Transport of Ca2+ and Ca2+-dependent permeability transition in heart mitochondria in the early stages of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive skeletal muscle disease that is associated with severe cardiac complications in the late stages. Significant mitochondrial dysfunction is reportedly responsible for the development of cardiomyopathy with age. At the same time, adaptive changes in mitochondrial metabolism in cardiomyocytes were identified in the early stages of DMD. In this work, we evaluate the functioning of calcium transport systems (MCU and NCLX), and MPT pore in the heart mitochondria of young dystrophin-deficient mice. As compared to wild-type animals, heart mitochondria of mdx mice have been found to be more efficient both in respect to Ca²⁺ uniport and Na⁺-dependent Ca²⁺ efflux. The data obtained indicate that the increased rate of Ca²⁺ uptake by heart mitochondria of mdx mice may be due to an increase in the ratio of MCU and MCUb subunits. In turn, an increase in the rate of Ca²⁺ efflux from organelles in DMD may be the result of a significant increase in the level of NCLX. Moreover, the heart mitochondria of mdx mice were more resistant to MPT pore opening, which may be due to an increase in the microviscosity of mitochondrial membranes of DMD mice. At the same time, the level of putative MPT pore proteins did not change. The paper discusses the effect of rearrangements of the mitochondrial proteome involved in the transport and accumulation of calcium on the adaptation of this organ to DMD.     

Age-dependent changes in diastolic Ca and Na concentrations in dystrophic cardiomyopathy: Role of Ca entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca²⁺ concentration ([Ca²⁺]_d) and diastolic Na⁺ concentration ([Na⁺]_d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd³⁺)-sensitive Ca²⁺ entry and inositol triphosphate (IP₃) signaling pathways in abnormal [Ca²⁺]_d and [Na⁺]_d were investigated. Our results showed an age-dependent increase in both [Ca²⁺]_d and [Na⁺]_d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd³⁺ treatment significantly reduced both [Ca²⁺]_d and [Na⁺]_d at all ages. In addition, blockade of the IP₃-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd³⁺ normalized both [Ca²⁺]_d and [Na⁺]_d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca²⁺ and Na⁺ overload mediated at least in part by enhanced Ca²⁺ entry through Gd³⁺ sensitive transient receptor potential channels (TRPC), and by IP₃ receptors.     

Nifedipine Treatment Reduces Resting Calcium Concentration,Oxidative and Apoptotic Gene Expression,and Improves Muscle Function in Dystrophic mdx Mice

Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca²⁺]_r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca²⁺]_r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca²⁺]_r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91^phox/p47^phox NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca²⁺]_r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD.     

Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition

Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca²⁺ homeostasis. This work examines the kinetic parameters of Ca²⁺ transport and the opening of the Ca²⁺-dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca²⁺ uniport, with the kinetics of Na⁺-dependent Ca²⁺ efflux not changing. The data obtained indicate that the decreased rate of Ca²⁺ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca²⁺ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca²⁺ homeostasis of skeletal-muscle mitochondria.     

Defects in Mitochondrial ATP Synthesis in Dystrophin-Deficient Mdx Skeletal Muscles May Be Caused by Complex I Insufficiency

Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca²⁺ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca²⁺ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca²⁺ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn) we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb’s cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca²⁺ load in comparison to controls, and 400 nM extramitochondrial Ca²⁺ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.     

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca²⁺ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca²⁺ deregulation and the Ca²⁺ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca²⁺ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca²⁺ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca²⁺ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca²⁺ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca²⁺ store contributes to the disrupted Ca²⁺ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.     

