Nanodysferlins support membrane repair and binding to TRIM72/MG53 but do not localize to t-tubules or stabilize Ca2+ signaling.

Mutations in the DYSF gene, encoding the protein dysferlin, lead to several forms of muscular dystrophy. In healthy skeletal muscle, dysferlin concentrates in the transverse tubules and is involved in repairing the sarcolemma and stabilizing Ca2+ signaling after membrane disruption. The DYSF gene encodes 7-8 C2 domains, several Fer and Dysf domains, and a C-terminal transmembrane sequence. Because its coding sequence is too large to package in adeno-associated virus, the full-length sequence is not amenable to current gene delivery methods. Thus, we have examined smaller versions of dysferlin, termed "nanodysferlins," designed to eliminate several C2 domains, specifically C2 domains D, E, and F; B, D, and E; and B, D, E, and F. We also generated a variant by replacing eight amino acids in C2G in the nanodysferlin missing domains D through F. We electroporated dysferlin-null A/J mouse myofibers with Venus fusion constructs of these variants, or as untagged nanodysferlins together with GFP, to mark transfected fibers We found that, although these nanodysferlins failed to concentrate in transverse tubules, three of them supported membrane repair after laser wounding while all four bound the membrane repair protein, TRIM72/MG53, similar to WT dysferlin. By contrast, they failed to suppress Ca2+ waves after myofibers were injured by mild hypoosmotic shock. Our results suggest that the internal C2 domains of dysferlin are required for normal t-tubule localization and Ca2+ signaling and that membrane repair does not require these C2 domains.

Optimization of Xenografting Methods for Generating Human Skeletal Muscle in Mice.

Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl2 is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl2 and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice.

Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlin-null skeletal muscle.

Dysferlin-null A/J myofibers generate abnormal Ca2+ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca2+ waves (Lukyanenko et al., J. Physiol., 2017). Ca2+ waves are typically associated with Ca2+-induced Ca2+ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca2+ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca2+ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca2+ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca2+ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca2+. EGTA, which has an affinity for Ca2+ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6fu in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca2+ signaling. GCaMP6fu introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca2+ signaling. Our results suggest that leak of Ca2+ into the triad junctional cleft underlies dysregulation of Ca2+ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca2+ signaling and protects muscle against injury by binding Ca2+ in the cleft.

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.

µ-Crystallin in Mouse Skeletal Muscle Promotes a Shift from Glycolytic toward Oxidative Metabolism.

µ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6-147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, ß-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that µ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of µ-crystallin are associated with a shift toward greater fat metabolism.

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.

Biomechanical Properties of the Sarcolemma and Costameres of Skeletal Muscle Lacking Desmin.

Intermediate filaments (IFs), composed primarily by desmin and keratins, link the myofibrils to each other, to intracellular organelles, and to the sarcolemma. There they may play an important role in transfer of contractile force from the Z-disks and M-lines of neighboring myofibrils to costameres at the membrane, across the membrane to the extracellular matrix, and ultimately to the tendon ("lateral force transmission"). We measured the elasticity of the sarcolemma and the connections it makes at costameres with the underlying contractile apparatus of individual fast twitch muscle fibers of desmin-null mice. By positioning a suction pipet to the surface of the sarcolemma and applying increasing pressure, we determined the pressure at which the sarcolemma separated from nearby sarcomeres, Pseparation, and the pressure at which the isolated sarcolemma burst, Pbursting. We also examined the time required for the intact sarcolemma-costamere-sarcomere complex to reach equilibrium at lower pressures. All measurements showed the desmin-null fibers to have slower equilibrium times and lower Pseparation and Pbursting than controls, suggesting that the sarcolemma and its costameric links to nearby contractile structures were weaker in the absence of desmin. Comparisons to earlier values determined for muscles lacking dystrophin or synemin suggest that the desmin-null phenotype is more stable than the former and less stable than the latter. Our results are consistent with the moderate myopathy seen in desmin-null muscles and support the idea that desmin contributes significantly to sarcolemmal stability and lateral force transmission.

µ-Crystallin: A thyroid hormone binding protein.

µ-Crystallin is a NADPH-regulated thyroid hormone binding protein encoded by the CRYM gene in humans. It is primarily expressed in the brain, muscle, prostate, and kidney, where it binds thyroid hormones, which regulate metabolism and thermogenesis. It also acts as a ketimine reductase in the lysine degradation pathway when it is not bound to thyroid hormone. Mutations in CRYM can result in non-syndromic deafness, while its aberrant expression, predominantly in the brain but also in other tissues, has been associated with psychiatric, neuromuscular, and inflammatory diseases. CRYM expression is highly variable in human skeletal muscle, with 15% of individuals expressing ≥13 fold more CRYM mRNA than the median level. Ablation of the Crym gene in murine models results in the hypertrophy of fast twitch muscle fibers and an increase in fat mass of mice fed a high fat diet. Overexpression of Crym in mice causes a shift in energy utilization away from glycolysis towards an increase in the catabolism of fat via ß-oxidation, with commensurate changes of metabolically involved transcripts and proteins. The history, attributes, functions, and diseases associated with CRYM, an important modulator of metabolism, are reviewed.

