TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells.

TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1-5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3-5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general.

Divergent modification of low-dose 56Fe-particle and proton radiation on skeletal muscle.

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 Ca²âº ([Ca²âº]i) homeostasis. 56Fe-particle irradiation also caused a reduction of depolarization-evoked Ca²âº release from the sarcoplasmic reticulum (SR). The increase in the [Ca²âº]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 [Ca²âº]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 Ca²âº 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.

Mitochondrial dysfunction in skeletal muscle of amyloid precursor protein-overexpressing mice.

Inclusion body myositis, the most common muscle disorder in the elderly, is partly characterized by abnormal expression of amyloid precursor protein (APP) and intracellular accumulation of its proteolytic fragments collectively known as ß-amyloid. The present study examined the effects of ß-amyloid accumulation on mitochondrial structure and function of skeletal muscle from transgenic mice (MCK-ßAPP) engineered to accumulate intramyofiber ß-amyloid. Electron microscopic analysis revealed that a large fraction of myofibers from 2-3-month-old MCK-ßAPP mice contained numerous, heterogeneous alterations in mitochondria, and other cellular organelles. [(1)H-decoupled](13)C NMR spectroscopy showed a substantial reduction in TCA cycle activity and indicated a switch from aerobic to anaerobic glucose metabolism in the MCK-ßAPP muscle. Isolated muscle fibers from the MCK-ßAPP mice also exhibited a reduction in cytoplasmic pH, an increased rate of ROS production, and a partially depolarized plasmalemma. Treatment of MCK-ßAPP muscle cells with Ru360, a mitochondrial Ca(2+) uniporter antagonist, reversed alterations in the plasmalemmal membrane potential (V(m)) and pH. Consistent with altered redox state of the cells, treatment of MCK-ßAPP muscle cells with glutathione reversed the effects of ß-amyloid accumulation on Ca(2+) transient amplitudes. We conclude that structural and functional alterations in mitochondria precede the reported appearance of histopathological and clinical features in the MCK-ßAPP mice and may represent key early events in the pathogenesis of inclusion body myositis.

Altered Ca2+ homeostasis in the skeletal muscle of DJ-1 null mice.

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 Ca(2+) 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 [Ca(2+)] ([Ca(2+)](i)) and depolarization-evoked Ca(2+) release in the mouse skeletal muscle. The loss of DJ-1 resulted in a more than two-fold increase in resting [Ca(2+)](i). While there was no alteration in the resting membrane potential, there was a significant decrease in depolarization-evoked Ca(2+) 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 Ca(2+) homeostasis. These results provide evidence of DJ-1's association with Ca(2+) regulatory pathways in mouse skeletal muscle, and suggest the potential benefit of resveratrol to functionally compensate for the loss of DJ-1.

Intracellular ß-amyloid accumulation leads to age-dependent progression of Ca2+ dysregulation in skeletal muscle.

Intramyofiber accumulation of ß-amyloid fragments (Aß) 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 [Ca(2+)] ([Ca(2+)](i)) as well as the depolarization-evoked Ca(2+) release from the sarcoplasmic reticulum in skeletal muscle from transgenic mice expressing human ßAPP (MCK-ßAPP). MCK-ßAPP mice show an age-dependent increase in [Ca(2+)](i) along with a reduction in depolarization-evoked Ca(2+) 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-ßAPP animals the increase in resting [Ca(2+)](i) can be attributed largely to Ca(2+) influx through nifedipine-sensitive Ca(2+) channels. In the adult MCK-ßAPP mice, in addition to the nifedipine-sensitive pathway, there is also a substantial contribution by the intracellular compartments to the increase in [Ca(2+)](i). These results suggest that ß-amyloid-induced disuption of Ca(2+) handling may represent an early event in the pathogenesis of IBM.

Amyloid-ß protein impairs Ca2+ release and contractility in skeletal muscle.

Inclusion body myositis (IBM), the most common muscle disorder in the elderly, is partly characterized by dysregulation of ß-amyloid precursor protein (ßAPP) expression and abnormal, intracellular accumulation of full-length ßAPP and ß-amyloid epitopes. The present study examined the effects of ß-amyloid accumulation on force generation and Ca(2+) release in skeletal muscle from transgenic mice harboring human ßAPP and assessed the consequence of Aß(1-42) modulation of the ryanodine receptor Ca(2+) release channels (RyRs). ß-Amyloid laden muscle produced less peak force and exhibited Ca(2+) transients with smaller amplitude. To determine whether modification of RyRs by ß-amyloid underlie the effects observed in muscle, in vitro Ca(2+) release assays and RyR reconstituted in planar lipid bilayer experiments were conducted in the presence of Aß(1-42). Application of Aß(1-42) to RyRs in bilayers resulted in an increased channel open probability and changes in gating kinetics, while addition of Aß(1-42) to the rabbit SR vesicles resulted in RyR-mediated Ca(2+) release. These data may relate altered ßAPP metabolism in IBM to reductions in RyR-mediated Ca(2+) release and muscle contractility.