nNOS regulation of skeletal muscle fatigue and exercise performance

Neuronal nitric oxide synthases (nNOS) are Ca²⁺/calmodulin-activated enzymes that synthesize the gaseous messenger nitric oxide (NO). nNOSμ and the recently described nNOSβ, both spliced nNOS isoforms, are important enzymatic sources of NO in skeletal muscle, a tissue long considered to be a paradigmatic system for studying NO-dependent redox signaling. nNOS is indispensable for skeletal muscle integrity and contractile performance, and deregulation of nNOSμ signaling is a common pathogenic feature of many neuromuscular diseases. Recent evidence suggests that both nNOSμ and nNOSβ regulate skeletal muscle size, strength, and fatigue resistance, making them important players in exercise performance. nNOSμ acts as an activity sensor and appears to assist skeletal muscle adaptation to new functional demands, particularly those of endurance exercise. Prolonged inactivity leads to nNOS-mediated muscle atrophy through a FoxO-dependent pathway. nNOS also plays a role in modulating exercise performance in neuromuscular disease. In the mdx mouse model of Duchenne muscular dystrophy, defective nNOS signaling is thought to restrict contractile capacity of working muscle in two ways: loss of sarcolemmal nNOSμ causes excessive ischemic damage while residual cytosolic nNOSμ contributes to hypernitrosylation of the ryanodine receptor, causing pathogenic Ca²⁺ leak. This defect in Ca²⁺ handling promotes muscle damage, weakness, and fatigue. This review addresses these recent advances in the understanding of nNOS-dependent redox regulation of skeletal muscle function and exercise performance under physiological and neuromuscular disease conditions.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Amelioration of muscular dystrophy by transgenic expression of Niemann-Pick C1

Duchenne muscular dystrophy (DMD) and other types of muscular dystrophies are caused by the loss or alteration of different members of the dystrophin protein complex. Understanding the molecular mechanisms by which dystrophin-associated protein abnormalities contribute to the onset of muscular dystrophy may identify new therapeutic approaches to these human disorders. By examining gene expression alterations in mouse skeletal muscle lacking α-dystrobrevin (Dtna^−/−), we identified a highly significant reduction of the cholesterol trafficking protein, Niemann-Pick C1 (NPC1). Mutations in NPC1 cause a progressive neurodegenerative, lysosomal storage disorder. Transgenic expression of NPC1 in skeletal muscle ameliorates muscular dystrophy in the Dtna^−/− mouse (which has a relatively mild dystrophic phenotype) and in the mdx mouse, a model for DMD. These results identify a new compensatory gene for muscular dystrophy and reveal a potential new therapeutic target for DMD.     

Adeno-associated Virus Serotype 8 (AAV8) Delivery of Recombinant A20 to Skeletal Muscle Reduces Pathological Activation of Nuclear Factor (NF)-κB in Muscle of mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic muscle disease caused by the absence of a functional dystrophin protein. Lack of dystrophin protein disrupts the dystrophin-glycoprotein complex causing muscle membrane instability and degeneration. One of the secondary manifestations resulting from lack of functional dystrophin in muscle tissue is an increased level of cytokines that recruit inflammatory cells, leading to chronic upregulation of the nuclear factor (NF)-κB. Negative regulators of the classical NF-κB pathway improve muscle health in the mdx mouse model for DMD. We have previously shown in vitro that a negative regulator of the NF-κB pathway, A20, plays a role in muscle regeneration. Here, we show that overexpression of A20 by using a muscle-specific promoter delivered with an adeno-associated virus serotype 8 (AAV8) vector to the mdx mouse decreases activation of the NF-κB pathway in skeletal muscle. Recombinant A20 expression resulted in a reduction in number of fibers with centrally placed nuclei and a reduction in the number of T cells infiltrating muscle transduced with the AAV8–A20 vector. Taken together, we conclude that overexpression of A20 in mdx skeletal muscle provides improved muscle health by reduction of chronic inflammation and muscle degeneration. These results suggest A20 is a potential therapeutic target to ameliorate symptoms of DMD.     

Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity,Inflammation Response and Oxidative Stress

The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca²⁺]i. The mdx group showed significant increase in H₂O₂ production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.     

