Senescence Is Associated With Elevated Intracellular Resting [Ca2 +] in Mice Skeletal Muscle Fibers. An in vivo Study.

Aging causes skeletal muscles to become atrophied, weak, and easily fatigued. Here, we have tested the hypothesis that normal aging in skeletal muscle cells is associated with Ca2+ intracellular dyshomeostasis and oxidative stress. Intracellular Ca2+ concentration ([Ca2+]i), resting intracellular Na+ concentration ([Na+]i) and reactive oxygen species (ROS) production were measured in vivo (superficial gastrocnemius fibers) using double-barreled ion-selective microelectrodes, and in vitro [isolated single flexor digitorum brevis fibers] using fluorescent ROS sensor CM-H2DCFDA in young (3 months of age), middle-aged (12 months of age), and aged (24 months of age) mice. We found an age-related increase in [Ca2+]i from 121 ± 4 nM in young muscle cells which rose to 255 ± 36 nM in middle-aged and to 409 ± 25 nM in aged cells. [Na+]i also showed an age-dependent elevation, increasing from 8 ± 0.5 mM in young muscle fibers, to 12 ± 1 mM in middle-aged and to 17 ± 1 mM in old muscle fibers. Using the fluorescent ROS sensor CM-H2DCFDA we found that these increases in intracellular cation concentrations were associated with significantly increased basal ROS production as demonstrated by age related increases in the rate of dichlorodihydrofluorescein fluorescence. To determine is this could be modified by reducing ROS and/or blocking sarcolemmal Ca2+ influx we administered flufenamic acid (FFA), a non-steroidal anti-inflammatory drug which is also a non-selective blocker of the transient receptor potential canonical channels (TRPCs), for 4 weeks to determine if this would have a beneficial effect. FFA treatment reduced both basal ROS production and muscle [Ca2+]i and [Na+]i in middle-aged and aged muscle fibers compared to fibers and muscles of untreated 12 and 24-months old mice. [Ca2+]i was reduced to 134 ± 8 nM in middle-aged muscle and to 246 ± 40 nM in muscle from aged mice. Likewise [Na+]i was reduced to 9 ± 0.7 mM in middle-aged muscles and to 13 ± 1 mM in muscle from aged mice. FFA treatment also reduced age associated increases in plasma interleukin 6 and tumor necrosis factor-alpha (TNF-α) concentrations which were elevated in 12 and 24-months old mice compared to young mice and decreased age-related muscle damage as indicated by a reduction in serum creatine kinase (CK) activity. Our data provides a direct demonstration that normal aging is associated with a significant elevation [Ca2+]i, [Na+]i, and intracellular ROS production in skeletal muscle fibers. Furthermore, the fact that FFA reduced the intracellular [Ca2+], [Na+], and ROS production as well as the elevated IL6, TNF-α, and CK levels, led us to suggest that its pharmacological effect may be related to its action both as a TRPC channel blocker and as an anti-inflammatory.

Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells.

Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 µM H2O2 increased NF-κB activity; after administration of 100 and 200 µM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 µM and 200 µM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca2+ release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.

Increased resting intracellular calcium modulates NF-κB-dependent inducible nitric-oxide synthase gene expression in dystrophic mdx skeletal myotubes.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca(2+)](rest)) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca(2+)](rest) was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca(2+) entry (low Ca(2+) solution, Ca(2+)-free solution, and Gd(3+)) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca(2+)](rest). Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca(2+)](rest) was reduced. Levels of mRNA for TNFα, IL-1ß, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca(2+)](rest) using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca(2+)](rest), is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells.

[Dysfunction of diastolic [Ca²âº] in cardiomyocytes isolated from chagasic patients]. / Disfunción de la [Ca(2+)] diastólica en cardiomiocitos aislados de pacientes chagásicos.

INTRODUCTION AND OBJECTIVES: Chagas is an endemic disease in Latin America, caused by the parasite Trypanosoma cruzi, which usually affects the functioning of the heart. We have studied the regulation of intracellular calcium in cardiomyocytes isolated from chagasic patients with different degrees of heart dysfunction. METHODS: Calcium selective microelectrodes were used to simultaneously measure diastolic calcium concentration ([Ca²âº](d)) and resting membrane potential in endomyocardial biopsies obtained from chagasic patients and controls. RESULTS: The [Ca²âº](d) increased by 123%, 295%, and 738% in chagasic patients in functional class I, II, and III, respectively, in relation to controls. Membrane potential showed a partial depolarization of 6% in functional class I, 10% in functional class II, and 22% in functional class III, compared to control values. Alteration in the [Ca²âº](d) was partially reverted by 1-[6-[[(17ß)-3-metoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), a ß-phospholipase C antagonist, and by 2-aminoethoxydiphenyl-borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker. Phenylephrine, an agent that induces a rapid transient increase in 1,4,5-trisphosphate intracellular content, produced a rise in [Ca²âº](d), higher in chagasic cardiomyocytes than in controls, and its effect was fully inhibited by 2-APB. CONCLUSIONS: In cardiomyocytes from chagasic patients there is a dysfunction of the regulation of the [Ca²âº](d), which correlates with the cardiac abnormalities observed in the different stages of the disease. This disturbance in the regulation of intracellular calcium appears to be associated with alterations in the regulation of intracellular messenger inositol 1,4,5-trisphosphate.

Heart and skeletal muscle are targets of dengue virus infection.

BACKGROUND: Dengue fever is one of the most significant re-emerging tropical diseases, despite our expanding knowledge of the disease, viral tropism is still not known to target heart tissues or muscle. METHODS: A prospective pediatric clinical cohort of 102 dengue hemorrhagic fever patients from Colombia, South America, was followed for 1 year. Clinical diagnosis of myocarditis was routinely performed. Electrocardiograph and echocardiograph analysis were performed to confirm those cases. Immunohistochemistry for detection of dengue virus and inflammatory markers was performed on autopsied heart tissue. In vitro studies of human striated skeletal fibers (myotubes) infected with dengue virus were used as a model for myocyte infection. Measurements of intracellular Ca2+ concentration as well as immunodetection of dengue virus and inflammation markers in infected myotubes were performed. RESULTS: Eleven children with dengue hemorrhagic fever presented with symptoms of myocarditis. Widespread viral infection of the heart, myocardial endothelium, and cardiomyocytes, accompanied by inflammation was observed in 1 fatal case. Immunofluorescence confocal microscopy showed that myotubes were infected by dengue virus and had increased expression of the inflammatory genes and protein IP-10. The infected myotubes also had increases in intracellular Ca2+ concentration. CONCLUSIONS: Vigorous infection of heart tissues in vivo and striated skeletal cells in vitro are demonstrated. Derangements of Ca2+ storage in the infected cells may directly contribute to the presentation of myocarditis in pediatric patients.

Cardiac gene expression profiling provides evidence for cytokinopathy as a molecular mechanism in Chagas' disease cardiomyopathy.

Chronic Chagas' disease cardiomyopathy is a leading cause of congestive heart failure in Latin America, affecting more than 3 million people. Chagas' cardiomyopathy is more aggressive than other cardiomyopathies, but little is known of the molecular mechanisms responsible for its severity. We characterized gene expression profiles of human Chagas' cardiomyopathy and dilated cardiomyopathy to identify selective disease pathways and potential therapeutic targets. Both our customized cDNA microarray (Cardiochip) and real-time reverse transcriptase-polymerase chain reaction analysis showed that immune response, lipid metabolism, and mitochondrial oxidative phosphorylation genes were selectively up-regulated in myocardial tissue of the tested Chagas' cardiomyopathy patients. Interferon (IFN)-gamma-inducible genes represented 15% of genes specifically up-regulated in Chagas' cardiomyopathy myocardial tissue, indicating the importance of IFN-gamma signaling. To assess whether IFN-gamma can directly modulate cardio-myocyte gene expression, we exposed fetal murine cardiomyocytes to IFN-gamma and the IFN-gamma-inducible chemokine monocyte chemoattractant protein-1. Atrial natriuretic factor expression increased 15-fold in response to IFN-gamma whereas combined IFN-gamma and monocyte chemoattractant protein-1 increased atrial natriuretic factor expression 400-fold. Our results suggest IFN-gamma and chemokine signaling may directly up-regulate cardiomyocyte expression of genes involved in pathological hypertrophy, which may lead to heart failure. IFN-gamma and other cytokine pathways may thus be novel therapeutic targets in Chagas' cardiomyopathy.

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.

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i.

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca(2+) concentration ([Ca(2+)](i)) using double-barreled, Ca(2+)-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca(2+)](i) was approximately twofold in MHS compared with MHN quiescent myoballs (232 +/- 35 vs. 112 +/- 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca(2+)](i) in both groups; however, the concentration required to cause a rise in [Ca(2+)](i) elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca(2+) buffer BAPTA reduced [Ca(2+)](i), raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca(2+)](i), and reduced the amount of Ca(2+) release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca(2+)](i) levels in these cells.

Estudo da funçäo simpático-adrenal em crianças submetidas a cirurgia cardíaca com hipotermia de superfície, perfusäo limitada e parada circulatória / Simpathoadrenal function during cardiac surgery in children using the technique of surface cooling: limited cardiopulmonary bypass and circulatory arrest

Os níveis de catecolamina plasmática foram medidos em 20 crianças (idade média de 6,00 ñ 5,86 meses; peso médio 5,3 ñ 1,82 kg), durante a correçäo de defeitos cardíacos congênitos, usando-se a hipotermia de superfície (26-C), perfusäo cardiopulmonar limitada e parada circulatória (15-C). Adrenalina e noradrenalina plasmática foram dosadas em amostras sangüíneas arteriais seriadas, usando-se a cromatografia. A hipotermia de superfície produziu um significante aumento de ambas as catecolaminas. Durante o resfriamento central, os níveis caíram devido à hemodiluiçäo. Após o período de parada circulatória (23/64 minutos, média de 41,3), ocorreu um aumento das catecolaminas plasmáticas, que persistiu durante o reaquecimento. Após o reaquecimento, as catecolaminas plasmáticas permaneceram elevadas até o final do ato cirúrgico. Nossos resultados mostram que a técnica de hipotermia de superfície, perfusäo cardiopulmonar limitada e parada circulatória, sob as nossas condiçöes de anestesia, produziu significante aumento da concentraçäo de adrenalina e noradrenalina plasmática, porém o significado biológico é, ainda, inseguro