Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca²⁺ dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca²⁺ depending on cellular activity. Resting intracellular calcium ([Ca²⁺]_r) and sodium ([Na⁺]_r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na⁺]_e elevates [Ca²⁺]_r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca²⁺ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca²⁺]_r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca²⁺]_r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca²⁺]_r in MHS muscle fibers and decreases the amplitude of [Ca²⁺]_r rise triggered by halothane, but had no effect on [Ca²⁺]_r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca²⁺ transient elicited by high [K⁺]_e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca²⁺]_r and the Ca²⁺ transient area induced by high [K⁺]_e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca²⁺ transients associated with K⁺-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.     

IP3 receptor blockade restores autophagy and mitochondrial function in skeletal muscle fibers of dystrophic mice

Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive destruction of muscle fibers associated with altered Ca²⁺ homeostasis. We have previously shown that the IP₃ receptor (IP₃R) plays a role in elevating basal cytoplasmic Ca²⁺ and that pharmacological blockade of IP₃R restores muscle function. Moreover, we have shown that the IP₃R pathway negatively regulates autophagy by controlling mitochondrial Ca²⁺ levels. Nevertheless, it remains unclear whether IP₃R is involved in abnormal mitochondrial Ca²⁺ levels, mitochondrial dynamics, or autophagy and mitophagy observed in adult DMD skeletal muscle. Here, we show that the elevated basal autophagy and autophagic flux levels were normalized when IP₃R was downregulated in mdx fibers. Pharmacological blockade of IP₃R in mdx fibers restored both increased mitochondrial Ca²⁺ levels and mitochondrial membrane potential under resting conditions. Interestingly, mdx mitochondria changed from a fission to an elongated state after IP₃R knockdown, and the elevated mitophagy levels in mdx fibers were normalized. To our knowledge, this is the first study associating IP₃R1 activity with changes in autophagy, mitochondrial Ca²⁺ levels, mitochondrial membrane potential, mitochondrial dynamics, and mitophagy in adult mouse skeletal muscle. Moreover, these results suggest that increased IP₃R activity in mdx fibers plays an important role in the pathophysiology of DMD. Overall, these results lead us to propose the use of specific IP₃R blockers as a new pharmacological treatment for DMD, given their ability to restore both autophagy/mitophagy and mitochondrial function.     

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).     

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.     

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.     

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.     

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.     

Mutation Types and Aging Differently Affect Revertant Fiber Expansion in Dystrophic Mdx and Mdx52 Mice

Duchenne muscular dystrophy (DMD), one of the most common and lethal genetic disorders, and the mdx mouse myopathies are caused by a lack of dystrophin protein. These dystrophic muscles contain sporadic clusters of dystrophin-expressing revertant fibers (RFs), as detected by immunohistochemistry. RFs are known to arise from muscle precursor cells with spontaneous exon skipping (alternative splicing) and clonally expand in size with increasing age through the process of muscle degeneration/regeneration. The expansion of revertant clusters is thought to represent the cumulative history of muscle regeneration and proliferation of such precursor cells. However, the precise mechanisms by which RFs arise and expand are poorly understood. Here, to test the effects of mutation types and aging on RF expansion and muscle regeneration, we examined the number of RFs in mdx mice (containing a nonsense mutation in exon 23) and mdx52 mice (containing deletion mutation of exon 52) with the same C57BL/6 background at 2, 6, 12, and 18months of age. Mdx mice displayed a significantly higher number of RFs compared to mdx52 mice in all age groups, suggesting that revertant fiber expansion largely depends on the type of mutation and/or location in the gene. A significant increase in the expression and clustering levels of RFs was found beginning at 6months of age in mdx mice compared with mdx52 mice. In contrast to the significant expansion of RFs with increasing age, the number of centrally nucleated fibers and embryonic myosin heavy chain-positive fibers (indicative of cumulative and current muscle regeneration, respectively) decreased with age in both mouse strains. These results suggest that mutation types and aging differently affect revertant fiber expansion in mdx and mdx52 mice.     

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.     