Increased intraneuronal resting [Ca2+] in adult Alzheimer's disease mice.

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 beta-amyloid proteins. Resting free Ca2+ concentration ([Ca2+](i)), as measured with Ca2+-selective microelectrodes, was greatly elevated in neurons from 3xTg-AD and APP(SWE) 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.

Transgenic expression of beta-APP in fast-twitch skeletal muscle leads to calcium dyshomeostasis and IBM-like pathology.

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.

Altered Ca2+ homeostasis in human uremic skeletal muscle: possible involvement of cADPR in elevation of intracellular resting [Ca2+].

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 beta-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+]i) in skeletal muscle obtained from control subjects and uremic patients (UP). [Ca2+]i was measured using double-barreled Ca2+-selective microelectrodes in muscle fibers, prior to and after microinjections of cADPR. RESULTS: Resting [Ca2+]i 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+]i in UP or control fibers. Microinjection of cADPR produced an elevation of [Ca2+]i in both groups of cells. This elevation was not mediated by Ca2+ influx, or inhibited by heparin or ryanodine. [cADPR]i 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+]i in UP muscle fibers and blocked the cADPR-elicited elevation in [Ca2+]i in both groups of muscle cells. CONCLUSION: Skeletal muscles of the UP exhibit chronic elevation of [Ca2+]i 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.

Calcium dyshomeostasis in beta-amyloid and tau-bearing skeletal myotubes.

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 beta-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 (C(2)C(12)) expressed beta-amyloid-42 (Abeta(42)) and fetal Tau peptides, as human transgenes encoded by herpes simplex virus, either individually or concurrently. Co-expression of Abeta(42) in C(2)C(12) myotubes resulted in hyperphosphorylation of Tau protein that was not observed when Tau was expressed alone. Resting calcium concentration and agonist-induced RyR-mediated Ca(2+) release were examined using calcium-specific microelectrodes and Fluo-4 epifluorescence, respectively. Co-expression of Abeta(42) and Tau cooperatively elevated basal levels of myoplasmic-free calcium, an effect that was accompanied by depolarization of the plasma membrane. Sarcoplasmic reticulum (SR) calcium release, induced by KCl depolarization, was not affected by Abeta(42) or Tau. In contrast, expression of Abeta(42), Tau, or Abeta(42) together with Tau resulted in enhanced sensitivity of ryanodine receptors to activation by caffeine. Notably, expression of beta-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 Ca(2+) leak and Tau hyperphosphorylation in skeletal muscle.

Ca2+ influx through alpha1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle.

Differentiated primary myotubes isolated from wild-type mice exhibit ryanodine-sensitive, spontaneous global Ca2+ oscillations as well as spontaneous depolarizations in the plasma membrane. Immunolabeling of these myotubes showed expression of both alpha1S dihydropyridine receptors (DHPRs) and ryanodine-sensitive Ca2+-release channel 1 (RyR1), the two key proteins in skeletal excitation-contraction (E-C) coupling. Spontaneous global Ca2+ oscillations could be inhibited by addition of 0.1 mM CdCl2/0.5 mM LaCl3 or 5 microM nifedipine to the extracellular bathing solution. After either treatment, Ca2+ oscillations could be restored upon extensive washing. Although exposure to DHPR antagonists completely blocked Ca2+ 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 Ca2+ oscillations were never present in cultured mdg myotubes, which lack the expression of alpha1SDHPRs. These results suggest that under physiological conditions in conjunction with the mechanical coupling between the alpha1SDHPRs and RyR1, the initiation of Ca2+ oscillations in myotubes may be facilitated, in part, by the Ca2+ influx through the alpha1s-subunit of the DHPR.

All three ryanodine receptor isoforms generate rapid cooling responses in muscle cells.

The rapid cooling (RC) response in muscle is an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) that is probably caused by Ca2+ release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR Ca2+ 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 [Ca2+]i and perfused with either cold ( approximately 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 Ca2+ 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. Ca2+ release from RyRs is likely caused by a RC-induced conformational change of the channel from the closed to the open state.

Interdomain interactions within ryanodine receptors regulate Ca2+ spark frequency in skeletal muscle.

DP4 is a 36-residue synthetic peptide that corresponds to the Leu(2442)-Pro(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(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(2)+ release events (Ca(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(2)+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca(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(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(2)+ release channel(s) generating the sparks. DP4 also increased [(3)H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca(2)+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca(2)+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg(17) to Cys(17) replacement (DP4mut), which corresponds to the Arg(2458)-to-Cys(2458) mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg(2)+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg(2)+-free RyR(s), thus promoting channel opening and production of Ca(2)+ sparks.