Pannexin 1 dysregulation in Duchenne muscular dystrophy and its exacerbation of dystrophic features in mdx mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is associated with impaired muscle regeneration, progressive muscle weakness, damage, and wasting. While the cause of DMD is an X-linked loss of function mutation in the gene encoding dystrophin, the exact mechanisms that perpetuate the disease progression are unknown. Our laboratory has demonstrated that pannexin 1 (Panx1 in rodents; PANX1 in humans) is critical for the development, strength, and regeneration of male skeletal muscle. In normal skeletal muscle, Panx1 is part of a multiprotein complex with dystrophin. We and others have previously shown that Panx1 levels and channel activity are dysregulated in various mouse models of DMD. METHODS: We utilized myoblast cell lines derived from DMD patients to assess PANX1 expression and function. To investigate how Panx1 dysregulation contributes to DMD, we generated a dystrophic (mdx) mouse model that lacks Panx1 (Panx1-/-/mdx). In depth characterization of this model included histological analysis, as well as locomotor, and physiological tests such as muscle force and grip strength assessments. RESULTS: Here, we demonstrate that PANX1 levels and channel function are reduced in patient-derived DMD myoblast cell lines. Panx1-/-/mdx mice have a significantly reduced lifespan, and decreased body weight due to lean mass loss. Their tibialis anterior were more affected than their soleus muscles and displayed reduced mass, myofiber loss, increased centrally nucleated myofibers, and a lower number of muscle stem cells compared to that of Panx1+/+/mdx mice. These detrimental effects were associated with muscle and locomotor functional impairments. In vitro, PANX1 overexpression in patient-derived DMD myoblasts improved their differentiation and fusion. CONCLUSIONS: Collectively, our findings suggest that PANX1/Panx1 dysregulation in DMD exacerbates several aspects of the disease. Moreover, our results suggest a potential therapeutic benefit to increasing PANX1 levels in dystrophic muscles.

A century of exercise physiology: effects of muscle contraction and exercise on skeletal muscle Na+,K+-ATPase, Na+ and K+ ions, and on plasma K+ concentration-historical developments.

This historical review traces key discoveries regarding K+ and Na+ ions in skeletal muscle at rest and with exercise, including contents and concentrations, Na+,K+-ATPase (NKA) and exercise effects on plasma [K+] in humans. Following initial measures in 1896 of muscle contents in various species, including humans, electrical stimulation of animal muscle showed K+ loss and gains in Na+, Cl- and H20, then subsequently bidirectional muscle K+ and Na+ fluxes. After NKA discovery in 1957, methods were developed to quantify muscle NKA activity via rates of ATP hydrolysis, Na+/K+ radioisotope fluxes, [3H]-ouabain binding and phosphatase activity. Since then, it became clear that NKA plays a central role in Na+/K+ homeostasis and that NKA content and activity are regulated by muscle contractions and numerous hormones. During intense exercise in humans, muscle intracellular [K+] falls by 21 mM (range - 13 to - 39 mM), interstitial [K+] increases to 12-13 mM, and plasma [K+] rises to 6-8 mM, whilst post-exercise plasma [K+] falls rapidly, reflecting increased muscle NKA activity. Contractions were shown to increase NKA activity in proportion to activation frequency in animal intact muscle preparations. In human muscle, [3H]-ouabain-binding content fully quantifies NKA content, whilst the method mainly detects α2 isoforms in rats. Acute or chronic exercise affects human muscle K+, NKA content, activity, isoforms and phospholemman (FXYD1). Numerous hormones, pharmacological and dietary interventions, altered acid-base or redox states, exercise training and physical inactivity modulate plasma [K+] during exercise. Finally, historical research approaches largely excluded female participants and typically used very small sample sizes.

The potassium-glycogen interaction on force and excitability in mouse skeletal muscle: implications for fatigue.

A reduced muscle glycogen content and potassium (K+ ) disturbances across muscle membranes occur concomitantly during repeated intense exercise and together may contribute to skeletal muscle fatigue. Therefore, we examined whether raised extracellular K+ concentration ([K+ ]o ) (4 to 11 mM) interacts with lowered glycogen to reduce force production. Isometric contractions were evoked in isolated mouse soleus muscles (37°C) using direct supramaximal field stimulation. (1) Glycogen declined markedly in non-fatigued muscle with >2 h exposure in glucose-free physiological saline compared with control solutions (11 mM glucose), i.e. to <45% control. (2) Severe glycogen depletion was associated with increased 5'-AMP-activated protein kinase activity, indicative of metabolic stress. (3) The decline of peak tetanic force at 11 mM [K+ ]o was exacerbated from 67% initial at normal glycogen to 22% initial at lowered glycogen. This was due to a higher percentage of inexcitable fibres (71% vs. 43%), yet without greater sarcolemmal depolarisation or smaller amplitude action potentials. (4) Returning glucose while at 11 mM [K+ ]o increased both glycogen and force. (5) Exposure to 4 mM [K+ ]o glucose-free solutions (15 min) did not increase fatiguability during repeated tetani; however, after recovery there was a greater force decline at 11 mM [K+ ]o at lower than normal glycogen. (6) An important exponential relationship was established between relative peak tetanic force at 11 mM [K+ ]o and muscle glycogen content. These findings provide direct evidence of a synergistic interaction between raised [K+ ]o and lowered muscle glycogen as the latter shifts the peak tetanic force-resting EM relationship towards more negative resting EM due to lowered sarcolemmal excitability, which hence may contribute to muscle fatigue. KEY POINTS: Diminished muscle glycogen levels and raised extracellular potassium concentrations ([K+ ]o ) occur simultaneously during intense exercise and together may contribute to muscle fatigue. Prolonged exposure of isolated non-fatigued soleus muscles of mice to glucose-free physiological saline solutions markedly lowered muscle glycogen levels, as does fatigue then recovery in glucose-free solutions. For both approaches, the subsequent decline of maximal force at 11 mM [K+ ]o , which mimics interstitial [K+ ] levels during intense exercise, was exacerbated at lowered compared with normal glycogen. This was mainly due to many more muscle fibres becoming inexcitable. We established an important relationship that provides evidence of a synergistic interaction between raised [K+ ]o and lowered glycogen content to reduce force production. This paper indicates that partially lowered muscle glycogen (and/or metabolic stress) together with elevated interstitial [K+ ] interactively lowers muscle force, and hence may diminish performance especially during repeated high-intensity exercise.

Exercise and fatigue: integrating the role of K+, Na+ and Cl- in the regulation of sarcolemmal excitability of skeletal muscle.

Perturbations in K+ have long been considered a key factor in skeletal muscle fatigue. However, the exercise-induced changes in K+ intra-to-extracellular gradient is by itself insufficiently large to be a major cause for the force decrease during fatigue unless combined to other ion gradient changes such as for Na+. Whilst several studies described K+-induced force depression at high extracellular [K+] ([K+]e), others reported that small increases in [K+]e induced potentiation during submaximal activation frequencies, a finding that has mostly been ignored. There is evidence for decreased Cl- ClC-1 channel activity at muscle activity onset, which may limit K+-induced force depression, and large increases in ClC-1 channel activity during metabolic stress that may enhance K+ induced force depression. The ATP-sensitive K+ channel (KATP channel) is also activated during metabolic stress to lower sarcolemmal excitability. Taking into account all these findings, we propose a revised concept in which K+ has two physiological roles: (1) K+-induced potentiation and (2) K+-induced force depression. During low-moderate intensity muscle contractions, the K+-induced force depression associated with increased [K+]e is prevented by concomitant decreased ClC-1 channel activity, allowing K+-induced potentiation of sub-maximal tetanic contractions to dominate, thereby optimizing muscle performance. When ATP demand exceeds supply, creating metabolic stress, both KATP and ClC-1 channels are activated. KATP channels contribute to force reductions by lowering sarcolemmal generation of action potentials, whilst ClC-1 channel enhances the force-depressing effects of K+, thereby triggering fatigue. The ultimate function of these changes is to preserve the remaining ATP to prevent damaging ATP depletion.

