Failure to Resolve Inflammation Contributes to Juvenile-Onset Cardiomyopathy in a Mouse Model of Duchenne Muscular Dystrophy.

The absence of dystrophin protein causes cardiac dysfunction in boys with Duchenne Muscular Dystrophy (DMD). However, the common mouse model of DMD (B10-mdx) does not manifest cardiac deficits until late adulthood limiting our understanding of the mechanism and therapeutic approaches to target the pediatric-onset cardiac pathology in DMD. We show the mdx mouse model on the DBA/2J genetic background (D2-mdx) displays juvenile-onset cardiomyopathy. Molecular and histological analysis revealed heightened leukocyte chemotactic signaling and failure to resolve inflammation, leading to chronic inflammation and extracellular matrix (ECM) fibrosis, causing cardiac pathology in juvenile D2-mdx mice. We show that pharmacologically activating the N-formyl peptide receptor 2 (FPR2) - a receptor that physiologically resolves acute inflammation, mitigated chronic cardiac inflammation and fibrosis, and prevented juvenile onset cardiomyopathy in the D2-mdx mice. These studies offer insights into pediatric onset of cardiac damage in DMD, a new therapeutic target, and identify a drug-based potential therapy.

SERCA1 Overexpression in Skeletal Muscle Attenuates Muscle Atrophy and Improves Motor Function in a Mouse Model of ALS.

Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of muscle mass and muscle function. Previous work from our lab demonstrated that skeletal muscles from a mouse model of ALS show elevated intracellular calcium (Ca2+) levels and heightened endoplasmic reticulum (ER) stress. Objective: To investigate whether overexpression of sarcoplasmic reticulum (SR) Ca2+ ATPase 1 (SERCA1) in skeletal muscle would improve intracellular Ca2+ handling, attenuate ER stress, and improve motor function ALS transgenic mice. Methods: B6SJL-Tg (SOD1*G93A)1Gur/J (ALS-Tg) mice were bred with skeletal muscle α-actinin SERCA1 overexpressing mice to generate wild type (WT), SERCA1 overexpression (WT/+SERCA1), ALS-Tg, and SERCA1 overexpressing ALS-Tg (ALS-Tg/+SERCA1) mice. Motor function (grip test) was assessed weekly and skeletal muscles were harvested at 16 weeks of age to evaluate muscle mass, SR-Ca2+ ATPase activity, levels of SERCA1 and ER stress proteins - protein disulfide isomerase (PDI), Grp78/BiP, and C/EBP homologous protein (CHOP). Single muscle fibers were also isolated from the flexor digitorum brevis muscle to assess changes in resting and peak Fura-2 ratios. Results: ALS-Tg/+SERCA1 mice showed improved motor function, delayed onset of disease, and improved muscle mass compared to ALS-Tg. Further, ALS-Tg/+SERCA1 mice returned levels of SERCA1 protein and SR-Ca2+ ATPase activity back to levels in WT mice. Unexpectedly, SERCA-1 overexpression increased levels of the ER stress maker Grp78/BiP in both WT and ALS-Tg mice, while not altering protein levels of PDI or CHOP. Lastly, single muscle fibers from ALS-Tg/+SERCA1 had similar resting but lower peak Fura-2 levels (at 30 Hz and 100 Hz) compared to ALS-Tg mice. Conclusions: These data indicate that SERCA1 overexpression attenuates the progressive loss of muscle mass and maintains motor function in ALS-Tg mice while not lowering resting Ca2+ levels or ER stress.

Single-cell transcriptomic analysis of the identity and function of fibro/adipogenic progenitors in healthy and dystrophic muscle.

Fibro/adipogenic progenitors (FAPs) are skeletal muscle stromal cells that support regeneration of injured myofibers and their maintenance in healthy muscles. FAPs are related to mesenchymal stem cells (MSCs/MeSCs) found in other adult tissues, but there is poor understanding of the extent of similarity between these cells. Using single-cell RNA sequencing (scRNA-seq) datasets from multiple mouse tissues, we have performed comparative transcriptomic analysis. This identified remarkable transcriptional similarity between FAPs and MeSCs, confirmed the suitability of PDGFRα as a reporter for FAPs, and identified extracellular proteolysis as a new FAP function. Using PDGFRα as a cell surface marker, we isolated FAPs from healthy and dysferlinopathic mouse muscles and performed scRNA-seq analysis. This revealed decreased FAP-mediated Wnt signaling as a potential driver of FAP dysfunction in dysferlinopathic muscles. Analysis of FAPs in dysferlin- and dystrophin-deficient muscles identified a relationship between the nature of muscle pathology and alteration in FAP gene expression.

