Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Stimulation of fat oxidation in rat muscle by unacylated ghrelin persists for 2-3 hours, but is independent of fatty acid transporter translocation.

While a definitive mechanism-of-action remains to be identified, recent findings indicate that ghrelin, particularly the unacylated form (UnAG), stimulates skeletal muscle fatty acid oxidation. The biological importance of UnAG-mediated increases in fat oxidation remains unclear, as UnAG peaks in the circulation before mealtimes, and decreases rapidly during the postprandial situation before increases in postabsorptive circulating lipids. Therefore, we aimed to determine if the UnAG-mediated stimulation of fat oxidation would persist long enough to affect the oxidation of meal-derived fatty acids, and if UnAG stimulated the translocation of fatty acid transporters to the sarcolemma as a mechanism-of-action. In isolated soleus muscle strips from male rats, short-term pre-treatment with UnAG elicited a persisting stimulus on fatty acid oxidation 2 h after the removal of UnAG. UnAG also caused an immediate phosphorylation of AMPK, but not an increase in plasma membrane FAT/CD36 or FABPpm. There was also no increase in AMPK signaling or increased FAT/CD36 or FABPpm content at the plasma membrane at 2 h which might explain the sustained increase in fatty acid oxidation. These findings confirm UnAG as a stimulator of fatty acid oxidation and provide evidence that UnAG may influence the handling of postprandial lipids. The underlying mechanisms are not known.

T-tubule recovery after detubulation in isolated mouse cardiomyocytes.

Remodeling of cardiac t-tubules in normal and pathophysiological conditions is an important process contributing to the functional performance of the heart. While it is well documented that deterioration of t-tubule network associated with various pathological conditions can be reversed under certain conditions, the mechanistic understanding of the recovery process is essentially lacking. Accordingly, in this study we investigated some aspects of the recovery of t-tubules after experimentally-induced detubulation. T-tubules of isolated mouse ventricular myocytes were first sealed using osmotic shock approach, and their recovery under various experimental conditions was then characterized using electrophysiologic and imaging techniques. The data show that t-tubule recovery is a strongly temperature-dependent process involving reopening of previously collapsed t-tubular segments. T-tubule recovery is slowed by (1) metabolic inhibition of cells, (2) reducing influx of extracellular Ca2+ as well as by (3) both stabilization and disruption of microtubules. Overall, the data show that t-tubule recovery is a highly dynamic process involving several central intracellular structures and processes and lay the basis for more detailed investigations in this area.

Consequences of sarcolemma fatigue on maximal muscle strength production in patients with myalgic encephalomyelitis/chronic fatigue syndrome.

BACKGROUND: Myalgic encephalomyelitis is an invalidating chronic disease often associated with exercise-induced alterations of muscle membrane excitability (M wave). No simultaneous measurements of maximal isometric force production and sarcolemma fatigue in the same muscle group have been previously reported. We hypothesized that M wave alterations could be partly responsible for the reduced muscle force present in this invalidating disease. METHODS: This retrospective study compared two groups of patients who presented (n = 30) or not (n = 28) alterations of M waves evoked by direct muscle stimulation during and after a cycling exercise bout. The maximal handgrip strength was measured before and after exercise, concomitantly with electromyogram recordings from flexor digitorum longus muscle. The patients also answered a questionnaire to identify eventual exacerbation of their clinical symptoms following the exercise test. FINDINGS: The M wave amplitude significantly decreased in muscles and the M wave duration significantly increased in the group of patients with M wave alterations after exercise. Resting values of handgrip were significantly lower in patients with exercise-induced M-wave alterations than in patients without M-wave abnormalities. In patients with exercise-induced M-wave alterations, handgrip significantly decreased after exercise and the changes in handgrip and M wave were positively correlated. The frequency of post-exertion malaise, increased fatigue, myalgia, headache and cognitive dysfunction was significantly higher in patients with M-wave alterations and variations in handgrip after exercise. INTERPRETATION: These data suggest that post-exercise sarcolemma fatigue often measured in patients with myalgic encephalomyelitis could be the cause of muscle failure.

Unusually severe muscular dystrophy upon in-frame deletion of the dystrophin rod domain and lack of compensation by membrane-localized utrophin.