Angiotensin 1-7 increases fiber cross-sectional area and force in juvenile mouse skeletal muscle.

Recent studies reported that in skeletal muscle angiotensin 1-7 (Ang 1-7), via its receptor Mas (MasR), prevents the atrophy induced by angiotensin II and by cast immobilization; it also improves muscle integrity and function in the mdx mouse, a muscular dystrophy model. The objectives of this study were to document 1) the extent of the Ang 1-7's hypertrophic effect in terms of muscle mass and muscle fiber cross-sectional area (CSA), 2) how Ang 1-7 affects muscle contractile function in terms of twitch and tetanic force, force-frequency relationship, and 3) whether the effect involves MasR. Wild-type and MasR-deficient [Mas receptor knockout mouse model (MasR-/-)] mice were treated with Ang 1-7 (100 ng/kg body wt·min using an osmotic pump) for 4 or 16 wk. Ang 1-7 significantly increased skeletal muscle/body weight ratio of soleus, tibialis, and gastrocnemius, but not of extensor digitorum longus (EDL). It significantly increased fiber cross-sectional area in the order of type I > IIA > IIB. In EDL and soleus muscles, it significantly increased twitch and tetanic force while causing a shift in the force-frequency relationship toward lower stimulation frequencies. It had no effect on fiber type composition. None of the Ang 1-7 effects observed in wild-type mice were observed in MasR-/- muscles. It caused a transient increase in phosphorylated protein kinase B (Akt) and 4EBP proteins while having no effect on S6 phosphorylation, MuRF-1, and atrogin-1 and a decrease in PAX7 expression in satellite cells. This is the first study demonstrating the hypertrophic effects of Ang 1-7 in normal muscle acting via its MasR.

The peak force-resting membrane potential relationships of mouse fast- and slow-twitch muscle.

We endeavored to understand the factors determining the peak force-resting membrane potential (EM) relationships of isolated slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles from mice (25°C), especially in relation to fatigue. Interrelationships between intracellular K+ activity ([Formula: see text]), extracellular K+ concentration ([K+]o), resting EM, action potentials, and force were studied. The large resting EM variation was mainly due to the variability of [Formula: see text]. Action potential overshoot-resting EM relationships determined at 4 and 8-10 mM [K+]o after short (<5 min) and prolonged (>50 min) depolarization periods revealed a constant overshoot from -90 to -70 mV providing a safety margin. Overshoot decline with depolarization beyond -70 mV was less after short than prolonged depolarization. Inexcitable fibers occurred only with prolonged depolarization. The overshoot decline during action potential trains (2 s) exceeded that during short depolarizations. Concomitant lower extracellular [Na+] and raised [K+]o depressed the overshoot in an additive manner and peak force in a synergistic manner. Raised [K+]o-induced force loss was exacerbated with transverse wire versus parallel plate stimulation in soleus, implicating action potential propagation failure in the surface membrane. Increasing stimulus pulse parameters restored tetanic force at 9-10 mM [K+]o in soleus but not EDL, indicative of action potential failure within trains. The peak tetanic force-resting EM relationships (determined with resting EM from deeper rather than surface fibers) were dynamic and showed pronounced force depression over -69 to -60 mV in both muscle types, implicating that such depolarization contributes to fatigue. The K+-Na+ interaction shifted this relationship toward less depolarized potentials, suggesting that the combined ionic effect is physiologically important during fatigue.

GCN5 maintains muscle integrity by acetylating YY1 to promote dystrophin expression.