Altered muscle niche contributes to myogenic deficit in the D2-mdx model of severe DMD.

Lack of dystrophin expression is the underlying genetic basis for Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2-mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2-mdx muscles is associated with an enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports the excessive accumulation of fibroadipogenic progenitors (FAPs), leading to increased fibrosis. Unexpectedly, the extent of damage and degeneration in juvenile D2-mdx muscle is significantly reduced in adults, and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance regenerative myogenesis in the adult D2-mdx muscle, reaching levels comparable to the milder B10-mdx model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with juvenile D2-mdx FAPs reduces their fusion efficacy. Wild-type juvenile D2 mice also manifest regenerative myogenic deficit and glucocorticoid treatment improves their muscle regeneration. Our findings indicate that aberrant stromal cell responses contribute to poor regenerative myogenesis and greater muscle degeneration in juvenile D2-mdx muscles and reversal of this reduces pathology in adult D2-mdx muscle, identifying these responses as a potential therapeutic target for the treatment of DMD.

Altered muscle niche contributes to myogenic deficit in the D2- mdx model of severe DMD.

Lack of dystrophin is the genetic basis for the Duchenne muscular dystrophy (DMD). However, disease severity varies between patients, based on specific genetic modifiers. D2- mdx is a model for severe DMD that exhibits exacerbated muscle degeneration and failure to regenerate even in the juvenile stage of the disease. We show that poor regeneration of juvenile D2- mdx muscles is associated with enhanced inflammatory response to muscle damage that fails to resolve efficiently and supports excessive accumulation of fibroadipogenic progenitors (FAPs). Unexpectedly, the extent of damage and degeneration of juvenile D2- mdx muscle is reduced in adults and is associated with the restoration of the inflammatory and FAP responses to muscle injury. These improvements enhance myogenesis in the adult D2- mdx muscle, reaching levels comparable to the milder (B10- mdx ) mouse model of DMD. Ex vivo co-culture of healthy satellite cells (SCs) with the juvenile D2- mdx FAPs reduced their fusion efficacy and in vivo glucocorticoid treatment of juvenile D2 mouse improved muscle regeneration. Our findings indicate that aberrant stromal cell response contributes to poor myogenesis and greater muscle degeneration in dystrophic juvenile D2- mdx muscles and reversal of this reduces pathology in adult D2- mdx mouse muscle, identifying these as therapeutic targets to treat dystrophic DMD muscles.

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics.

BACKGROUND: A common polymorphism (R577X) in the ACTN3 gene results in the complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~ 16% of the world's population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0 + 20% stretch did not cause eccentric damage. In contrast, L0 + 30% stretch produced a significant ~ 40% deficit in maximum force; here, we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this. METHODS: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA. RESULTS: We show that the absence of α-actinin-3 does not affect the visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically stretched. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar branching. CONCLUSIONS: The absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0 + 30%, but not L0 + 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3-deficient humans have been reported to have a faster decline in muscle function with increasing age, that is, as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibre breakages.

Pathogenic role and therapeutic potential of fibro-adipogenic progenitors in muscle disease.

Aside from myofibers, numerous mononucleated cells reside in the skeletal muscle. These include the mesenchymal cells called fibro-adipogenic progenitors (FAPs), that support muscle development and regeneration in adult muscles. Recent evidence shows that defects in FAP function contributes to chronic muscle diseases and targeting FAPs offers avenues for treating these diseases.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies -Eteplirsen, Golodirsen, Viltolarsen, and Casimersen -for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Human muscle stem cells are refractory to aging.

Age-related loss of muscle mass and strength is widely attributed to limitation in the capacity of muscle resident satellite cells to perform their myogenic function. This idea contains two notions that have not been comprehensively evaluated by experiment. First, it entails the idea that we damage and lose substantial amounts of muscle in the course of our normal daily activities. Second, it suggests that mechanisms of muscle repair are in some way exhausted, thus limiting muscle regeneration. A third potential option is that the aged environment becomes inimical to the conduct of muscle regeneration. In the present study, we used our established model of human muscle xenografting to test whether muscle samples taken from cadavers, of a range of ages, maintained their myogenic potential after being transplanted into immunodeficient mice. We find no measurable difference in regeneration across the range of ages investigated up to 78 years of age. Moreover, we report that satellite cells maintained their myogenic capacity even when muscles were grafted 11 days postmortem in our model. We conclude that the loss of muscle mass with increasing age is not attributable to any intrinsic loss of myogenicity and is most likely a reflection of progressive and detrimental changes in the muscle microenvironment such as to disfavor the myogenic function of these cells.