BACKGROUND: Utrophin, a dystrophin homolog, is consistently upregulated in muscles of patients with Duchenne muscular dystrophy (DMD) and is believed to partially compensate for the lack of dystrophin in dystrophic muscle. Even though several animal studies support the idea that utrophin can modulate DMD disease severity, human clinical data are scarce. METHODS: We describe a patient with the largest reported in-frame deletion in the DMD gene, including exons 10-60 and thus encompassing the entire rod domain. FINDINGS: The patient presented with an unusually early and severe progressive weakness, initially suggesting congenital muscular dystrophy. Immunostaining of his muscle biopsy showed that the mutant protein was able to localize at the sarcolemma and stabilize the dystrophin-associated complex. Strikingly, utrophin protein was absent from the sarcolemmal membrane despite the upregulation of utrophin mRNA. CONCLUSIONS: Our results suggest that the internally deleted and dysfunctional dystrophin lacking the entire rod domain may exert a dominant-negative effect by preventing upregulated utrophin protein from reaching the sarcolemmal membrane and thus blocking its partial rescue of muscle function. This unique case may set a lower size limit for similar constructs in potential gene therapy approaches. FUNDING: This work was supported by a grant from MDA USA (MDA3896) and by grant number R01AR051999 from NIAMS/NIH to C.G.B.

Full-length human dystrophin on human artificial chromosome compensates for mouse dystrophin deficiency in a Duchenne muscular dystrophy mouse model.

Dystrophin maintains membrane integrity as a sarcolemmal protein. Dystrophin mutations lead to Duchenne muscular dystrophy, an X-linked recessive disorder. Since dystrophin is one of the largest genes consisting of 79 exons in the human genome, delivering a full-length dystrophin using virus vectors is challenging for gene therapy. Human artificial chromosome is a vector that can load megabase-sized genome without any interference from the host chromosome. Chimeric mice carrying a 2.4-Mb human dystrophin gene-loaded human artificial chromosome (DYS-HAC) was previously generated, and dystrophin expression from DYS-HAC was confirmed in skeletal muscles. Here we investigated whether human dystrophin expression from DYS-HAC rescues the muscle phenotypes seen in dystrophin-deficient mice. Human dystrophin was normally expressed in the sarcolemma of skeletal muscle and heart at expected molecular weights, and it ameliorated histological and functional alterations in dystrophin-deficient mice. These results indicate that the 2.4-Mb gene is enough for dystrophin to be correctly transcribed and translated, improving muscular dystrophy. Therefore, this technique using HAC gives insight into developing new treatments and novel humanized Duchenne muscular dystrophy mouse models with human dystrophin gene mutations.

Analysis of Dysferlin Direct Interactions with Putative Repair Proteins Links Apoptotic Signaling to Ca2+ Elevation via PDCD6 and FKBP8.

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases. Like otoferlin, dysferlin directly interacted via its carboxy terminus with FKBP8, an anti-apoptotic outer mitochondrial membrane protein, and via its C2DE domain with apoptosis-linked gene (ALG-2/PDCD6), linking anti-apoptosis with apoptosis. Confocal Z-stack immunofluorescence confirmed co-compartmentalization of PDCD6 and FKBP8 at the sarcolemmal membrane. Our evidence supports the hypothesis that prior to injury, dysferlin C2 domains self-interact and give rise to a folded, compact structure as indicated for otoferlin. With elevation of intracellular Ca2+ in injury, dysferlin would unfold and expose the cC2A domain for interaction with annexin A1, calpain-3, mitsugumin 53, affixin, and caveolin-3, and dysferlin would realign from its interactions with PDCD6 at basal calcium levels to interact strongly with FKBP8, an intramolecular rearrangement facilitating membrane repair.

Is the fundamental pathology in Duchenne's muscular dystrophy caused by a failure of glycogenolysis-glycolysis in costameres?

Duchenne muscular dystrophy (DMD) is the most common form of progressive childhood muscular dystrophy associated with weakness of limbs, loss of ambulation, heart weakness and early death. The mutations causing either loss-of-expression or function of the full-length protein dystrophin (Dp427) from the DMD gene are responsible for the disease pathology. Dp427 forms a part of the large dystroglycan complex, called DAPC, in the sarcolemma, and its absence derails muscle contraction. Muscle biopsies from DMD patients show an overactivation of excitation-contraction-coupling (ECC) activable calcium incursion, sarcolemmal ROS production, NHE1 activation, IL6 secretion, etc. The signalling pathways, like Akt/PBK, STAT3, p38MAPK, and ERK1/2, are also hyperactive in DMD. These pathways are responsible for post-mitotic trophic growth and metabolic adaptation, in response to exercise in healthy muscles, but cause atrophy and cell death in dystrophic muscles. We hypothesize that the metabolic background of repressed glycolysis in DMD, as opposed to excess glycolysis seen in cancers or healthy contracting muscles, changes the outcome of these 'growth pathways'. The reduced glycolysis has been considered a secondary outcome of the cytoskeletal disruptions seen in DMD. Given the cytoskeleton-crosslinking ability of the glycolytic enzymes, we hypothesize that the failure of glycogenolytic and glycolytic enzymes to congregate is the primary pathology, which then affects the subsarcolemmal cytoskeletal organization in costameres and initiates the pathophysiology associated with DMD, giving rise to the tissue-specific differences in disease progression between muscle, heart and brain. The lacunae in the regulation of the key components of the hypothesized metabolome, and the limitations of this theory are deliberated. The considerations for developing future therapies based on known pathological processes are also discussed.

Ion Channels of the Sarcolemma and Intracellular Organelles in Duchenne Muscular Dystrophy: A Role in the Dysregulation of Ion Homeostasis and a Possible Target for Therapy.

Duchenne muscular dystrophy (DMD) is caused by the absence of the dystrophin protein and a properly functioning dystrophin-associated protein complex (DAPC) in muscle cells. DAPC components act as molecular scaffolds coordinating the assembly of various signaling molecules including ion channels. DMD shows a significant change in the functioning of the ion channels of the sarcolemma and intracellular organelles and, above all, the sarcoplasmic reticulum and mitochondria regulating ion homeostasis, which is necessary for the correct excitation and relaxation of muscles. This review is devoted to the analysis of current data on changes in the structure, functioning, and regulation of the activity of ion channels in striated muscles in DMD and their contribution to the disruption of muscle function and the development of pathology. We note the prospects of therapy based on targeting the channels of the sarcolemma and organelles for the correction and alleviation of pathology, and the problems that arise in the interpretation of data obtained on model dystrophin-deficient objects.

Orientation of the Dysferlin C2A Domain is Responsive to the Composition of Lipid Membranes.

Dysferlin is a 230 kD protein that plays a critical function in the active resealing of micron-sized injuries to the muscle sarcolemma by recruiting vesicles to patch the injured site via vesicle fusion. Muscular dystrophy is observed in humans when mutations disrupt this repair process or dysferlin is absent. While lipid binding by dysferlin's C2A domain (dysC2A) is considered fundamental to the membrane resealing process, the molecular mechanism of this interaction is not fully understood. By applying nonlinear surface-specific vibrational spectroscopy, we have successfully demonstrated that dysferlin's N-terminal C2A domain (dysC2A) alters its binding orientation in response to a membrane's lipid composition. These experiments reveal that dysC2A utilizes a generic electrostatic binding interaction to bind to most anionic lipid surfaces, inserting its calcium binding loops into the lipid surface while orienting its ß-sheets 30-40° from surface normal. However, at lipid surfaces, where PI(4,5)P2 is present, dysC2A tilts its ß-sheets more than 60° from surface normal to expose a polybasic face, while it binds to the PI(4,5)P2 surface. Both lipid binding mechanisms are shown to occur alongside dysC2A-induced lipid clustering. These different binding mechanisms suggest that dysC2A could provide a molecular cue to the larger dysferlin protein as to signal whether it is bound to the sarcolemma or another lipid surface.

Identifying FDA-Approved Drugs that Upregulate Utrophin A as a Therapeutic Strategy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations and deletions within the DMD gene, which result in a lack of dystrophin protein at the sarcolemma of skeletal muscle fibers. The absence of dystrophin fragilizes the sarcolemma and compromises its integrity during cycles of muscle contraction, which, progressively, leads to reductions in muscle mass and function. DMD is thus a progressive muscle-wasting disease that results in a loss of ambulation, cardiomyopathy , respiratory impairment, and death. Although there is presently no cure for DMD, recent advances have led to many promising treatments. One such approach entails increasing expression of a homologous protein to dystrophin, named utrophin A, which is endogenously expressed in both healthy and DMD muscle fibers. Upregulation of utrophin A all along the sarcolemma of DMD muscle fibers can, in part, compensate for the absence of dystrophin. Over the years, our laboratory has focused a significant portion of our efforts in identifying and characterizing drugs and small molecules for their ability to target utrophin A and cause its overexpression. As part of these efforts, we have recently developed a novel ELISA-based high-throughput drug screen, to identify FDA-approved drugs that increase the expression of utrophin A in muscle cells in culture as well as in dystrophic mice. Here, we describe our overall strategy to identify and characterize several FDA-approved drugs that upregulate utrophin A expression and provide details on all experimental approaches. Such strategy has the potential to lead to the rapid development of novel therapeutics for DMD.

Deficient Sarcolemma Repair in ALS: A Novel Mechanism with Therapeutic Potential.

The plasma membrane (sarcolemma) of skeletal muscle myofibers is susceptible to injury caused by physical and chemical stresses during normal daily movement and/or under disease conditions. These acute plasma membrane disruptions are normally compensated by an intrinsic membrane resealing process involving interactions of multiple intracellular proteins including dysferlin, annexin, caveolin, and Mitsugumin 53 (MG53)/TRIM72. There is new evidence for compromised muscle sarcolemma repair mechanisms in Amyotrophic Lateral Sclerosis (ALS). Mitochondrial dysfunction in proximity to neuromuscular junctions (NMJs) increases oxidative stress, triggering MG53 aggregation and loss of its function. Compromised membrane repair further worsens sarcolemma fragility and amplifies oxidative stress in a vicious cycle. This article is to review existing literature supporting the concept that ALS is a disease of oxidative-stress induced disruption of muscle membrane repair that compromise the integrity of the NMJs and hence augmenting muscle membrane repair mechanisms could represent a viable therapeutic strategy for ALS.

The role of the dystrophin glycoprotein complex in muscle cell mechanotransduction.

Dystrophin is the central protein of the dystrophin-glycoprotein complex (DGC) in skeletal and heart muscle cells. Dystrophin connects the actin cytoskeleton to the extracellular matrix (ECM). Severing the link between the ECM and the intracellular cytoskeleton has a devastating impact on the homeostasis of skeletal muscle cells, leading to a range of muscular dystrophies. In addition, the loss of a functional DGC leads to progressive dilated cardiomyopathy and premature death. Dystrophin functions as a molecular spring and the DGC plays a critical role in maintaining the integrity of the sarcolemma. Additionally, evidence is accumulating, linking the DGC to mechanosignalling, albeit this role is still less understood. This review article aims at providing an up-to-date perspective on the DGC and its role in mechanotransduction. We first discuss the intricate relationship between muscle cell mechanics and function, before examining the recent research for a role of the dystrophin glycoprotein complex in mechanotransduction and maintaining the biomechanical integrity of muscle cells. Finally, we review the current literature to map out how DGC signalling intersects with mechanical signalling pathways to highlight potential future points of intervention, especially with a focus on cardiomyopathies.

Caveolin 3 suppresses phosphorylation-dependent activation of sarcolemmal nNOS.

Mutations of the caveolin 3 gene cause autosomal dominant limb-girdle muscular dystrophy (LGMD)1C. In mice, overexpression of mutant caveolin 3 leads to loss of caveolin 3 and results in myofiber hypotrophy in association with activation of neuronal nitric oxide synthase (nNOS) at the sarcolemma. Here, we show that caveolin 3 directly bound to nNOS and suppressed its phosphorylation-dependent activation at a specific residue, Ser1412 in the nicotinamide adenine dinucleotide phosphate (NADPH)-flavin adenine dinucleotide (FAD) module near the C-terminus of the reduction domain, in vitro. Constitutively active nNOS enhanced myoblast fusion, but not myogenesis, in vitro. Phosphorylation-dependent activation of nNOS occurred in muscles from caveolin 3-mutant mice and LGMD1C patients. Mating with nNOS-mutant mice exacerbated myofiber hypotrophy in the caveolin 3-mutant mice. In nNOS-mutant mice, regenerating myofibers after cardiotoxin injury became hypotrophic with reduced myoblast fusion. Administration of NO donor increased myofiber size and the number of myonuclei in the caveolin 3-mutant mice. Exercise also increased myofiber size accompanied by phosphorylation-dependent activation of nNOS in wild-type and caveolin 3-mutant mice. These data indicate that caveolin 3 inhibits phosphorylation-dependent activation of nNOS, which leads to myofiber hypertrophy via enhancing myoblast fusion. Hypertrophic signaling by nNOS phosphorylation could act in a compensatory manner in caveolin 3-deficient muscles.