Chronic losartan administration reduces mortality and preserves cardiac but not skeletal muscle function in dystrophic mice

Duchenne muscular dystrophy (DMD) is a degenerative disorder affecting skeletal and cardiac muscle for which there is no effective therapy. Angiotension receptor blockade (ARB) has excellent therapeutic potential in DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6–9 months of therapy in the mdx mouse model of DMD. Since cardiac-related death is major cause of mortality in DMD, it is important to evaluate the effect of any novel treatment on the heart. Therefore, we evaluated the long-term impact of ARB on both the skeletal muscle and cardiac phenotype of the mdx mouse. Mdx mice received either losartan (0.6 g/L) (n = 8) or standard drinking water (n = 9) for two years, after which echocardiography was performed to assess cardiac function. Skeletal muscle weight, morphology, and function were assessed. Fibrosis was evaluated in the diaphragm and heart by Trichrome stain and by determination of tissue hydroxyproline content. By the study endpoint, 88% of treated mice were alive compared to only 44% of untreated (p = 0.05). No difference in skeletal muscle morphology, function, or fibrosis was noted in losartan-treated animals. Cardiac function was significantly preserved with losartan treatment, with a trend towards reduction in cardiac fibrosis. We saw no impact on the skeletal muscle disease progression, suggesting that other pathways that trigger fibrosis dominate over angiotensin II in skeletal muscle long term, unlike the situation in the heart. Our study suggests that ARB may be an important prophylactic treatment for DMD-associated cardiomyopathy, but will not impact skeletal muscle disease.     

Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7⁺ and Myf5⁻) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.KEY WORDS: Muscular dystrophy, Notch signaling, Stem cell     

Ca2+ Overload and Sarcoplasmic Reticulum Instability in tric-a Null Skeletal Muscle

The sarcoplasmic reticulum (SR) of skeletal muscle contains K⁺, Cl⁻, and H⁺ channels may facilitate charge neutralization during Ca²⁺ release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca²⁺ release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a^−/− skeletal muscle showed Ca²⁺ overload inside the SR with frequent formation of Ca²⁺ deposits compared with the wild type muscle. This elevated SR Ca²⁺ pool in the tric-a^−/− muscle could be released by caffeine, whereas the elemental Ca²⁺ release events, e.g. osmotic stress-induced Ca²⁺ spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of “alternan” behavior with isolated tric-a^−/− skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca^{2+ ATPase} function could lead to aggravation of the stress-induced alternans in the tric-a^−/− muscle. Our data suggests that absence of TRIC-A may lead to Ca²⁺ overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca²⁺ movement across the SR membrane. The observed alternan behavior with the tric-a^−/− muscle may reflect a skeletal muscle version of store overload-induced Ca²⁺ release that has been reported in the cardiac muscle under stress conditions.     

Inhibition of the angiotensin-converting enzyme decreases skeletal muscle fibrosis in dystrophic mice by a diminution in the expression and activity of connective tissue growth factor (CTGF/CCN-2)

The renin-angiotensin system (RAS), through angiotensin II and the angiotensin-converting enzyme (ACE), is involved in the genesis and progression of fibrotic diseases characterized by the replacement of normal tissue by an accumulation of an extracellular matrix (ECM). Duchenne muscular dystrophy (DMD) presents fibrosis and a decrease in muscle strength produced by chronic damage. The mdx mouse is a murine model of DMD and develops the same characteristics as dystrophic patients when subjected to chronic exercise. The connective tissue growth factor (CTGF/CCN2) and transforming growth factor type beta (TGF-β), which are overexpressed in muscular dystrophies, play a major role in many progressive scarring conditions. We have tested the hypothesis that ACE inhibition decreases fibrosis in dystrophic skeletal muscle by treatment of mdx mice with the ACE inhibitor enalapril. Both sedentary and exercised mdx mice treated with enalapril showed improvement in gastrocnemius muscle strength explained by a reduction in both muscle damage and ECM accumulation. ACE inhibition decreased CTGF expression in sedentary or exercised mdx mice and diminished CTGF-induced pro-fibrotic activity in a model of CTGF overexpression by adenoviral infection. Enalapril did not have an effect on TGF-β1 expression or its signaling activity in sedentary or exercised dystrophic mice. Thus, ACE inhibition might improve muscle strength and decrease fibrosis by diminishing specifically CTGF expression and activity without affecting TGF-β1 signaling. Our data provide insights into the pathogenic events in dystrophic muscle. We propose ACE as a target for developing therapies for DMD and related diseases.     