Coping with the calcium overload caused by cell injury: ER to the rescue.

Cells maintain their cytosolic calcium (Ca2+) in nanomolar range and use controlled increase in Ca2+ for intracellular signaling. With the extracellular Ca2+ in the millimolar range, there is a steep Ca2+ gradient across the plasma membrane (PM). Thus, injury that damages PM, leads to a cytosolic Ca2+ overload, which helps activate PM repair (PMR) response. However, in order to survive, the cells must cope with the Ca2+ overload. In a recent study (Chandra et al. J Cell Biol, doi: 10.1083/jcb.202006035) we have examined how cells cope with injury-induced cytosolic Ca2+ overload. By monitoring Ca2+ dynamics in the cytosol and endoplasmic reticulum (ER), we found that PM injury-triggered increase in cytosolic Ca2+ is taken up by the ER. Pharmacological inhibition of ER Ca2+ uptake interferes with this process and compromises the repair ability of the injured cells. Muscle cells from patients and mouse model for the muscular dystrophy showed that lack of Anoctamin 5 (ANO5)/Transmembrane protein 16E (TMEM16E), an ER-resident putative Ca2+-activated chloride channel (CaCC), are poor at coping with cytosolic Ca2+ overload. Pharmacological inhibition of CaCC and lack of ANO5, both prevent Ca2+ uptake into ER. These studies identify a requirement of Cl- uptake by the ER in sequestering injury-triggered cytosolic Ca2+ increase in the ER. Further, these studies show that ER helps injured cells cope with Ca2+ overload during PMR, lack of which contributes to muscular dystrophy due to mutations in the ANO5 protein.

Endoplasmic reticulum maintains ion homeostasis required for plasma membrane repair.

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.

Dysregulation of Mitochondrial Ca2+ Uptake and Sarcolemma Repair Underlie Muscle Weakness and Wasting in Patients and Mice Lacking MICU1.

Muscle function is regulated by Ca2+, which mediates excitation-contraction coupling, energy metabolism, adaptation to exercise, and sarcolemmal repair. Several of these actions rely on Ca2+ delivery to the mitochondrial matrix via the mitochondrial Ca2+ uniporter, the pore of which is formed by mitochondrial calcium uniporter (MCU). MCU's gatekeeping and cooperative activation are controlled by MICU1. Loss-of-protein mutation in MICU1 causes a neuromuscular disease. To determine the mechanisms underlying the muscle impairments, we used MICU1 patient cells and skeletal muscle-specific MICU1 knockout mice. Both these models show a lower threshold for MCU-mediated Ca2+ uptake. Lack of MICU1 is associated with impaired mitochondrial Ca2+ uptake during excitation-contraction, aerobic metabolism impairment, muscle weakness, fatigue, and myofiber damage during physical activity. MICU1 deficit compromises mitochondrial Ca2+ uptake during sarcolemmal injury, which causes ineffective repair of the damaged myofibers. Thus, dysregulation of mitochondrial Ca2+ uptake hampers myofiber contractile function, likely through energy metabolism and membrane repair.

Vamorolone targets dual nuclear receptors to treat inflammation and dystrophic cardiomyopathy.

Cardiomyopathy is a leading cause of death for Duchenne muscular dystrophy. Here, we find that the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) can share common ligands but play distinct roles in dystrophic heart and skeletal muscle pathophysiology. Comparisons of their ligand structures indicate that the Δ9,11 modification of the first-in-class drug vamorolone enables it to avoid interaction with a conserved receptor residue (N770/N564), which would otherwise activate transcription factor properties of both receptors. Reporter assays show that vamorolone and eplerenone are MR antagonists, whereas prednisolone is an MR agonist. Macrophages, cardiomyocytes, and CRISPR knockout myoblasts show vamorolone is also a dissociative GR ligand that inhibits inflammation with improved safety over prednisone and GR-specific deflazacort. In mice, hyperaldosteronism activates MR-driven hypertension and kidney phenotypes. We find that genetic dystrophin loss provides a second hit for MR-mediated cardiomyopathy in Duchenne muscular dystrophy model mice, as aldosterone worsens fibrosis, mass and dysfunction phenotypes. Vamorolone successfully prevents MR-activated phenotypes, whereas prednisolone activates negative MR and GR effects. In conclusion, vamorolone targets dual nuclear receptors to treat inflammation and cardiomyopathy with improved safety.