Protein lysine acetylation is a post-translational modification that regulates protein structure and function. It is targeted to proteins by lysine acetyltransferases (KATs) or removed by lysine deacetylases. This work identifies a role for the KAT enzyme general control of amino acid synthesis protein 5 (GCN5; KAT2A) in regulating muscle integrity by inhibiting DNA binding of the transcription factor/repressor Yin Yang 1 (YY1). Here we report that a muscle-specific mouse knockout of GCN5 (Gcn5skm-/-) reduces the expression of key structural muscle proteins, including dystrophin, resulting in myopathy. GCN5 was found to acetylate YY1 at two residues (K392 and K393), disrupting the interaction between the YY1 zinc finger region and DNA. These findings were supported by human data, including an observed negative correlation between YY1 gene expression and muscle fiber diameter. Collectively, GCN5 positively regulates muscle integrity through maintenance of structural protein expression via acetylation-dependent inhibition of YY1. This work implicates the role of protein acetylation in the regulation of muscle health and for consideration in the design of novel therapeutic strategies to support healthy muscle during myopathy or aging.

Angiotensin 1-7 prevents the excessive force loss resulting from 14- and 28-day denervation in mouse EDL and soleus muscle.

Denervation leads to muscle atrophy, which is described as muscle mass and force loss, the latter exceeding expectation from mass loss. The objective of this study was to determine the efficiency of angiotensin (Ang) 1-7 at reducing muscle atrophy in mouse extensor digitorum longus (EDL) and soleus following 14- and 28-d denervation periods. Some denervated mice were treated with Ang 1-7 or diminazene aceturate (DIZE), an ACE2 activator, to increase Ang 1-7 levels. Ang 1-7/DIZE treatment had little effect on muscle mass loss and fiber cross-sectional area reduction. Ang 1-7 and DIZE fully prevented the loss of tetanic force normalized to cross-sectional area and accentuated the increase in twitch force in denervated muscle. However, they did not prevent the shift of the force-frequency relationship toward lower stimulation frequencies. The Ang 1-7/DIZE effects on twitch and tetanic force were completely blocked by A779, a MasR antagonist, and were not observed in MasR-/- muscles. Ang 1-7 reduced the extent of membrane depolarization, fully prevented the loss of membrane excitability, and maintained the action potential overshoot in denervated muscles. Ang 1-7 had no effect on the changes in α-actin, myosin, or MuRF-1, atrogin-1 protein content or the content of total or phosphorylated Akt, S6, and 4EPB. This is the first study that provides evidence that Ang 1-7 maintains normal muscle function in terms of maximum force and membrane excitability during 14- and 28-d periods after denervation.

Lower Ca2+ enhances the K+-induced force depression in normal and HyperKPP mouse muscles.

Hyperkalemic periodic paralysis (HyperKPP) manifests as stiffness or subclinical myotonic discharges before or during periods of episodic muscle weakness or paralysis. Ingestion of Ca2+ alleviates HyperKPP symptoms, but the mechanism is unknown because lowering extracellular [Ca2+] ([Ca2+]e) has no effect on force development in normal muscles under normal conditions. Lowering [Ca2+]e, however, is known to increase the inactivation of voltage-gated cation channels, especially when the membrane is depolarized. Two hypotheses were tested: (1) lowering [Ca2+]e depresses force in normal muscles under conditions that depolarize the cell membrane; and (2) HyperKPP muscles have a greater sensitivity to low Ca2+-induced force depression because many fibers are depolarized, even at a normal [K+]e. In wild type muscles, lowering [Ca2+]e from 2.4 to 0.3 mM had little effect on tetanic force and membrane excitability at a normal K+ concentration of 4.7 mM, whereas it significantly enhanced K+-induced depression of force and membrane excitability. In HyperKPP muscles, lowering [Ca2+]e enhanced the K+-induced loss of force and membrane excitability not only at elevated [K+]e but also at 4.7 mM K+. Lowering [Ca2+]e increased the incidence of generating fast and transient contractures and gave rise to a slower increase in unstimulated force, especially in HyperKPP muscles. Lowering [Ca2+]e reduced the efficacy of salbutamol, a ß2 adrenergic receptor agonist and a treatment for HyperKPP, to increase force at elevated [K+]e. Replacing Ca2+ by an equivalent concentration of Mg2+ neither fully nor consistently reverses the effects of lowering [Ca2+]e. These results suggest that the greater Ca2+ sensitivity of HyperKPP muscles primarily relates to (1) a greater effect of Ca2+ in depolarized fibers and (2) an increased proportion of depolarized HyperKPP muscle fibers compared with control muscle fibers, even at normal [K+]e.

Identification of therapeutics that target eEF1A2 and upregulate utrophin A translation in dystrophic muscles.

Up-regulation of utrophin in muscles represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy. We previously demonstrated that eEF1A2 associates with the 5'UTR of utrophin A to promote IRES-dependent translation. Here, we examine whether eEF1A2 directly regulates utrophin A expression and identify via an ELISA-based high-throughput screen, FDA-approved drugs that upregulate both eEF1A2 and utrophin A. Our results show that transient overexpression of eEF1A2 in mouse muscles causes an increase in IRES-mediated translation of utrophin A. Through the assessment of our screen, we reveal 7 classes of FDA-approved drugs that increase eEF1A2 and utrophin A protein levels. Treatment of mdx mice with the 2 top leads results in multiple improvements of the dystrophic phenotype. Here, we report that IRES-mediated translation of utrophin A via eEF1A2 is a critical mechanism of regulating utrophin A expression and reveal the potential of repurposed drugs for treating DMD via this pathway.

Motor transmission defects with sex differences in a new mouse model of mild spinal muscular atrophy.

BACKGROUND: Mouse models of mild spinal muscular atrophy (SMA) have been extremely challenging to generate. This paucity of model systems has limited our understanding of pathophysiological events in milder forms of the disease and of the effect of SMN depletion during aging. METHODS: A mild mouse model of SMA, termed Smn2B/-;SMN2+/-, was generated by crossing Smn-/-;SMN2 and Smn2B/2B mice. This new model was characterized using behavioral testing, histology, western blot, muscle-nerve electrophysiology as well as ultrasonography to study classical SMA features and extra-neuronal involvement. FINDINGS: Smn2B/-;SMN2+/- mice have normal survival, mild but sustained motor weakness, denervation and neuronal/neuromuscular junction (NMJ) transmission defects, and neurogenic muscle atrophy that are more prominent in male mice. Increased centrally located nuclei, intrinsic contractile and relaxation muscle defects were also identified in both female and male mice, with some male predominance. There was an absence of extra-neuronal pathology. INTERPRETATION: The Smn2B/-;SMN2+/- mouse provides a model of mild SMA, displaying some hallmark features including reduced weight, sustained motor weakness, electrophysiological transmission deficit, NMJ defects, and muscle atrophy. Early and prominent increase central nucleation and intrinsic electrophysiological deficits demonstrate the potential role played by muscle in SMA disease. The use of this model will allow for the understanding of the most susceptible pathogenic molecular changes in motor neurons and muscles, investigation of the effects of SMN depletion in aging, sex differences and most importantly will provide guidance for the currently aging SMA patients treated with the recently approved genetic therapies. FUNDING: This work was supported by Cure SMA/Families of SMA Canada (grant numbers KOT-1819 and KOT-2021); Muscular Dystrophy Association (USA) (grant number 575466); and Canadian Institutes of Health Research (CIHR) (grant number PJT-156379).

EGFR-Aurka Signaling Rescues Polarity and Regeneration Defects in Dystrophin-Deficient Muscle Stem Cells by Increasing Asymmetric Divisions.

Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Critical Assessment of the mdx Mouse with Ex Vivo Eccentric Contraction of the Diaphragm Muscle.

Muscle function and health progressively deteriorate during the progression of muscle dystrophies. The ability to objectively characterize muscle function and muscle damage is useful not only when comparing variants of dystrophy models, but also for characterizing the effects of interventions aiming to improve or halt the progressive decline of muscle function and muscle health. The protocols in this chapter describe the use of ex vivo eccentric contraction of the diaphragm muscle as a measure of muscle susceptibility to damage. Because muscle has a robust regenerative capacity, unhealthy muscle may be functionally close to normal; therefore, protocols for ex vivo characterization of muscle are often essential for assessing the effects of interventions. Additional methods that can be applied for assessment of dystrophic muscle are also highlighted. © 2018 by John Wiley & Sons, Inc.

Muscle-specific expression of the RNA-binding protein Staufen1 induces progressive skeletal muscle atrophy via regulation of phosphatase tensin homolog.

Converging lines of evidence have now highlighted the key role for post-transcriptional regulation in the neuromuscular system. In particular, several RNA-binding proteins are known to be misregulated in neuromuscular disorders including myotonic dystrophy type 1, spinal muscular atrophy and amyotrophic lateral sclerosis. In this study, we focused on the RNA-binding protein Staufen1, which assumes multiple functions in both skeletal muscle and neurons. Given our previous work that showed a marked increase in Staufen1 expression in various physiological and pathological conditions including denervated muscle, in embryonic and undifferentiated skeletal muscle, in rhabdomyosarcomas as well as in myotonic dystrophy type 1 muscle samples from both mouse models and humans, we investigated the impact of sustained Staufen1 expression in postnatal skeletal muscle. To this end, we generated a skeletal muscle-specific transgenic mouse model using the muscle creatine kinase promoter to drive tissue-specific expression of Staufen1. We report that sustained Staufen1 expression in postnatal skeletal muscle causes a myopathy characterized by significant morphological and functional deficits. These deficits are accompanied by a marked increase in the expression of several atrophy-associated genes and by the negative regulation of PI3K/AKT signaling. We also uncovered that Staufen1 mediates PTEN expression through indirect transcriptional and direct post-transcriptional events thereby providing the first evidence for Staufen1-regulated PTEN expression. Collectively, our data demonstrate that Staufen1 is a novel atrophy-associated gene, and highlight its potential as a biomarker and therapeutic target for neuromuscular disorders and conditions.

KATP channel deficiency in mouse FDB causes an impairment of energy metabolism during fatigue.

The skeletal muscle ATP-sensitive K+ (KATP) channel is crucial in preventing fiber damage and contractile dysfunction, possibly by preventing damaging ATP depletion. The objective of this study was to investigate changes in energy metabolism during fatigue in wild-type and inwardly rectifying K+ channel (Kir6.2)-deficient (Kir6.2-/-) flexor digitorum brevis (FDB), a muscle that lacks functional KATP channels. Fatigue was elicited with one tetanic contraction every second. Decreases in ATP and total adenylate levels were significantly greater in wild-type than Kir6.2-/- FDB during the last 2 min of the fatigue period. Glycogen depletion was greater in Kir6.2-/- FDB for the first 60 s, but not by the end of the fatigue period, while there was no difference in glucose uptake. The total amount of glucosyl units entering glycolysis was the same in wild-type and Kir6.2-/- FDB. During the first 60 s, Kir6.2-/- FDB generated less lactate and more CO2; in the last 120 s, Kir6.2-/- FDB stopped generating CO2 and produced more lactate. The ATP generated during fatigue from phosphocreatine, glycolysis (lactate), and oxidative phosphorylation (CO2) was 3.3-fold greater in Kir6.2-/- than wild-type FDB. Because ATP and total adenylate were significantly less in Kir6.2-/- FDB, it is suggested that Kir6.2-/- FDB has a greater energy deficit, despite a greater ATP production, which is further supported by greater glucose uptake and lactate and CO2 production in Kir6.2-/- FDB during the recovery period. It is thus concluded that a lack of functional KATP channels results in an impairment of energy metabolism.

Combinatorial therapeutic activation with heparin and AICAR stimulates additive effects on utrophin A expression in dystrophic muscles.

Upregulation of utrophin A is an attractive therapeutic strategy for treating Duchenne muscular dystrophy (DMD). Over the years, several studies revealed that utrophin A is regulated by multiple transcriptional and post-transcriptional mechanisms, and that pharmacological modulation of these pathways stimulates utrophin A expression in dystrophic muscle. In particular, we recently showed that activation of p38 signaling causes an increase in the levels of utrophin A mRNAs and protein by decreasing the functional availability of the destabilizing RNA-binding protein called K-homology splicing regulatory protein, thereby resulting in increases in the stability of existing mRNAs. Here, we treated 6-week-old mdx mice for 4 weeks with the clinically used anticoagulant drug heparin known to activate p38 mitogen-activated protein kinase, and determined the impact of this pharmacological intervention on the dystrophic phenotype. Our results show that heparin treatment of mdx mice caused a significant ∼1.5- to 3-fold increase in utrophin A expression in diaphragm, extensor digitorum longus and tibialis anterior (TA) muscles. In agreement with these findings, heparin-treated diaphragm and TA muscle fibers showed an accumulation of utrophin A and ß-dystroglycan along their sarcolemma and displayed improved morphology and structural integrity. Moreover, combinatorial drug treatment using both heparin and 5-amino-4-imidazolecarboxamide riboside (AICAR), the latter targeting 5' adenosine monophosphate-activated protein kinase and the transcriptional activation of utrophin A, caused an additive effect on utrophin A expression in dystrophic muscle. These findings establish that heparin is a relevant therapeutic agent for treating DMD, and illustrate that combinatorial treatment of heparin with AICAR may serve as an effective strategy to further increase utrophin A expression in dystrophic muscle via activation of distinct signaling pathways.

Understanding the physiology of the asymptomatic diaphragm of the M1592V hyperkalemic periodic paralysis mouse.

The diaphragm muscle of hyperkalemic periodic paralysis (HyperKPP) patients and of the M1592V HyperKPP mouse model rarely suffers from the myotonic and paralytic symptoms that occur in limb muscles. Enigmatically, HyperKPP diaphragm expresses the mutant NaV1.4 channel and, more importantly, has an abnormally high Na(+) influx similar to that in extensor digitorum longus (EDL) and soleus, two hindlimb muscles suffering from the robust HyperKPP abnormalities. The objective was to uncover the physiological mechanisms that render HyperKPP diaphragm asymptomatic. A first mechanism involves efficient maintenance of resting membrane polarization in HyperKPP diaphragm at various extracellular K(+) concentrations compared with larger membrane depolarizations in HyperKPP EDL and soleus. The improved resting membrane potential (EM) results from significantly increased Na(+) K(+) pump electrogenic activity, and not from an increased protein content. Action potential amplitude was greater in HyperKPP diaphragm than in HyperKPP soleus and EDL, providing a second mechanism for the asymptomatic behavior of the HyperKPP diaphragm. One suggested mechanism for the greater action potential amplitude is lower intracellular Na(+) concentration because of greater Na(+) K(+) pump activity, allowing better Na(+) current during the action potential depolarization phase. Finally, HyperKPP diaphragm had a greater capacity to generate force at depolarized EM compared with wild-type diaphragm. Action potential amplitude was not different between wild-type and HyperKPP diaphragm. There was also no evidence for an increased activity of the Na(+)-Ca(2+) exchanger working in the reverse mode in the HyperKPP diaphragm compared with the wild-type diaphragm. So, a third mechanism remains to be elucidated to fully understand how HyperKPP diaphragm generates more force compared with wild type. Although the mechanism for the greater force at depolarized resting EM remains to be determined, this study provides support for the modulation of the Na(+) K(+) pump as a component of therapy to alleviate weakness in HyperKPP.

Physiological basis for muscle stiffness and weakness in a knock-in M1592V mouse model of hyperkalemic periodic paralysis.

The mechanisms responsible for the onset and progressive worsening of episodic muscle stiffness and weakness in hyperkalemic periodic paralysis (HyperKPP) are not fully understood. Using a knock-in HyperKPP mouse model harboring the M1592V NaV1.4 channel mutant, we interrogated changes in physiological defects during the first year, including tetrodotoxin-sensitive Na(+) influx, hindlimb electromyographic (EMG) activity and immobility, muscle weakness induced by elevated [K(+)]e, myofiber-type composition, and myofiber damage. In situ EMG activity was greater in HyperKPP than wild-type gastrocnemius, whereas spontaneous muscle contractions were observed in vitro. We suggest that both the greater EMG activity and spontaneous contractions are related to periods of hyperexcitability during which fibers generate action potentials by themselves in the absence of any stimulation and that these periods are the cause of the muscle stiffness reported by patients. HyperKPP muscles had a greater sensitivity to the K(+)-induced force depression than wild-type muscles. So, an increased interstitial K(+) concentration locally near subsets of myofibers as a result of the hyperexcitability likely produced partial loss of force rather than complete paralysis. NaV1.4 channel protein content reached adult level by 3 weeks postnatal in both wild type and HyperKPP and apparent symptoms did not worsen after the first month of age suggesting (i) that the phenotypic behavior of M1592V HyperKPP muscles results from defective function of mutant NaV1.4 channels rather than other changes in protein expression after the first month and (ii) that the lag in onset during the first decade and the progression of human HyperKPP symptoms during adolescence are a function of NaV1.4 channel content.

Changes in myoplasmic Ca2+ during fatigue differ between FDB fibers, between glibenclamide-exposed and Kir6.2-/- fibers and are further modulated by verapamil.

One objective of this study was to document how individual FDB muscle fibers depend on the myoprotection of KATP channels during fatigue. Verapamil, a CaV1.1 channel blocker, prevents large increases in unstimulated force during fatigue in KATP-channel-deficient muscles. A second objective was to determine if verapamil reduces unstimulated [Ca(2+)]i in KATP-channel-deficient fibers. We measured changes in myoplasmic [Ca(2+)] ([Ca(2+)]i) using two KATP-channel-deficient models: (1) a pharmacological approach exposing fibers to glibenclamide, a channel blocker, and (2) a genetic approach using fibers from null mice for the Kir6.2 gene. Fatigue was elicited with one tetanic contraction every sec for 3 min. For all conditions, large differences in fatigue kinetics were observed from fibers which had greater tetanic [Ca(2+)]i at the end than at the beginning of fatigue to fibers which eventually completely failed to release Ca(2+) upon stimulation. Compared to control conditions, KATP-channel-deficient fibers had a greater proportion of fiber with large decreases in tetanic [Ca(2+)]i, fade and complete failure to release Ca(2+) upon stimulation. There was, however, a group of KATP-channel-deficient fibers that had similar fatigue kinetics to those of the most fatigue-resistant control fibers. For the first time, differences in fatigue kinetics were observed between Kir6.2(-/-) and glibenclamide-exposed muscle fibers. Verapamil significantly reduced unstimulated and tetanic [Ca(2+)]i. It is concluded that not all fibers are dependent on the myoprotection of KATP channels and that the decrease in unstimulated force by verapamil reported in a previous studies in glibenclamide-exposed fibers is due to a reduction in Ca(2+) load by reducing Ca(2+) influx through CaV1.1 channels between and during contractions.