Reduced Sarcolemmal Membrane Repair Exacerbates Striated Muscle Pathology in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.

CMYA5 establishes cardiac dyad architecture and positioning.

Cardiac excitation-contraction coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse tubules and junctional sarcoplasmic reticulum. Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac development, homeostasis, and disease. Here, we use proximity proteomics in intact, living hearts to identify proteins enriched near dyads. Among these proteins is CMYA5, an under-studied striated muscle protein that co-localizes with Z-lines, junctional sarcoplasmic reticulum proteins, and transverse tubules in mature cardiomyocytes. During cardiac development, CMYA5 positioning adjacent to Z-lines precedes junctional sarcoplasmic reticulum positioning or transverse tubule formation. CMYA5 ablation disrupts dyad architecture, dyad positioning at Z-lines, and junctional sarcoplasmic reticulum Ca2+ release, leading to cardiac dysfunction and inability to tolerate pressure overload. These data provide mechanistic insights into cardiomyopathy pathogenesis by demonstrating that CMYA5 anchors junctional sarcoplasmic reticulum to Z-lines, establishes dyad architecture, and regulates dyad Ca2+ release.

Evaluating Therapeutic Activity of Galectin-1 in Sarcolemma Repair of Skeletal Muscle.

Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models.

Zebrafish integrin a3b is required for cardiac contractility and cardiomyocyte proliferation.

In cardiac muscle cells, heterodimeric integrin transmembrane receptors are known to serve as mechanotransducers, translating mechanical force to biochemical signaling. However, the roles of many individual integrins have still not been delineated. In this report, we demonstrate that Itga3b is localized to the sarcolemma of cardiomyocytes from 24 to 96 hpf. We further show that heterozygous and homozygous itga3b/bdf mutant embryos display a cardiomyopathy phenotype, with decreased cardiac contractility and reduced cardiomyocyte number. Correspondingly, proliferation of ventricular and atrial cardiomyoctyes and ventricular epicardial cells is decreased in itga3b mutant hearts. The contractile dysfunction of itga3b mutants can be attributed to cardiomyocyte sarcomeric disorganization, including thin myofilaments with blurred and shortened Z-discs. Together, our results reveal that Itga3b localizes to the myocardium sarcolemma, and it is required for cardiac contractility and cardiomyocyte proliferation.

Co-overexpression of CD36 and FABPpm increases fatty acid transport additively, not synergistically, within muscle.

We aimed to determine the combined effects of overexpressing plasma membrane fatty acid binding protein (FABPpm) and fatty acid translocase (CD36) on skeletal muscle fatty acid transport to establish if these transport proteins function collaboratively. Electrotransfection with either FABPpm or CD36 increased their protein content at the plasma membrane (+75% and +64%), increased fatty acid transport rates by +24% for FABPpm and +62% for CD36, resulting in a calculated transport efficiency of ∼0.019 and ∼0.053 per unit protein change for FABPpm and CD36, respectively. We subsequently used these data to determine if increasing both proteins additively or synergistically increased fatty acid transport. Cotransfection of FABPpm and CD36 simultaneously increased protein content in whole muscle (FABPpm, +46%; CD36, +45%) and at the sarcolemma (FABPpm, +41%; CD36, +42%), as well as fatty acid transport rates (+50%). Since the relative effects of changing FABPpm and CD36 content had been independently determined, we were able to a predict a change in fatty acid transport based on the overexpression of plasmalemmal transporters in the cotransfection experiments. This prediction yielded an increase in fatty acid transport of +0.984 and +1.722 pmol/mg prot/15 s for FABPpm and CD36, respectively, for a total increase of +2.96 pmol/mg prot/15 s. This calculated determination was remarkably consistent with the measured change in transport, namely +2.89 pmol/mg prot/15 s. Altogether, these data indicate that increasing CD36 and FABPpm alters fatty acid transport rates additively, but not synergistically, suggesting an independent mechanism of action within muscle for each transporter. This conclusion was further supported by the observation that plasmalemmal CD36 and FABPpm did not coimmunoprecipitate.

The C2 domains of dysferlin: roles in membrane localization, Ca2+ signalling and sarcolemmal repair.

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.