Longitudinal Sarcoplasmic Reticulum Research Articles

Sarcoplasmic Reticulum Ca2+ Buffer Proteins: A Focus on the Yet-To-Be-Explored Role of Sarcalumenin in Skeletal Muscle Health and Disease.

Sarcalumenin (SAR) is a luminal Ca2+ buffer protein with high capacity but low affinity for calcium binding found predominantly in the longitudinal sarcoplasmic reticulum (SR) of fast- and slow-twitch skeletal muscles and the heart. Together with other luminal Ca2+ buffer proteins, SAR plays a critical role in modulation of Ca2+ uptake and Ca2+ release during excitation-contraction coupling in muscle fibers. SAR appears to be important in a wide range of other physiological functions, such as Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) stabilization, Store-Operated-Calcium-Entry (SOCE) mechanisms, muscle fatigue resistance and muscle development. The function and structural features of SAR are very similar to those of calsequestrin (CSQ), the most abundant and well-characterized Ca2+ buffer protein of junctional SR. Despite the structural and functional similarity, very few targeted studies are available in the literature. The present review provides an overview of the role of SAR in skeletal muscle physiology, as well as of its possible involvement and dysfunction in muscle wasting disorders, in order to summarize the current knowledge on SAR and drive attention to this important but still underinvestigated/neglected protein.

Desmin interacts with STIM1 and coordinates Ca2+ signaling in skeletal muscle.

Stromal interaction molecule 1 (STIM1), the sarcoplasmic reticulum (SR) transmembrane protein, activates store-operated Ca2+ entry (SOCE) in skeletal muscle and, thereby, coordinates Ca2+ homeostasis, Ca2+-dependent gene expression, and contractility. STIM1 occupies space in the junctional SR membrane of the triads and the longitudinal SR at the Z-line. How STIM1 is organized and is retained in these specific subdomains of the SR is unclear. Here, we identified desmin, the major type III intermediate filament protein in muscle, as a binding partner for STIM1 based on a yeast 2-hybrid screen. Validation of the desmin-STIM1 interaction by immunoprecipitation and immunolocalization confirmed that the CC1-SOAR domains of STIM1 interact with desmin to enhance STIM1 oligomerization yet limit SOCE. Based on our studies of desmin-KO mice, we developed a model wherein desmin connected STIM1 at the Z-line in order to regulate the efficiency of Ca2+ refilling of the SR. Taken together, these studies showed that desmin-STIM1 assembles a cytoskeletal-SR connection that is important for Ca2+ signaling in skeletal muscle.

Cylindrical spirals in two families: Clinical and genetic investigations

Cylindrical spirals are a rare ultrastructural finding on muscle biopsy, with fewer than 20 reported cases since its first description in 1979. These structures are sometimes observed with tubular aggregates and are thought to comprise longitudinal sarcoplasmic reticulum. While mutations in genes encoding key components of Ca2+ handling (ORAI1 and STIM1) underlie tubular aggregate myopathy, no causative genes have been associated with cylindrical spirals. Here we describe two families with cylindrical spirals on muscle biopsy with a suspected genetic cause. In one family we identified a known truncating variant in EBF3, previously associated with a neurodevelopmental disorder. The affected individuals in this family present with clinical features overlapping with those described for EBF3 disease. An isolated proband in the second family harbours bi-allelic truncating variants in TTN and her clinical course and other features on biopsy are highly concordant for titinopathy. From experimental studies, EBF3 is known to be involved in Ca2+ regulation in muscle, thus EBF3 dysregulation may represent a novel mechanism of impaired Ca2+ handling leading to cylindrical spirals. Additional cases of EBF3 disease or titinopathy with cylindrical spirals need to be identified to support the involvement of these genes in the pathogenesis of cylindrical spirals.

Transport of the alpha subunit of the voltage gated L‐type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles

Contraction of skeletal muscle is initiated by excitation-contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca2+ release. EC coupling requires L-type voltage gated Ca2+ channels (the dihydropyridine receptor or DHPR) located at triads, which are junctions between the transverse (T) tubule and sarcoplasmic reticulum (SR) membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers, we find that DHPR is transported along the longitudinal SR in a microtubule-independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRβ, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads.

Nanospan, an alternatively spliced isoform of sarcospan, localizes to the sarcoplasmic reticulum in skeletal muscle and is absent in limb girdle muscular dystrophy 2F

BackgroundSarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models.MethodsStandard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections.ResultsNanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd−/−) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies.ConclusionsAlternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865–71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes.