Na+ Dysregulation Coupled with Ca2+ Entry through NCX1 Promotes Muscular Dystrophy in Mice

Unregulated Ca²⁺ entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na⁺-Ca²⁺ exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd^−/−), Dysf^−/−, and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd^−/− mice. Measured increases in baseline Na⁺ and Ca²⁺ in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca²⁺ influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca²⁺ levels. Indeed, Atp1a2^+/− (encoding Na⁺-K⁺ ATPase α2) mice, which have reduced Na⁺ clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na⁺-K⁺ ATPase inhibitor digoxin. Treatment of Sgcd^−/− mice with ranolazine, a broadly acting Na⁺ channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology.     

Pathways of abnormal stress-induced Ca influx into dystrophic mdx cardiomyocytes

In Duchenne muscular dystrophy, deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle, but also to dilated cardiomyopathy, accounting for about 20% of the mortality. Mechanisms leading to cardiomyocyte cell death and cardiomyopathy are not well understood. One hypothesis suggests that the lack of dystrophin leads to membrane instability during mechanical stress and to activation of Ca²⁺ entry pathways. Using cardiomyocytes isolated from dystrophic mdx mice we dissected the contribution of various putative Ca²⁺ influx pathways with pharmacological tools. Cytosolic Ca²⁺ and Na⁺ signals as well as uptake of membrane impermeant compounds were monitored with fluorescent indicators using confocal microscopy and photometry. Membrane stress was applied as moderate osmotic challenges while membrane current was quantified using the whole-cell patch-clamp technique. Our findings suggest a major contribution of two primary Ca²⁺ influx pathways, stretch-activated membrane channels and short-lived microruptures. Furthermore, we found evidence for a secondary Ca²⁺ influx pathway, the Na⁺–Ca²⁺ exchange (NCX), which in cardiac muscle has a large transport capacity. After stress it contributes to Ca²⁺ entry in exchange for Na⁺ which had previously entered via primary stress-induced pathways, representing a previously not recognized mechanism contributing to subsequent cellular damage. This complexity needs to be considered when targeting abnormal Ca²⁺ influx as a treatment option for dystrophy.     

The effects of mesenchymal stem cells on c-kit up-regulation and cell-cycle re-entry of neonatal cardiomyocytes are mediated by activation of insulin-like growth factor 1 receptor

Canonical-type transient receptor potential cation channel type 3 (TRPC3) allows the entry of extracellular Ca²⁺ and Na⁺ into various cells. In mouse skeletal myotubes, functional interaction between TRPC3 and RyR1 (ryanodine receptor type 1/Ca²⁺-release channel on sarcoplasmic reticulum membrane) regulates the gain of excitation–contraction coupling. Junctophilin-2 (JP2) is a TRPC3-interacting protein in mouse skeletal myotubes. Based on these knowledge from bona-fide TRPC3-expressing cells, to identify critical binding region(s) of JP2 that participate in binding to TRPC3, various JP2 portions were subjected to co-immunoprecipitation assay with intact TRPC3 from rabbit skeletal muscle. A region covering 143 to 234 amino acids of JP2 (F1-2) was the most efficient portion binding to TRPC3. Through mutational studies, we found that the binding ability of JP2 to TRPC3 was mainly due to glutamate in the F1-2 region (E227). This substantial binding between JP2 and TRPC3 suggests that JP2 can be a regulatory protein of TRPC3 and/or TRPC3-mediated Ca²⁺ homeostasis in skeletal muscle.     

ACE2 Is Augmented in Dystrophic Skeletal Muscle and Plays a Role in Decreasing Associated Fibrosis

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype.     

Phospholipase A2-derived lysophosphatidylcholine triggers Ca entry in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca²⁺ entry through sarcolemmal cationic channels. Using the Mn²⁺ quench method, we show here that Mn²⁺ entry triggered by Ca²⁺ store depletion but not basal Mn²⁺ entry relies on Ca²⁺-independent PLA₂ (iPLA₂) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx^5cv mouse. iPLA₂ was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA₂ lipid product lysophosphatidylcholine was found to trigger Ca²⁺ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA₂ and lysophospholipid production may be of interest to reduce Ca²⁺ entry and subsequent degeneration of dystrophic muscle.