0.01). Glycogen markedly recovered after 4 h of recovery with CHO (61% ± 2% of preexercise) and SR Ca release rate returned to preexercise level. However, in the absence of CHO during the first 4 h of recovery, glycogen and SR Ca release rate remained depressed, with the normalization of both parameters at the end of the 24 h of recovery after receiving a CHO-enriched diet. Linear regression demonstrated a significant correlation between SR Ca release rate and muscle glycogen content (P < 0.01, r = 0.30). The 4 h of cycling exercise reduced Wpeak by 5.5%-8.9% at different cadences (P < 0.05), and Wpeak was normalized after 4 h of recovery with CHO, whereas Wpeak remained depressed (P < 0.05) after water provision. Wpeak was fully recovered after 24 h in both the H2O and the CHO group. In conclusion, the present results suggest that low muscle glycogen depresses muscle SR Ca release rate, which may contribute to fatigue and delayed recovery of Wpeak 4 h postexercise.

Eudistomin D and penaresin derivatives as modulators of ryanodine receptor channels and sarcoplasmic reticulum Ca2+ ATPase in striated muscle.

Eudistomin D (EuD) and penaresin (Pen) derivatives are bioactive alkaloids from marine sponges found to induce Ca(2+) release from striated muscle sarcoplasmic reticulum (SR). Although these alkaloids are believed to affect ryanodine receptor (RyR) gating in a "caffeine-like" manner, no single-channel study confirmed this assumption. Here, EuD and MBED (9-methyl-7-bromoeudistomin D) were contrasted against caffeine on their ability to modulate the SR Ca(2+) loading/leak from cardiac and skeletal muscle SR microsomes as well as the function of RyRs in planar bilayers. The effects of these alkaloids on [(3)H]ryanodine binding and SR Ca(2+) ATPase (SERCA) activity were also tested. MBED (1-5 μM) fully mimicked maximal activating effects of caffeine (20 mM) on SR Ca(2+) leak. At the single-channel level, MBED mimicked the agonistic action of caffeine on cardiac RyR gating (i.e., stabilized long openings characteristic of "high-open-probability" mode). EuD was a partial agonist at the maximal doses tested. The tested Pen derivatives displayed mild to no agonism on RyRs, SR Ca(2+) leak, or [(3)H]ryanodine binding studies. Unlike caffeine, EuD and some Pen derivatives significantly inhibited SERCA at concentrations required to modulate RyRs. Instead, MBED's affinity for RyRs (EC50 ∼ 0.5 μM) was much larger than for SERCA (IC50 > 285 μM). In conclusion, MBED is a potent RyR agonist and, potentially, a better choice than caffeine for microsomal and cell studies due to its reported lack of effects on adenosine receptors and phosphodiesterases. As a high-affinity caffeine-like probe, MBED could also help identify the caffeine-binding site in RyRs.

RNAi mediated knockdown of the ryanodine receptor gene decreases chlorantraniliprole susceptibility in Sogatella furcifera

The diamide insecticides activate ryanodine receptors (RyRs) to release and deplete intracellular calcium stores from the sarcoplasmic reticulum of muscles and the endoplasmic reticulum of many types of cells. They rapidly interrupt feeding of the target pest and eventually kill the pest due to starvation. However, information about the structure and function of insect RyRs is still limited. In this study, we isolated a 15,985bp full-length cDNA (named SfRyR) from Sogatella furcifera, a serious rice planthopper pest throughout Asia. SfRyR encodes a 5140-amino acid protein, which shares 78–97% sequence identities with other insect homologues, and less than 50% identities with Homo sapiens RyR1–3. All hallmarks of the RyR proteins are conserved in SfRyR. In the N-terminus, SfRyR has a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeated domain and a RIH-associated domain. In the C-terminus, SfRyR possesses two consensus calcium ion-binding EF-hand motifs, and six transmembrane helices. Temporal and spatial expression analysis showed that SfRyR was widely found in all development stages including egg, first through fifth instar nymphs, macropterous adult females and males. On day 2 fifth-instar nymphs, SfRyR was ubiquitously expressed in the head, thorax and abdomen. Dietary ingestion of dsSfRyR1 and dsSfRyR2 significantly reduced the mRNA level of SfRyR in the treated nymphs by 77.9% and 81.8% respectively, and greatly decreased chlorantraniliprole-induced mortality. Thus, our results suggested that SfRyR gene encoded a functional RyR that mediates chlorantraniliprole toxicity to S. furcifera.

The Mboat Family Protein Mitsugumin 56 Contributes to Postnatal Maturation in the Muscle Sarcoplasmic Reticulum

The sarcoplasmic reticulum (SR) is a highly-specialized form of the smooth endoplasmic reticulum (ER), and functions intensively as an intracellular Ca2+ store in muscle cells. Although major proteins contributing to Ca2+ handling have been extensively studied, there are still as-yet-unknown SR components, which would potentially open new fields in muscle physiology. We currently identified mitsugumin 56 (MG56), that is a novel SR membrane protein expressed in striated muscle. MG56 belongs to the MBOAT (membrane-bound O-acyltransferase) family, and is specifically localized to the junctional SR composing the triad in skeletal muscle. Although Mg56-knockout mice normally grew until postnatal day 7 (P7), they exhibited suckling failure and died within two weeks under starvation conditions. In skeletal muscle from Mg56-knockout mice, SR elements began to swell near the Z-line on P5, and further developed enormous vacuoles spreading over sarcomeres afterwards. In tension measurements, Mg56-knockout extensor digitorum longus extensor digitorum longus muscle retained apparently normal contractile features on P7, but attenuated its vital contractility on P9. On the other hand, spontaneous Ca2+ sparks/waves under resting conditions were observed in isolated flexor digitarium breris fibers of Mg56-knockout mice on P7-9. Therefore, MG56 essentially contributes to postnatal SR maturation in skeletal muscle, although its contribution to SR Ca2+ handling remains to be defined at the molecular level.