Novel Local RYR1 Junctional Calcium Responses and Dynamics by Improved Calcium Sensor Catcher

The precise spatio-temporal characteristics of Ca2+ transient are critical for the physiological regulation of Ca2+-dependent signaling processes. Because determining Ca2+ dynamics occurring in the myoplasm and sarcoplasmic reticulum (SR) under normal conditions is essential in order to fully understand changes that may occur under stressful or pathological conditions, we designed a new generation of Ca2+ sensors. Here, we report the optimization of a genetically-encoded Ca2+ sensor called “CatchER” which exhibits rapid kinetics and specific targeting to the endoplasmic reticulum (ER). We also describe the characteristics of CatchER-T’, a sensor variant with significantly improved specificities, including enhanced fluorescence at 37°C and a greater calcium dependent dynamic range. CatchER-T’ is targeted to the ER via the signal peptide of calreticulin and the ER retention sequence KDEL. Differential drug induced Ca2+ releases and ER refilling are observed for various cell types. Furthermore in electroporated mouse flexor digitorum brevis (FDB) muscle fibers, targeting CatchER-T’ close to the RyR1 channel via a sequence obtained from the junctional SR protein JP-45, revealed that local Ca2+ release via electrical stimulation is significantly greater and faster than that occurring in the SR lumen as assessed using CatchER-T’, i.e. the probe targeted to the lumen of the longitudinal SR. The impact of local ER/SR calcium dynamics in controlling cellular signaling and excitation contraction coupling will be presented.

The Transmembrane Domain is Sufficient to Direct Junctophilin-1 Localization at the Junctional SR

The sarcoplasmic reticulum (SR) of striated muscle cells is mainly dedicated to Ca2+ homeostasis and regulation of muscle contraction. The SR is organized in longitudinal and junctional SR (j-SR). In skeletal muscle, this latter domain together with the T-tubules form specific junctions called triads, where proteins regulating the excitation-contraction coupling mechanism assemble. Junctophilins (JPs) are directly involved in the formation and maintenance of triads. Basically, they are anchored to the SR via their C-terminal transmembrane domain (TMD), while their N-terminus contains eight MORN motifs, which associate with the phospholipids of the T-tubules. Nevertheless, how JPs are targeted to triads is not known. The roles of the N-terminal and the C-terminal regions of JP1 in this process were investigated. Expression in primary myotubes and/or muscle fibers of JP1 deletion mutants lacking the TMD resulted in protein distribution at both the surface sarcolemma and the T-tubules, confirming that MORN motifs are involved in JP1 interaction with the sarcolemma, but are not sufficient to restrict its localization at the T-tubules.On the other hand, progressive deletion of MORN motifs I-VI, MORN motifs I-VIII or of the entire region 1-635, did no affect JP1 localization at triads, indicating that the presence of the TMD is sufficient for JP1 localization at the j-SR.These results indicate that the localization of JP1 at the triads appears to be mainly directed by mechanisms acting at the level of the SR, rather than at T-tubules. FRAP analysis performed on a GFP-TMD fusion protein expressed in myotubes indicated that this protein has a high mobility, suggesting the absence of strong protein-protein interactions occurring at the j-SR. Further work is needed to better understand the molecular mechanisms driving TMD-mediated JP1 localization at triads.

Organization of junctional sarcoplasmic reticulum proteins in skeletal muscle fibers.

The sarcoplasmic reticulum (SR) of striated muscles is specialized for releasing Ca(2+) following sarcolemma depolarization in order to activate muscle contraction. To this end, the SR forms a network of longitudinal tubules and cisternae that surrounds the myofibrils and, at the same time, participates to the assembly of the triadic junctional membrane complexes formed by the close apposition of one t-tubule, originated from the sarcolemma, and two SR terminal cisternae. Advancements in understanding the molecular basis of the SR structural organization have identified an interaction between sAnk1, a transmembrane protein located on the longitudinal SR (l-SR) tubules, and obscurin, a myofibrillar protein. The direct interaction between these two proteins results in molecular contacts that have the overall effect to stabilize the l-SR tubules along myofibrils in skeletal muscle fibers. Less known is the structural organization of the sites in the SR that are specialized for Ca(2+) release and are positioned at the junctional SR (j-SR), i.e. the region of the terminal cisternae that faces the t-tubule at triads. At the j-SR, several trans-membrane proteins like triadin, junctin, or intra-luminal SR proteins like calsequestrin, are assembled together with the ryanodine receptor, the SR Ca(2+) release channel, into a macromolecular complex specialized in releasing Ca(2+). At triads, the 12nm-wide gap between the t-tubule and the j-SR allows the ryanodine receptor on the j-SR to be functionally coupled with the voltage-gated L-type calcium channel on the t-tubule in order to allow the transduction of the voltage-induced signal into Ca(2+) release through the ryanodine receptor channels. The muscle-specific junctophilin isoforms (JPH1 and JPH2) are anchored to the j-SR with a trans-membrane segment present at the C-terminus and are capable to bind the sarcolemma with a series of phospholipid-binding motifs localized at the N-terminus. Accordingly, through this dual interaction, JPH1 and JPH2 are responsible for the assembly of the triadic junctional membrane complexes. Recent data indicate that junctophilins seem also to interact with other proteins of the excitation-contraction machinery, suggesting that they may contribute to hold excitation-contraction coupling proteins to the sites where the j-SR is being organized.

Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers.

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγ charge and L-type Ca(2+) current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

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.

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues, heart failure and atherosclerosis

The sarco(endo)plasmic reticulum Ca2+ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis).Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~91% of total SERCA and SERCA2b for ~5%. Among SERCA3, SERCA3b was the most expressed (~3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b.In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively.In conclusion: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation–contraction coupling in both CMs and contractile VSMCs.

The Intraluminal Domain of Junctin Contains Junctional Sarcoplasmic Reticulum Targeting Sequences

Junctin is an integral membrane protein of the sarcoplasmic reticulum (SR) encoded by the Aspartyl beta-hydroxylase (AβH) gene. The AβH gene can undergo different alternative splicing events, generating at least three distinct proteins, the Aspartyl beta-hydroxylase, junctin and junctate, all sharing the same transmembrane domain and a short highly charged acidic luminal region, but carrying distinct intraluminal tails. Interstingly, junctin and junctate are both expressed in skeletal muscle, but only junctin was shown to interact and co-localize with the Ca2+ release complex in the junctional SR, whereas junctate displays a typical longitudinal SR distribution. These two proteins may thus represent an ideal molecular model to investigate the presence of specific regions responsible for protein targeting to the junctional SR.To this aim, junctin deletion mutants were tagged with a green fluorescent protein (GFP) and expressed in primary myoblasts. The results obtained showed that even deletion of small regions within intraluminal domain of junctin resulted in protein mislocalization, thus indicating that the entire luminal sequence is required for protein targeting to the junctional SR. Interestingly, the same region, if inserted downstream the cytoplasmic and trasmembrane domains of sarcolipin, a resident protein of the longitudinal SR, is able to completely re-localize the protein to the junctional SR, further confirming that the intraluminal domain of junctin may actually contain minimal junctional SR targeting sequences.

High Levels of Calsequestrin in Some Snake Muscles. Why?

Calsequestrin (CSQ) is the most abundant Ca2+ binding protein in sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It is characterized by a high capacity and a moderate affinity for Ca2+ and thus it maintains luminal free Ca2+ in the junctional SR cisternae (jSR) at the physiological concentration of 1mM, while storing up to 20mM total calcium. Thin-section TEMs of superficial epaxial muscles from Nerodia sipedon and Boa constrictor show at least two structurally distinguishable types of fibers, probably twitch and tonic. Twitch fibers have unusually wide jSR cisternae completely filled with a finely granular matrix of CSQ polymers. The visible luminal content also extends far from the triads into the longitudinal SR. This is also the case in anterior intermandibular muscles from the same snakes. The percentage of fiber volume occupied by SR in twitch fibers from superficial epaxial muscles was calculated, from morphometry of thin sections, to be 9.5% in Nerodia, 10.6% in Boa and 7.5% in leg muscles from the lizard Anolis carolinensis, another lepidosaur. Comparable data from the literature for mouse indicate 5.5% in EDL and 2.5% in soleus. So, overall, the snake muscles have considerably higher Ca2+ storage capacity than lizard and mouse, two species that move frequently. We suggest that this may be related to prolonged periods of inactivity of these snakes, interrupted by the very infrequent use of muscles during prey capture and locomotion.Supported by NIH HL048093 (CFA)

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

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

Slowed relaxation and preserved maximal force in soleus muscles of mice with targeted disruption of theSerca2gene in skeletal muscle