Skeletal muscle cell transplantation: models and methods.

Xenografts of skeletal muscle are used to study muscle repair and regeneration, mechanisms of muscular dystrophies, and potential cell therapies for musculoskeletal disorders. Typically, xenografting involves using an immunodeficient host that is pre-injured to create a niche for human cell engraftment. Cell type and method of delivery to muscle depend on the specific application, but can include myoblasts, satellite cells, induced pluripotent stem cells, mesangioblasts, immortalized muscle precursor cells, and other multipotent cell lines delivered locally or systemically. Some studies follow cell engraftment with interventions to enhance cell proliferation, migration, and differentiation into mature muscle fibers. Recently, several advances in xenografting human-derived muscle cells have been applied to study and treat Duchenne muscular dystrophy and Facioscapulohumeral muscular dystrophy. Here, we review the vast array of techniques available to aid researchers in designing future experiments aimed at creating robust muscle xenografts in rodent hosts.

Keratin 18 is an integral part of the intermediate filament network in murine skeletal muscle.

Intermediate filaments (IFs) contribute to force transmission, cellular integrity, and signaling in skeletal muscle. We previously identified keratin 19 (Krt19) as a muscle IF protein. We now report the presence of a second type I muscle keratin, Krt18. Krt18 mRNA levels are about half those for Krt19 and only 1:1,000th those for desmin; the protein was nevertheless detectable in immunoblots. Muscle function, measured by maximal isometric force in vivo, was moderately compromised in Krt18-knockout (Krt18-KO) or dominant-negative mutant mice (Krt18 DN), but structure was unaltered. Exogenous Krt18, introduced by electroporation, was localized in a reticulum around the contractile apparatus in wild-type muscle and to a lesser extent in muscle lacking Krt19 or desmin or both proteins. Exogenous Krt19, which was either reticular or aggregated in controls, became reticular more frequently in Krt19-null than in Krt18-null, desmin-null, or double-null muscles. Desmin was assembled into the reticulum normally in all genotypes. Notably, all three IF proteins appeared in overlapping reticular structures. We assessed the effect of Krt18 on susceptibility to injury in vivo by electroporating siRNA into tibialis anterior (TA) muscles of control and Krt19-KO mice and testing 2 wk later. Results showed a 33% strength deficit (reduction in maximal torque after injury) compared with siRNA-treated controls. Conversely, electroporation of siRNA to Krt19 into Krt18-null TA yielded a strength deficit of 18% after injury compared with controls. Our results suggest that Krt18 plays a complementary role to Krt19 in skeletal muscle in both assembling keratin-based filaments and transducing contractile force.

Muscle xenografts reproduce key molecular features of facioscapulohumeral muscular dystrophy.

Aberrant expression of DUX4, a gene unique to humans and primates, causes Facioscapulohumeral Muscular Dystrophy-1 (FSHD), yet the pathogenic mechanism is unknown. As transgenic overexpression models have largely failed to replicate the genetic changes seen in FSHD, many studies of endogenously expressed DUX4 have been limited to patient biopsies and myogenic cell cultures, which never fully differentiate into mature muscle fibers. We have developed a method to xenograft immortalized human muscle precursor cells from patients with FSHD and first-degree relative controls into the tibialis anterior muscle compartment of immunodeficient mice, generating human muscle xenografts. We report that FSHD cells mature into organized and innervated human muscle fibers with minimal contamination of murine myonuclei. They also reconstitute the satellite cell niche within the xenografts. FSHD xenografts express DUX4 and DUX4 downstream targets, retain the 4q35 epigenetic signature of their original donors, and express a novel protein biomarker of FSHD, SLC34A2. Ours is the first scalable, mature in vivo human model of FSHD. It should be useful for studies of the pathogenic mechanism of the disease as well as for testing therapeutic strategies targeting DUX4 expression.

Absence of synemin in mice causes structural and functional abnormalities in heart.

Cardiomyopathies have been linked to changes in structural proteins, including intermediate filament (IF) proteins located in the cytoskeleton. IFs associate with the contractile machinery and costameres of striated muscle and with intercalated disks in the heart. Synemin is a large IF protein that mediates the association of desmin with Z-disks and stabilizes intercalated disks. It also acts as an A-kinase anchoring protein (AKAP). In murine skeletal muscle, the absence of synemin causes a mild myopathy. Here, we report that the genetic silencing of synemin in mice (synm -/-) causes left ventricular systolic dysfunction at 3months and 12-16months of age, and left ventricular hypertrophy and dilatation at 12-16months of age. Isolated cardiomyocytes showed alterations in calcium handling that indicate defects intrinsic to the heart. Although contractile and costameric proteins remained unchanged in the old synm -/- hearts, we identified alterations in several signaling proteins (PKA-RII, ERK and p70S6K) critical to cardiomyocyte function. Our data suggest that synemin plays an important regulatory role in the heart and that the consequences of its absence are profound.

Effect of Ibuprofen on Skeletal Muscle of Dysferlin-Null Mice.

Ibuprofen, a nonsteroidal anti-inflammatory drug, and nitric oxide (NO) donors have been reported to reduce the severity of muscular dystrophies in mice associated with the absence of dystrophin or α-sarcoglycan, but their effects on mice that are dystrophic due to the absence of dysferlin have not been examined. We have tested ibuprofen, as well as isosorbide dinitrate (ISDN), a NO donor, to learn whether used alone or together they protect dysferlin-null muscle in A/J mice from large strain injury (LSI) induced by a series of high strain lengthening contractions. Mice were maintained on chow containing ibuprofen and ISDN for 4 weeks. They were then subjected to LSI and maintained on the drugs for 3 additional days. We measured loss of torque immediately following injury and at day 3 postinjury, fiber necrosis, and macrophage infiltration at day 3 postinjury, and serum levels of the drugs at the time of euthanasia. Loss of torque immediately after injury was not altered by the drugs. However, the torque on day 3 postinjury significantly decreased as a function of ibuprofen concentration in the serum (range, 0.67-8.2 µg/ml), independent of ISDN. The effects of ISDN on torque loss at day 3 postinjury were not significant. In long-term studies of dysferlinopathic BlAJ mice, lower doses of ibuprofen had no effects on muscle morphology, but reduced treadmill running by 40%. Our results indicate that ibuprofen can have deleterious effects on dysferlin-null muscle and suggest that its use at pharmacological doses should be avoided by individuals with dysferlinopathies.

Coupling of excitation to Ca2+ release is modulated by dysferlin.

KEY POINTS: Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca2+ channels protect voltage-induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+ -free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca2+ leak. ABSTRACT: Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca2+ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca2+ leak from the SR into the cytoplasm.

Interactions between small ankyrin 1 and sarcolipin coordinately regulate activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1).

SERCA1, the sarco(endo)plasmic reticulum Ca2+-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca2+ levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca2+-ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca2+-ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity.

Deficiency of the intermediate filament synemin reduces bone mass in vivo.

While the type IV intermediate filament protein, synemin, has been shown to play a role in striated muscle and neuronal tissue, its presence and function have not been described in skeletal tissue. Here, we report that genetic ablation of synemin in 14-wk-old male mice results in osteopenia that includes a more than 2-fold reduction in the trabecular bone fraction in the distal femur and a reduction in the cross-sectional area at the femoral middiaphysis due to an attendant reduction in both the periosteal and endosteal perimeter. Analysis of serum markers of bone formation and static histomorphometry revealed a statistically significant defect in osteoblast activity and osteoblast number in vivo. Interestingly, primary osteoblasts isolated from synemin-null mice demonstrate markedly enhanced osteogenic capacity with a concomitant reduction in cyclin D1 mRNA expression, which may explain the loss of osteoblast number observed in vivo. In total, these data suggest an important, previously unknown role for synemin in bone physiology.

Neuromuscular electrical stimulation promotes development in mice of mature human muscle from immortalized human myoblasts.

BACKGROUND: Studies of the pathogenic mechanisms underlying human myopathies and muscular dystrophies often require animal models, but models of some human diseases are not yet available. Methods to promote the engraftment and development of myogenic cells from individuals with such diseases in mice would accelerate such studies and also provide a useful tool for testing therapeutics. Here, we investigate the ability of immortalized human myogenic precursor cells (hMPCs) to form mature human myofibers following implantation into the hindlimbs of non-obese diabetic-Rag1 (null) IL2rγ (null) (NOD-Rag)-immunodeficient mice. RESULTS: We report that hindlimbs of NOD-Rag mice that are X-irradiated, treated with cardiotoxin, and then injected with immortalized control hMPCs or hMPCs from an individual with facioscapulohumeral muscular dystrophy (FSHD) develop mature human myofibers. Furthermore, intermittent neuromuscular electrical stimulation (iNMES) of the peroneal nerve of the engrafted limb enhances the development of mature fibers in the grafts formed by both immortal cell lines. With control cells, iNMES increases the number and size of the human myofibers that form and promotes closer fiber-to-fiber packing. The human myofibers in the graft are innervated, fully differentiated, and minimally contaminated with murine myonuclei. CONCLUSIONS: Our results indicate that control and FSHD human myofibers can form in mice engrafted with hMPCs and that iNMES enhances engraftment and subsequent development of mature human muscle.

Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle.

Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ~2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca(2+) affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca(2+) in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca(2+) misregulation.

Myofiber damage precedes macrophage infiltration after in vivo injury in dysferlin-deficient A/J mouse skeletal muscle.

Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage.