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The rapid cooling (RC) response in muscle is an increase in cytoplasmic [Ca.sup.2+] concentration ([[Ca..sup.2+]].sub.i]) that is probably caused by [Ca.sup.2+] release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR [Ca.sup.2+] release channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and RyR3). In the current investigation, the RC response was studied in RyR-null muscle cells (1B5) before and after transduction with HSV-1 virions containing the cDNAs encoding for RyR1, RyR2, or RyR3. Cells were loaded with fluo 4-AM to monitor changes in [[Ca.sup.2+]].sub.i] and perfused with either cold (~0[degrees]C), room temperature (RT), or RT buffer containing 40 mM caffeine. Control cells showed no significant response to cold or caffeine, whereas robust [Ca.sup.2+] transients were recorded in response to both RC and caffeine in transduced cells expressing any one of the three RyR isoforms. Our data demonstrate directly that RyRs are responsible for the RC response and that all three isoforms respond in a similar manner. [Ca.sup.2+] release from RyRs is likely caused by a RC-induced conformational change of the channel from the closed to the open state. calcium release channel; sarcoplasmic reticulum; excitation-contraction coupling

Differentiated primary myotubes isolated from wild-type mice exhibit ryanodine-sensitive, spontaneous global [Ca.sup.2+] oscillations as well as spontaneous depolarizations in the plasma membrane. Immunolabeling of these myotubes showed expression of both [[alpha].sub.1s] dihydropyridine receptors (DHPRs) and ryanodine-sensitive [Ca.sup.2+]-release channel 1 (RyR1), the two key proteins in skeletal excitation-contraction (E-C) coupling. Spontaneous global [Ca.sup.2+] oscillations could be inhibited by addition of 0.1 mM Cd[Cl.sub.2]/0.5 mM La[Cl.sub.3] or 5 [micro]M nifedipine to the extracellular bathing solution. After either treatment, [Ca.sup.2+] oscillations could be restored upon extensive washing. Although exposure to DHPR antagonists completely blocked [Ca.sup.2+] oscillations, normal orthograde signaling between DHPRs and RyRs, such as that elicited by 80 mM KC1 depolarization, was still observed. In addition, we showed that spontaneous [Ca.sup.2+] oscillations were never present in cultured mdg myotubes, which lack the expression of [sub.[[alpha].sub.1s]]DHPRs. These results suggest that under physiological conditions in conjunction with the mechanical coupling between the [sub.[[alpha].sub.1s]]DHPRs and RyR1, the initiation of [Ca.sup.2+] oscillations in myotubes may be facilitated, in part, by the [Ca.sup.2+] influx through the [[alpha].sub.1s]-subunit of the DHPR. calcium-induced calcium release; dihydropyridine receptors; excitation-contraction coupling; ryanodine receptors; skeletal muscle

DP4 is a 36-residue synthetic peptide that corresponds to the [Leu.sup.2442]-[Pro.sup.2477] region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the [Ca.sup.2+] release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618-11625). We have investigated the effects of DP4 on local SR [Ca.sup.2+] release events ([Ca.sup.2+] sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR [Ca.sup.2+] release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in [Ca.sup.2+] spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the [Ca.sup.2+] sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR [Ca.sup.2+] release channel(s) generating the sparks. DP4 also increased [[sup.3]H] ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR [Ca.sup.2+] release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in [Ca.sup.2+] spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an [Arg.sup.17] to [Cys.sup.7] replacement (DP4mut), which corresponds to the [Arg.sup.2458]-to-[Cys.sup.2458] mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. [Mg.sup.2+] dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially [Mg.sup.2+]-free RyR(s), thus promoting channel opening and production of [Ca.sup.2+] sparks. KEY WORDS: E-C coupling * [Ca.sup.2+] release channel * domain peptides * [Ca.sup.2+]-induced [Ca.sup.2+] release * sarcoplasmic reticulum

It is unknown whether loss of skeletal muscle mass and function experienced by astronauts during space flight could be augmented by ionizing radiation (IR), such as low-dose high-charge and energy (HZE) particles or low-dose high-energy proton radiation. In the current study adult mice were irradiated whole-body with either a single dose of 15 cGy of 1 GeV/n 56Fe-particle or with a 90 cGy proton of 1 GeV/n proton particles. Both ionizing radiation types caused alterations in the skeletal muscle cytoplasmic Ca2+ ([Ca2+] i) homeostasis. 56Fe-particle irradiation also caused a reduction of depolarization-evoked Ca2+ release from the sarcoplasmic reticulum (SR). The increase in the [Ca2+]i was detected as early as 24 h after 56Fe-particle irradiation, while effects of proton irradiation were only evident at 72 h. In both instances [Ca2+]i returned to baseline at day 7 after irradiation. All 56Fe-particle irradiated samples revealed a significant number of centrally localized nuclei, a histologic manifestation of regenerating muscle, 7 days after irradiation. Neither unirradiated control or proton-irradiated samples exhibited such a phenotype. Protein analysis revealed significant increase in the phosphorylation of Akt, Erk1/2 and rpS6k on day 7 in 56Fe-particle irradiated skeletal muscle, but not proton or unirradiated skeletal muscle, suggesting activation of pro-survival signaling. Our findings suggest that a single low-dose 56Fe-particle or proton exposure is sufficient to affect Ca2+ homeostasis in skeletal muscle. However, only 56Fe-particle irradiation led to the appearance of central nuclei and activation of pro-survival pathways, suggesting an ongoing muscle damage/recovery process. [ABSTRACT FROM AUTHOR]

Abstract: Loss-of-function mutations in DJ−1 are associated with early-onset of Parkinson''s disease. Although DJ−1 is ubiquitously expressed, the functional pathways affected by it remain unresolved. Here we demonstrate an involvement of DJ−1 in the regulation of Ca2+ homeostasis in mouse skeletal muscle. Using enzymatically dissociated flexor digitorum brevis muscle fibers from wild-type (wt) and DJ−1 null mice, we examined the effects of DJ−1 protein on resting, cytoplasmic [Ca2+] ([Ca2+] i ) and depolarization-evoked Ca2+ release in the mouse skeletal muscle. The loss of DJ−1 resulted in a more than two-fold increase in resting [Ca2+] i . While there was no alteration in the resting membrane potential, there was a significant decrease in depolarization-evoked Ca2+ release from the sarcoplasmic reticulum in the DJ−1 null muscle cells. Consistent with the role of DJ−1 in oxidative stress regulation and mitochondrial functional maintenance, treatments of DJ−1 null muscle cells with resveratrol, a mitochondrial activator, or glutathione, a potent antioxidant, reversed the effects of the loss of DJ−1 on Ca2+ homeostasis. These results provide evidence of DJ−1''s association with Ca2+ regulatory pathways in mouse skeletal muscle, and suggest the potential benefit of resveratrol to functionally compensate for the loss of DJ−1. [Copyright &y& Elsevier]

Abstract: Inclusion body myositis (IBM), the most common muscle disorder in the elderly, is partly characterized by dysregulation of β-amyloid precursor protein (βAPP) expression and abnormal, intracellular accumulation of full-length βAPP and β-amyloid epitopes. The present study examined the effects of β-amyloid accumulation on force generation and Ca2+ release in skeletal muscle from transgenic mice harboring human βAPP and assessed the consequence of Aβ1–42 modulation of the ryanodine receptor Ca2+ release channels (RyRs). β-Amyloid laden muscle produced less peak force and exhibited Ca2+ transients with smaller amplitude. To determine whether modification of RyRs by β-amyloid underlie the effects observed in muscle, in vitro Ca2+ release assays and RyR reconstituted in planar lipid bilayer experiments were conducted in the presence of Aβ1–42. Application of Aβ1–42 to RyRs in bilayers resulted in an increased channel open probability and changes in gating kinetics, while addition of Aβ1–42 to the rabbit SR vesicles resulted in RyR-mediated Ca2+ release. These data may relate altered βAPP metabolism in IBM to reductions in RyR-mediated Ca2+ release and muscle contractility. [ABSTRACT FROM AUTHOR]