Effect of Adenosine and Intracellular GTP on KATP Channels of Mammalian Skeletal Muscle

We investigated the action of adenosine and GTP on K_ATP channels, using inside-out patch clamp recordings from dissociated single fibers of rat flexor digitorum brevis (FDB) skeletal muscle. In excised patches, K_ATP channels could be activated by a combination of an extracellular adenosine agonist and intracellular Mg²⁺-ATP and GTP or GTP-γ-S. The activation required hydrolyzable ATP and could be partially reversed with Mg²⁺, suggesting that it may involve a G-protein dependent phosphorylation of K_ATP channels. We found that K_ATP channels of the rat FDB could not be activated by Mg²⁺-ATP alone or by Mg²⁺-ATP in the presence of extracellular adenosine. Patches whose channel activity had been `rundown' by Ca²⁺ could not be recovered by adenosine, GTP or Mg²⁺-ATP. K_ATP channels activated by adenosine receptor agonists had a similar ATP sensitivity to those under control conditions; but adenosine appears to be able to switch these K_ATP channels from an inactive to an active mode. Received: 29 December 1995/Revised: 22 March 1996     

Whole Body Periodic Acceleration Is an Effective Therapy to Ameliorate Muscular Dystrophy in mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca²⁺ and Na⁺ overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca²⁺ and Na⁺ overload, diminished abnormal sarcolemmal Ca²⁺ entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.     

Characterization of Dystrophin Deficient Rats: A New Model for Duchenne Muscular Dystrophy

A few animal models of Duchenne muscular dystrophy (DMD) are available, large ones such as pigs or dogs being expensive and difficult to handle. Mdx (X-linked muscular dystrophy) mice only partially mimic the human disease, with limited chronic muscular lesions and muscle weakness. Their small size also imposes limitations on analyses. A rat model could represent a useful alternative since rats are small animals but 10 times bigger than mice and could better reflect the lesions and functional abnormalities observed in DMD patients. Two lines of Dmd mutated-rats (Dmd^mdx) were generated using TALENs targeting exon 23. Muscles of animals of both lines showed undetectable levels of dystrophin by western blot and less than 5% of dystrophin positive fibers by immunohistochemistry. At 3 months, limb and diaphragm muscles from Dmd^mdx rats displayed severe necrosis and regeneration. At 7 months, these muscles also showed severe fibrosis and some adipose tissue infiltration. Dmd^mdx rats showed significant reduction in muscle strength and a decrease in spontaneous motor activity. Furthermore, heart morphology was indicative of dilated cardiomyopathy associated histologically with necrotic and fibrotic changes. Echocardiography showed significant concentric remodeling and alteration of diastolic function. In conclusion, Dmd^mdx rats represent a new faithful small animal model of DMD.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (R _LIS), phase I response, followed by an increase in tight junctional resistance (R _TJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC₅₀ of 6 μM). The objective of the study was to determine the degree to which the changes in [Ca²⁺]_i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La³⁺, and Mn²⁺ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease inR _LIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La³⁺ also inhibited the ATP-induced phase II response, but Mn²⁺ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase inR _TJ. KCl-induced membrane depolarization stimulated an acute decrease inR _LIS and a late increase inR _TJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca²⁺]_i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases inR _TJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic ATP-like changes inR _TE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease inR _LIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase inR _TJ.     

Reduction of blood pressure by store‐operated calcium channel blockers

The voltage‐operated Ca²⁺ channels (VOCC), which allow Ca²⁺ influx from the extracellular space, are inhibited by anti‐hypertensive agents such as verapamil and nifedipine. The Ca²⁺ entering from outside into the cell triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca²⁺ stores in the SR, another type of Ca²⁺ channels in the cell membrane, known as store‐operated Ca²⁺ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd³⁺) sensitive. Both SK and Gd³⁺ have been shown to reduce [Ca²⁺]_i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd³⁺ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd³⁺ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca²⁺]_i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca²⁺]_i. On the other hand, Gd³⁺ inhibited the LPA and Ang II induced change in [Ca²⁺]_i, but had no effect on the ET‐1 and NE induced increase in [Ca²⁺]_i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca²⁺]_i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.     