Calcium dysregulation, functional calpainopathy, and endoplasmic reticulum stress in sporadic inclusion body myositis.

Sporadic inclusion body myositis (IBM) is the most common primary myopathy in the elderly, but its pathoetiology is still unclear. Perturbed myocellular calcium (Ca2+) homeostasis can exacerbate many of the factors proposed to mediate muscle degeneration in IBM, such as mitochondrial dysfunction, protein aggregation, and endoplasmic reticulum stress. Ca2+ dysregulation may plausibly be initiated in IBM by immune-mediated membrane damage and/or abnormally accumulating proteins, but no studies to date have investigated Ca2+ regulation in IBM patients. We first investigated protein expression via immunoblot in muscle biopsies from IBM, dermatomyositis, and non-myositis control patients, identifying several differentially expressed Ca2+-regulatory proteins in IBM. Next, we investigated the Ca2+-signaling transcriptome by RNA-seq, finding 54 of 183 (29.5%) genes from an unbiased list differentially expressed in IBM vs. controls. Using an established statistical approach to relate genes with causal transcription networks, Ca2+ abundance was considered a significant upstream regulator of observed whole-transcriptome changes. Post-hoc analyses of Ca2+-regulatory mRNA and protein data indicated a lower protein to transcript ratio in IBM vs. controls, which we hypothesized may relate to increased Ca2+-dependent proteolysis and decreased protein translation. Supporting this hypothesis, we observed robust (4-fold) elevation in the autolytic activation of a Ca2+-activated protease, calpain-1, as well as increased signaling for translational attenuation (eIF2a phosphorylation) downstream of the unfolded protein response. Finally, in IBM samples we observed mRNA and protein under-expression of calpain-3, the skeletal muscle-specific calpain, which broadly supports proper Ca2+ homeostasis. Together, these data provide novel insight into mechanisms by which intracellular Ca2+ regulation is perturbed in IBM and offer evidence of pathological downstream effects.

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation.

Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene's muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5'-diphosphate (ADP)-ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr-/- mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.

The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle.

BACKGROUND: The SH3 and cysteine-rich domain 3 (Stac3) gene is specifically expressed in the skeletal muscle. Stac3 knockout mice die perinatally. In this study, we determined the potential role of Stac3 in postnatal skeletal muscle growth, fiber composition, and contraction by generating conditional Stac3 knockout mice. METHODS: We disrupted the Stac3 gene in 4-week-old male mice using the Flp-FRT and tamoxifen-inducible Cre-loxP systems. RESULTS: RT-qPCR and western blotting analyses of the limb muscles of target mice indicated that nearly all Stac3 mRNA and more than 70 % of STAC3 protein were deleted 4 weeks after tamoxifen injection. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei, with no effect on the total myofiber number. Grip strength and grip time tests indicated that postnatal Stac3 deletion decreased limb muscle strength in mice. Muscle contractile tests revealed that postnatal Stac3 deletion reduced electrostimulation-induced but not the ryanodine receptor agonist caffeine-induced maximal force output in the limb muscles. Calcium imaging analysis of single flexor digitorum brevis myofibers indicated that postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. CONCLUSIONS: This study demonstrates that STAC3 is important to myofiber hypertrophy, myofiber-type composition, contraction, and excitation-induced calcium release from the sarcoplasmic reticulum in the postnatal skeletal muscle.

SERCA1 overexpression minimizes skeletal muscle damage in dystrophic mouse models.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting secondary to repeated muscle damage and inadequate repair. Elevations in intracellular free Ca²âº have been implicated in disease progression, and sarcoplasmic/endoplasmic reticulum Ca²âº-ATPase 1 (SERCA1) overexpression has been shown to ameliorate the dystrophic phenotype in mdx mice. The purpose of this study was to assess the effects of SERCA1 overexpression in the more severe mdx/Utr(-/-) mouse model of DMD. Mice overexpressing SERCA1 were crossed with mdx/Utr ± mice to generate mdx/Utr(-/-)/+SERCA1 mice and compared with wild-type (WT), WT/+SERCA1, mdx/+SERCA1, and genotype controls. Mice were assessed at ∼12 wk of age for changes in Ca²âº handling, muscle mass, quadriceps torque, markers of muscle damage, and response to repeated eccentric contractions. SERCA1-overexpressing mice had a two- to threefold increase in maximal sarcoplasmic reticulum Ca²âº-ATPase activity compared with WT which was associated with normalization in body mass for both mdx/+SERCA1 and mdx/Utr(-/-)/+SERCA1. Torque deficit in the quadriceps after eccentric injury was 2.7-fold greater in mdx/Utr(-/-) vs. WT mice, but only 1.5-fold greater in mdx/Utr(-/-)/+SERCA1 vs. WT mice, an attenuation of 44%. Markers of muscle damage (% centrally nucleated fibers, necrotic area, and serum creatine kinase levels) were higher in both mdx and mdx/Utr(-/-) vs. WT, and all were attenuated by overexpression of SERCA1. These data indicate that SERCA1 overexpression ameliorates functional impairments and cellular markers of damage in a more severe mouse model of DMD. These findings support targeting intracellular Ca²âº control as a therapeutic approach for DMD.

The role of proteases in excitation-contraction coupling failure in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is one of the most frequent types of muscular dystrophy. Alterations in intracellular calcium (Ca(2+)) handling are thought to contribute to the disease severity in DMD, possibly due to the activation of Ca(2+)-activated proteases. The purpose of this study was twofold: 1) to determine whether prolonged excitation-contraction (E-C) coupling disruption following repeated contractions is greater in animals lacking both dystrophin and utrophin (mdx/Utr(-/-)) compared with mice lacking only dystrophin (mdx); and 2) to assess whether protease inhibition can prevent E-C coupling failure following repeated tetani in these dystrophic mouse models. Excitation-contraction coupling was assessed using Fura-2 ratio, as an index of intracellular free Ca(2+) concentration, in response to electrical stimulation of single muscle fibers from the flexor digitorum brevis muscle. Resting Fura-2 ratio was higher in dystrophic compared with control (Con) fibers, but peak Fura-2 ratios during stimulation were similar in dystrophic and Con fibers. One hour after a series of repeated tetani, peak Fura-2 ratios were reduced by 30 ± 5.6%, 23 ± 2%, and 36 ± 3.1% in mdx, mdx/Utr(+/-), and mdx/Utr(-/-), respectively, with the greatest reduction in mdx/Utr(-/-) fibers (P < 0.05). Protease inhibition attenuated this decrease in peak Fura-2 ratio. These data indicate that E-C coupling impairment after repeated contractions is greatest in fibers lacking both dystrophin and utrophin and that prevention of protease activation can mitigate the prolonged E-C coupling impairment. These data further suggest that acute protease inhibition may be useful in reducing muscle weakness in DMD.

Perturbations in intracellular Ca2+ handling in skeletal muscle in the G93A*SOD1 mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by skeletal muscle atrophy and weakness, ultimately leading to respiratory failure. The purpose of this study was to assess changes in skeletal muscle excitation-contraction (E-C) coupling and intracellular Ca(2+) handling during disease progression in the G93A*SOD1 ALS transgenic (ALS Tg) mouse model. To assess E-C coupling, single muscle fibers were electrically stimulated (10-150 Hz), and intracellular free Ca(2+) concentration was assessed using fura-2. There were no differences in peak fura-2 ratio at any stimulation frequency at 70 days (early presymptomatic). However, at 90 days (late presymptomatic) and 120-140 days (symptomatic), fura-2 ratio was increased at 10 Hz in ALS Tg compared with wild-type (WT) fibers (0.670 ± 0.02 vs. 0.585 ± 0.02 for 120-140 days; P < 0.05). There was also a significant increase in resting fura-2 ratio at 90 days (0.351 ± 0.008 vs. 0.390 ± 0.009 in WT vs. ALS Tg; P < 0.05) and 120-140 days (0.374 ± 0.001 vs. 0.415 ± 0.003 in WT vs. ALS Tg; P < 0.05). These increases in intracellular Ca(2+) in ALS Tg muscle were associated with reductions in the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase proteins SERCA1 (to 54% and 19% of WT) and SERCA2 (to 56% and 11% of WT) and parvalbumin (to 80 and 62% of WT) in gastrocnemius muscle at 90 and 120-140 days, respectively. There was no change in dihydropyridine receptor/l-type Ca(2+) channel at any age. Overall, these data demonstrate minimal changes in electrically evoked Ca(2+) transients but elevations in intracellular Ca(2+) attributable to decreased Ca(2+)-clearance proteins. These data suggest that elevations in cellular Ca(2+) could contribute to muscle weakness during disease progression in ALS mice.