Intramyofiber accumulation of β-amyloid fragments (Aβ) is a pathologic hallmark of inclusion-body myositis (IBM), a progressive skeletal muscle disorder. We investigated the temporal pattern of alterations in the resting cytoplasmic [Ca2+] ([Ca2+]i) as well as the depolarization-evoked Ca2+ release from the sarcoplasmic reticulum in skeletal muscle from transgenic mice expressing human βAPP (MCK-βAPP). MCK-βAPP mice show an age-dependent increase in [Ca2+]i along with a reduction in depolarization-evoked Ca2+ release, which appear well before the other reported aspects of IBM, such as inclusion formation, inflammation, centralized nuclei, atrophy, and skeletal muscle weakness. In the young MCK-βAPP animals the increase in resting [Ca2+]i can be attributed largely to Ca2+ influx through nifedipine-sensitive Ca2+ channels. In the adult MCK-βAPP mice, in addition to the nifedipine-sensitive pathway, there is also a substantial contribution by the intracellular compartments to the increase in [Ca2+]i. These results suggest that β-amyloid-induced disuption of Ca2+ handling may represent an early event in the pathogenesis of IBM. Muscle Nerve, 2010 [ABSTRACT FROM AUTHOR]

Neurodegeneration in Alzheimer’s disease (AD) has been linked to intracellular accumulation of misfolded proteins and dysregulation of intracellular Ca2+. In the current work, we determined the contribution of specific Ca2+ pathways to an alteration in Ca2+ homeostasis in primary cortical neurons from an adult triple transgenic (3xTg-AD) mouse model of AD that exhibits intraneuronal accumulation of β-amyloid proteins. Resting free Ca2+ concentration ([Ca2+]i), as measured with Ca2+-selective microelectrodes, was greatly elevated in neurons from 3xTg-AD and APPSWE mouse strains when compared with their respective non-transgenic neurons, while there was no alteration in the resting membrane potential. In the absence of the extracellular Ca2+, the [Ca2+]i returned to near normal levels in 3xTg-AD neurons, demonstrating that extracellular Ca2+contributed to elevated [Ca2+]i. Application of nifedipine, or a non-L-type channel blocker, SKF-96365, partially reduced [Ca2+]i. Blocking the ryanodine receptors, with ryanodine or FLA-365 had no effect, suggesting that these channels do not contribute to the elevated [Ca2+]i. Conversely, inhibition of inositol trisphosphate receptors with xestospongin C produced a partial reduction in [Ca2+]i. These results demonstrate that an elevation in resting [Ca2+]i, contributed by aberrant Ca2+entry and release pathways, should be considered a major component of the abnormal Ca2+ homeostasis associated with AD. [ABSTRACT FROM AUTHOR]

Background: Patients with chronic renal failure may develop muscle weakness and fatigability due to disorders of skeletal muscle function, collectively known as the uremic myopathy. Cyclic adenosine diphosphate-ribose (cADPR), an endogenous metabolite of β-NAD+ activates Ca2+ release from intracellular stores in vertebrate and invertebrate cells. The current study investigated the possible role of cADPR in uremic myopathy. Methods: We have examined the effect of cADPR on myoplasmic resting Ca2+ concentration ([Ca2+]1) in skeletal muscle obtained from control subjects and uremic patients (UP). [Ca2+]1 was measured using double-barreled Ca2+-selective microelectrodes in muscle fibers, prior to and after microinjections of cADPR. Results: Resting [Ca2+]1 was elevated in UP fibers compared with fibers obtained from control subjects. Removal of extracellular Ca2+ or incubation of cells with nifedipine, did not modify [Ca2+]1 in UP or control fibers. Microinjection of cADPR produced an elevation of [Ca2+]1 in both groups of cells. This elevation was not mediated by Ca2+ influx, or inhibited by heparin or ryanodine. [cADPR]1 was determined to be higher in muscle fibers from UP compared to those from the control subjects. Incubation of cells with 8-bromo- cADPR, a cADPR antagonist, partially reduced [Ca2+]1 in UP muscle fibers and blocked the cADPR-elicited elevation in [Ca2+]1 in both groups of muscle cells. Conclusion: Skeletal muscles of the UP exhibit chronic elevation of [Ca2+]1 that can be partially reduced by application of 8- bromo-cADPR. cADPR was able to mobilize Ca2+ from intracellular stores, by a mechanism that is independent of ryanodine or inositol trisphosphate receptors. It can be postulated that an alteration in the cADPR-signaling pathway may exist in skeletal muscle of the patients suffering from uremic myopathy. [ABSTRACT FROM AUTHOR]

The relative scarcity of inclusion-affected muscle cells or markers of cell death in inclusion body myositis (IBM) is in distinction to the specific and early intracellular deposition of several Alzheimer's Disease (AD)-related proteins. The current study examined the possible correlation between myotube β-amyloid and/or Tau accumulations and a widespread mishandling of intracellular muscle calcium concentration that could potentially account for the unrelenting weakness in affected patients. Cultured myogenic cells (C2C12) expressed β-amyloid-42 (Aβ42) and fetal Tau peptides, as human transgenes encoded by herpes simplex virus, either individually or concurrently. Co-expression of Aβ42 in C2C12 myotubes resulted in hyperphosphorylation of Tau protein that was not observed when Tau was expressed alone. Resting calcium concentration and agonist-induced RyR-mediated Ca2+ release were examined using calcium-specific microelectrodes and FLuo-4 epifluorescence, respectively. Co-expression of Aβ42 and Tau cooperatively elevated basal levels of myoplasmic-free calcium, an effect that was accompanied by depolarization of the plasma membrane. Sareoplasmic reticulum (SR) calcium release, induced by KCl depolarization, was not affected by Aβ42 or Tau. In contrast, expression of Aβ42, Tau, or Aβ42 together with Tau resulted in enhanced sensitivity of ryanodine receptors to activation by caffeine. Notably, expression of β-amyloid, alone, was sufficient to result in an increased sensitivity to direct activation by caffeine. Current results indicate that amyloid proteins cooperate to raise resting calcium levels and that these effects are associated with a passive SR Ca2+ leak and Tau hyperphosphorylation in skeletal muscle. [ABSTRACT FROM AUTHOR]

Differentiated primary myotubes isolated from wild-type mice exhibit ryanodine sensitive, spontaneous global Ca[sup 2+] oscillations as well as spontaneous depolarizations in the plasma membrane. Immunolabeling of these myotubes showed expression of both Otis dihydropyridine receptors (DHPRs) and ryanodine-sensitive Ca[sup 2+]-release channel 1 (RyR1), the two key proteins in skeletal excitation-contraction (E-C) coupling. Spontaneous global Ca[sup 2+] oscillations could be inhibited by addition of 0.1 mM CdCl[sub 2]/0.5 mM LaCl[sub 3] or 5 µM nifedipine to the extracellular bathing solution. After either treatment, Ca[sup 2+] oscillations could be restored upon extensive washing. Although exposure to DHPR antagonists completely blocked Ca[sup 2+] oscillations, normal orthograde signaling between DHPRs and RyRs, such as that elicited by 80 mM KCl depolarization, was still observed. In addition, we showed that spontaneous Ca[sup 2+] oscillations were never present in cultured mdg myotubes, which lack the expression of α[sub 1S]DHPRs. These results suggest that under physiological conditions in conjunction with the mechanical coupling between the α[sub 1S]DHPRs and RyR1, the initiation of Ca[sup 2+] oscillations in myotubes may be facilitated, in part, by the Ca[sup 2+] influx through the α[sub 1s]-subunit of the DHPR. [ABSTRACT FROM AUTHOR]

Investigates the effects of domain peptide-4 (DP4) on local sarcoplasmic reticulum Calciumsup2+ release events in saponin-permeabilized frog skeletal muscle fibers. Concentration dependence of DP4 effects in muscle fibers; Modulation of DP4 effects in muscle fibers; Reaction scheme and molecular model for Calciumsup2+ spark activation with and without DP4.

The article presents a study which examined how the transgenic expression of β-APP in fast-twitch skeletal muscle leads to calcium dyshomeostasis and inclusion body myositis (IBM)-like pathology. A recognized pathological feature of human IBM is the intracellular deposition of the β-amyloid peptide in skeletal muscle fibers. Transgene integration was analyzed in total DNA extracted from distal tail samples through genomic Southern blot hybridizations and polymerase chain reaction.

Background: Patients with chronic renal failure may develop muscle weakness and fatigability due to disorders of skeletal muscle function, collectively known as the uremic myopathy. Cyclic adenosine diphosphate-ribose (cADPR), an endogenous metabolite of β-NAD+ activates Ca2+ release from intracellular stores in vertebrate and invertebrate cells. The current study investigated the possible role of cADPR in uremic myopathy. Methods: We have examined the effect of cADPR on myoplasmic resting Ca2+ concentration ([Ca2+]1) in skeletal muscle obtained from control subjects and uremic patients (UP). [Ca2+]1 was measured using double-barreled Ca2+-selective microelectrodes in muscle fibers, prior to and after microinjections of cADPR. Results: Resting [Ca2+]1 was elevated in UP fibers compared with fibers obtained from control subjects. Removal of extracellular Ca2+ or incubation of cells with nifedipine, did not modify [Ca2+]1 in UP or control fibers. Microinjection of cADPR produced an elevation of [Ca2+]1 in both groups of cells. This elevation was not mediated by Ca2+ influx, or inhibited by heparin or ryanodine. [cADPR]1 was determined to be higher in muscle fibers from UP compared to those from the control subjects. Incubation of cells with 8-bromo- cADPR, a cADPR antagonist, partially reduced [Ca2+]1 in UP muscle fibers and blocked the cADPR-elicited elevation in [Ca2+]1 in both groups of muscle cells. Conclusion: Skeletal muscles of the UP exhibit chronic elevation of [Ca2+]1 that can be partially reduced by application of 8- bromo-cADPR. cADPR was able to mobilize Ca2+ from intracellular stores, by a mechanism that is independent of ryanodine or inositol trisphosphate receptors. It can be postulated that an alteration in the cADPR-signaling pathway may exist in skeletal muscle of the patients suffering from uremic myopathy. [ABSTRACT FROM AUTHOR]

It is unknown whether loss of skeletal muscle mass and function experienced by astronauts during space flight could be augmented by ionizing radiation (IR), such as low-dose high-charge and energy (HZE) particles or low-dose high-energy proton radiation. In the current study adult mice were irradiated whole-body with either a single dose of 15 cGy of 1 GeV/n ⁵⁶Fe-particle or with a 90 cGy proton of 1 GeV/n proton particles. Both ionizing radiation types caused alterations in the skeletal muscle cytoplasmic Ca²⁺ ([Ca²⁺]i) homeostasis. ⁵⁶Fe-particle irradiation also caused a reduction of depolarization-evoked Ca²⁺ release from the sarcoplasmic reticulum (SR). The increase in the [Ca²⁺]i was detected as early as 24 h after ⁵⁶Fe-particle irradiation, while effects of proton irradiation were only evident at 72 h. In both instances [Ca²⁺]i returned to baseline at day 7 after irradiation. All ⁵⁶Fe-particle irradiated samples revealed a significant number of centrally localized nuclei, a histologic manifestation of regenerating muscle, 7 days after irradiation. Neither unirradiated control or proton-irradiated samples exhibited such a phenotype. Protein analysis revealed significant increase in the phosphorylation of Akt, Erk1/2 and rpS6k on day 7 in ⁵⁶Fe-particle irradiated skeletal muscle, but not proton or unirradiated skeletal muscle, suggesting activation of pro-survival signaling. Our findings suggest that a single low-dose ⁵⁶Fe-particle or proton exposure is sufficient to affect Ca²⁺ homeostasis in skeletal muscle. However, only ⁵⁶Fe-particle irradiation led to the appearance of central nuclei and activation of pro-survival pathways, suggesting an ongoing muscle damage/recovery process.

Intracellular deposition of the beta-amyloid (Abeta) peptide is an increasingly recognized pathological hallmark associated with neurodegeneration and muscle wasting in Alzheimer's disease (AD) and inclusion body myositis (IBM), respectively. Previous reports have implicated dysregulation of beta-amyloid precursor protein (betaAPP) expression in IBM. Accumulation of full-length betaAPP, its various proteolytic derivatives including Abeta, and phospho-tau into vacuolated inclusions is an early pathogenic event. We previously reported on a statistical tendency favoring fast twitch fiber involvement in IBM, reminiscent of the tissue specific patterns of misfolded protein deposition seen in neurodegenerative diseases. To test this principle, we generated an animal model in which human wild-type (WT) betaAPP expression was limited to postnatal type II skeletal muscle. Hemizygous transgenic mice harboring increased levels of holo betaAPP751 and Abeta in skeletal muscle fibers became significantly weaker with age compared with nontransgenic littermates and exhibited typical myopathic features. A subpopulation of dissociated muscle fibers from transgenic mice exhibited a 2-fold increase in resting calcium and membrane depolarization compared with nontransgenic littermates. Taken together, these data indicate that overexpression of human betaAPP in fast twitch skeletal muscle of transgenic mice is sufficient for the development of some features characteristic of IBM, including abnormal tau histochemistry. The increase in resting calcium and depolarization are novel findings, suggesting both a mechanism for the weakness and an avenue for therapeutic intervention in IBM.