Dystrophic mdx mouse myoblasts exhibit elevated ATP/UTP-evoked metabotropic purinergic responses and alterations in calcium signalling

Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive wasting of muscle fibres is a cause of muscle deterioration, there is a growing body of evidence that the triggering effects of DMD mutation are present at the earlier stage of muscle development and affect myogenic cells. Among these abnormalities, elevated activity of P2X7 receptors and increased store-operated calcium entry myoblasts have been identified in mdx mouse. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies linked these elevated purinergic responses to the increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. However, the ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation confirmed at the protein level, including a decrease of Gq11 subunit α, plasma membrane calcium ATP-ase, inositol-2,4,5-trisphosphate-receptor proteins and elevation of phospholipase Cβ, sarco-endoplamatic reticulum calcium ATP-ase and sodium‑calcium exchanger. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular nucleotides is determined by particular receptor overexpression, the intensity of such altered response depends on relative activities of downstream calcium regulators that are also affected by Dmd mutations. Furthermore, these phenotypic effects of DMD emerge as early as in undifferentiated muscle. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.     

The weak combined magnetic field,tuned to ion parametric resonance for Ca<Superscript>2+</Superscript>, stimulates dystrophin synthesis in the skeletal muscles of mdx mice subjected to cell therapy

The possibility of using bone marrow stem cells for treatment of Duchenne muscular dystrophy is intensely studied. Mdx mice are the most widely used laboratory model of Duchenne muscular dystrophy. One approach of cell therapy of muscular dystrophy is substitution of bone marrow in mdx mice after their X-ray irradiation. However, this method does not allow one to increase significantly dystrophin synthesis in muscular fibers of mdx mice. To improve the effect of transplanted cells on muscle regeneration, we additionally treated mdx mice subjected to transplantation of bone marrow cells with a weak combined magnetic field tuned to ion parametric resonance for Ca²⁺ (Ca²⁺-CMF). We found that, in irradiated chimeric 3 and 5 Gy mdx mice, additional treatment with Ca²⁺-CMF for 1 month resulted in significant increases in the portions of dystrophin-positive muscle fibers, by 15.8 and 18.3%, respectively, as compared to the control groups. Furthermore, the share of muscle fibers without centrally located nuclei also increased. We suggest that the magnetic field with these parameters may stimulate functioning of nuclei of donor cells, which were incorporated into muscle fibers.     

Unloaded speed of shortening in voltage-clamped intact skeletal muscle fibers from wt, mdx, and transgenic minidystrophin mice using a novel high-speed acquisition system

Skeletal muscle unloaded shortening has been indirectly determined in the past. Here, we present a novel high-speed optical tracking technique that allows recording of unloaded shortening in single intact, voltage-clamped mammalian skeletal muscle fibers with 2-ms time resolution. L-type Ca²⁺ currents were simultaneously recorded. The time course of shortening was biexponential: a fast initial phase, τ₁, and a slower successive phase, τ_2, with activation energies of 59 kJ/mol and 47 kJ/mol. Maximum unloaded shortening speed, v_u,max, was faster than that derived using other techniques, e.g., ∼14.0 L₀ s⁻¹ at 30°C. Our technique also allowed direct determination of shortening acceleration. We applied our technique to single fibers from C57 wild-type, dystrophic mdx, and minidystrophin-expressing mice to test whether unloaded shortening was affected in the pathophysiological mechanism of Duchenne muscular dystrophy. v_u,max and a_u,max values were not significantly different in the three strains, whereas τ₁ and τ₂ were increased in mdx fibers. The results were complemented by myosin heavy and light chain (MLC) determinations that showed the same myosin heavy chain IIA profiles in the interossei muscles from the different strains. In mdx muscle, MLC-1f was significantly increased and MLC-2f and MLC-3f somewhat reduced. Fast initial active shortening seems almost unaffected in mdx muscle.     

Allele-Specific Gene Silencing in Two Mouse Models of Autosomal Dominant Skeletal Myopathy

We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4–6 month old heterozygous Ryr1^Y524S/+ (YS/+) and Ryr1^I4895T/+ (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively. Treatment of IT/+ mice resulted in a modest rescue of deficits in the maximum rate (∼38% rescue) and magnitude (∼78%) of ligand-induced Ca²⁺ release that occurred in the absence of a change in the magnitude of electrically-evoked Ca²⁺ release. Compared to the difference between the caffeine sensitivity of Ca²⁺ release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC₅₀: 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC₅₀: 2.8 mM) and 4 week (EC₅₀: 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca²⁺ observed in FDB fibers from YS/+ mice was normalized to WT levels after 2 weeks of treatment with YS allele-specific siRNA. As determined by quantitative real time PCR, the degree of functional rescue in YS/+ and IT/+ mice correlated well with the relative increase in fractional WT allele expression.