Sarcoplasmic reticulum Ca(2+) ATPases (SERCAs) play a major role in muscle contractility by pumping Ca(2+) from the cytosol into the sarcoplasmic reticulum (SR) Ca(2+) store, allowing muscle relaxation and refilling of the SR with releasable Ca(2+). Decreased SERCA function has been shown to result in impaired muscle function and disease in human and animal models. In this study, we present a new mouse model with targeted disruption of the Serca2 gene in skeletal muscle (skKO) to investigate the functional consequences of reduced SERCA2 expression in skeletal muscle. SkKO mice were viable and basic muscle structure was intact. SERCA2 abundance was reduced in multiple muscles, and by as much as 95% in soleus muscle, having the highest content of slow-twitch fibres (40%). The Ca(2+) uptake rate was significantly reduced in SR vesicles in total homogenates. We did not find any compensatory increase in SERCA1 or SERCA3 abundance, or altered expression of several other Ca(2+)-handling proteins. Ultrastructural analysis revealed generally well-preserved muscle morphology, but a reduced volume of the longitudinal SR. In contracting soleus muscle in vitro preparations, skKO muscles were able to fully relax, but with a significantly slowed relaxation time compared to controls. Surprisingly, the maximal force and contraction rate were preserved, suggesting that skKO slow-twitch fibres may be able to contribute to the total muscle force despite loss of SERCA2 protein. Thus it is possible that SERCA-independent mechanisms can contribute to muscle contractile function.

Sarcalumenin plays a critical role in age-related cardiac dysfunction due to decreases in SERCA2a expression and activity

Impaired Ca2+ reuptake into the sarcoplasmic reticulum (SR) underlies a primary pathogenesis of heart failure in the aging heart. Sarcalumenin (SAR), a Ca2+-binding glycoprotein located in the longitudinal SR, regulates Ca2+ reuptake by interacting with SR Ca2+-ATPase (SERCA). Here we found that the expression levels of both SAR and SERCA2 proteins were significantly downregulated in senescent wild-type mice (18-month old) and that downregulation of SAR protein preceded downregulation of SERCA2 protein. The downregulation of SERCA2 protein was greater in senescent SARKO mice than in age-matched senescent wild-type mice, which was at least in part due to progressive degradation of SERCA2 protein in SARKO mice. Senescent SARKO mice exhibited typical findings of heart failure such as increased sympathetic activity, impaired exercise tolerance, and upregulation of biomarkers of cardiac stress. Consequently, cardiac function was progressively decreased in senescent SARKO. We also found that the expression levels of endoplasmic reticulum (ER) stress-related genes such as x-box binding protein 1 (XBP1) were significantly increased in senescent SARKO mice, indicating that senescent SARKO mice exhibited ER stress. Thus we uncovered the important role of SAR in maintaining Ca2+ transport activity of SERCA2a and cardiac function in the senescent population.

Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type

Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, VO2 max = 68 ± 5 ml kg-1 min-1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. .

Isoform-specific roles of protein phosphatase 1 catalytic subunits in sarcoplasmic reticulum-mediated Ca2+ cycling

protein phosphatase 1 (PP1) is the major isotype of serine/threonine phosphatase in cardiomyocytes, and its activity has been thought to be important for heart failure progression. The PP1 catalytic subunits consist of three distinct genes, PP1α, PP1β/δ, and PP1γ. To date, the function of each PP1 isoform is not well characterized in cardiomyocytes. We sought to determine the functional contribution of each PP1 isoform to sarcoplasmic reticulum (SR)-mediated Ca(2+) cycling in isolated adult rat cardiomyocytes. adenoviral vectors encoding short hairpin RNA for each PP1 isoform were transfected into isolated rat cardiomyocytes, and this was followed by analysis of cell shortening, Ca(2+) transients, and the phosphorylation levels of Ca(2+) regulatory proteins. Physical interactions between each PP1 isoform and SR Ca(2+) regulatory proteins were characterized in isolated cardiomyocytes expressing green fluorescent protein (GFP)-tagged PP1 catalytic subunits, and also in canine junctional and longitudinal SR preparations. Successful PP1 isoform knockdown was achieved for each isoform without affecting the expression of the other isoforms. PP1β knockdown most significantly enhanced the Ca(2+) transient and cell shortening by augmenting phospholamban (PLN) phosphorylation at baseline and with low-dose isoproterenol stimulation (10 nM). Interestingly, PP1β was preferentially associated with sarco-endoplasmic ATPase and PLN in GFP-PP1-transfected cardiomyocytes, as well as in canine longitudinal SR preparations. these findings indicate that PP1β is the most significant PP1 isoform involved in regulating SR Ca(2+) cycling in rat cardiomyocytes.

Transient Receptor Potential Canonical Type 1 (TRPC1) Operates as a Sarcoplasmic Reticulum Calcium Leak Channel in Skeletal Muscle

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca(2+) stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca(2+) influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca(2+) pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca(2+) pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca(2+) which was not observed in control cells. Ca(2+) measurements indicated that resting Ca(2+) and the rate of Ca(2+) increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca(2+) transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca(2+) leak channel in skeletal muscle.

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling