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1.
J Biol Chem ; 299(8): 104992, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37392848

RESUMEN

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.


Asunto(s)
Halotano , Respuesta al Choque Térmico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Hipertermia Maligna , Animales , Ratones , Calcio/metabolismo , Halotano/farmacología , Respuesta al Choque Térmico/genética , Hipertermia Maligna/genética , Hipertermia Maligna/metabolismo , Hipertermia Maligna/patología , Músculo Esquelético/metabolismo , Mutación , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética
2.
Br J Anaesth ; 133(5): 1093-1100, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-39304470

RESUMEN

BACKGROUND: As the primary Ca2+ release channel in skeletal muscle sarcoplasmic reticulum (SR), mutations in type 1 ryanodine receptor (RyR1) or its binding partners underlie a constellation of muscle disorders, including malignant hyperthermia (MH). In patients with MH mutations, triggering agents including halogenated volatile anaesthetics bias RyR1 to an open state resulting in uncontrolled Ca2+ release, increased sarcomere tension, and heat production. Propofol does not trigger MH and is commonly used for patients at risk of MH. The atomic-level interactions of any anaesthetic with RyR1 are unknown. METHODS: RyR1 opening was measured by [3H]ryanodine binding in heavy SR vesicles (wild type) and single-channel recordings of MH mutant R615C RyR1 in planar lipid bilayers, each exposed to propofol or the photoaffinity ligand analogue m-azipropofol (AziPm). Activator-mediated wild-type RyR1 opening as a function of propofol concentration was measured by Fura-2 Ca2+ imaging of human skeletal myotubes. AziPm binding sites, reflecting propofol binding, were identified on RyR1 using photoaffinity labelling. Propofol binding affinity to a photoadducted site was predicted using molecular dynamics (MD) simulation. RESULTS: Both propofol and AziPm decreased RyR1 opening in planar lipid bilayers (P<0.01) and heavy SR vesicles, and inhibited activator-induced Ca2+ release from human skeletal myotube SR. Several putative propofol binding sites on RyR1 were photoadducted by AziPm. MD simulation predicted propofol KD values of 55.8 µM and 1.4 µM in the V4828 pocket in open and closed RyR1, respectively. CONCLUSIONS: Propofol demonstrated direct binding and inhibition of RyR1 at clinically plausible concentrations, consistent with the hypothesis that propofol partially mitigates malignant hyperthermia by inhibition of induced Ca2+ flux through RyR1.


Asunto(s)
Anestésicos Intravenosos , Músculo Esquelético , Propofol , Canal Liberador de Calcio Receptor de Rianodina , Propofol/farmacología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Anestésicos Intravenosos/farmacología , Hipertermia Maligna/metabolismo , Hipertermia Maligna/genética , Sitios de Unión/efectos de los fármacos , Calcio/metabolismo , Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Simulación de Dinámica Molecular , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo
3.
J Biol Chem ; 295(45): 15226-15235, 2020 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-32826313

RESUMEN

Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism, which has directed research interest into oxidative phosphorylation and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ∼16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression, and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA-Seq data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared with WT controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I-facilitated oxidative phosphorylation in mutant muscle. Furthermore, we observed a gene-dose-specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively, these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.


Asunto(s)
Hipertermia/genética , Hipertermia/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Femenino , Masculino , Ratones
4.
FASEB J ; 34(6): 8721-8733, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32367593

RESUMEN

Malignant hyperthermia (MH) is characterized by induction of skeletal muscle hyperthermia in response to a dysregulated increase in myoplasmic calcium. Although altered energetics play a central role in MH, MH-susceptible humans and mouse models are often described as having no phenotype until exposure to a triggering agent. The purpose of this study was to determine the influence of the R163C ryanodine receptor 1 mutation, a common MH mutation in humans, on energy expenditure, and voluntary wheel running in mice. Energy expenditure was measured by indirect respiration calorimetry in wild-type (WT) and heterozygous R163C (HET) mice over a range of ambient temperatures. Energy expenditure adjusted for body weight or lean mass was increased (P < .05) in male, but not female, HET mice housed at 22°C or when housed at 28°C with a running wheel. In female mice, voluntary wheel running was decreased (P < .05) in the HET vs WT animals when analyzed across ambient temperatures. The thermoneutral zone was also widened in both male and female HET mice. The results of the study show that the R163C mutations alters energetics even at temperatures that do not typically induce MH.


Asunto(s)
Metabolismo Energético/fisiología , Hipertermia/patología , Hipertermia Maligna/patología , Actividad Motora/fisiología , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Femenino , Heterocigoto , Hipertermia/metabolismo , Masculino , Hipertermia Maligna/metabolismo , Potenciales de la Membrana/fisiología , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación/genética , Canal Liberador de Calcio Receptor de Rianodina/genética
5.
Anesthesiology ; 134(2): 234-247, 2021 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-33301562

RESUMEN

BACKGROUND: Pharmacologic modulation has previously shown that transient receptor potential canonical (TRPC) channels play an important role in the pathogenesis of malignant hyperthermia. This study tested the hypothesis that genetically suppressing the function of TRPC6 can partially ameliorate muscle cation dyshomeostasis and the response to halothane in a mouse model relevant to malignant hyperthermia. METHODS: This study examined the effect of overexpressing a muscle-specific nonconducting dominant-negative TRPC6 channel in 20 RYR1-p.R163C and 20 wild-type mice and an equal number of nonexpressing controls, using calcium- and sodium-selective microelectrodes and Western blots. RESULTS: RYR1-p.R163C mouse muscles have chronically elevated intracellular calcium and sodium levels compared to wild-type muscles. Transgenic expression of the nonconducting TRPC6 channel reduced intracellular calcium from 331 ± 34 nM (mean ± SD) to 190 ± 27 nM (P < 0.0001) and sodium from 15 ± 1 mM to 11 ± 1 mM (P < 0.0001). Its expression lowered the increase in intracellular Ca2+ of the TRPC6-specific activator hyperforin in RYR1-p.R163C muscle fibers from 52% (348 ± 37 nM to 537 ± 70 nM) to 14% (185 ± 11 nM to 210 ± 44 nM). Western blot analysis of TRPC3 and TRPC6 expression showed the expected increase in TRPC6 caused by overexpression of its dominant-negative transgene and a compensatory increase in expression of TRPC3. Although expression of the muscle-specific dominant-negative TRPC6 was able to modulate the increase in intracellular calcium during halothane exposure and prolonged life (35 ± 5 min vs. 15 ± 3 min; P < 0.0001), a slow, steady increase in calcium began after 20 min of halothane exposure, which eventually led to death. CONCLUSIONS: These data support previous findings that TRPC channels play an important role in causing the intracellular calcium and sodium dyshomeostasis associated with RYR1 variants that are pathogenic for malignant hyperthermia. However, they also show that modulating TRPC channels alone is not sufficient to prevent the lethal effect of exposure to volatile anesthetic malignant hyperthermia-triggering agents.


Asunto(s)
Calcio/metabolismo , Hipertermia Maligna/genética , Hipertermia Maligna/fisiopatología , Canal Catiónico TRPC6/genética , Canal Catiónico TRPC6/metabolismo , Animales , Modelos Animales de Enfermedad , Hipertermia Maligna/metabolismo , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo
6.
Anesthesiology ; 133(2): 364-376, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32665491

RESUMEN

BACKGROUND: Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca dyshomeostasis in a mouse model relevant to malignant hyperthermia. METHODS: This study examined the mechanisms responsible for Ca dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. RESULTS: RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene-dose-dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. CONCLUSIONS: Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia-susceptible muscle, both at rest and during the malignant hyperthermia crisis.


Asunto(s)
Calcio/metabolismo , Modelos Animales de Enfermedad , Hipertermia Maligna/metabolismo , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6/metabolismo , Animales , Femenino , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Indanos/farmacología , Masculino , Hipertermia Maligna/genética , Hipertermia Maligna/patología , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Canal Liberador de Calcio Receptor de Rianodina/biosíntesis , Canal Liberador de Calcio Receptor de Rianodina/genética , Canales Catiónicos TRPC/antagonistas & inhibidores , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6/antagonistas & inhibidores , Canal Catiónico TRPC6/genética
8.
Br J Anaesth ; 122(5): 613-621, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30916033

RESUMEN

BACKGROUND: Individuals genetically susceptible to malignant hyperthermia (MH) exhibit hypermetabolic reactions when exposed to volatile anaesthetics. Mitochondrial dysfunction has previously been associated with the MH-susceptible (MHS) phenotype in animal models, but evidence of this in human MH is limited. METHODS: We used high resolution respirometry to compare oxygen consumption rates (oxygen flux) between permeabilised human MHS and MH-negative (MHN) skeletal muscle fibres with or without prior exposure to halothane. A substrate-uncoupler-inhibitor titration protocol was used to measure the following components of the electron transport chain under conditions of oxidative phosphorylation (OXPHOS) or after uncoupling the electron transport system (ETS): complex I (CI), complex II (CII), CI+CII and, as a measure of mitochondrial mass, complex IV (CIV). RESULTS: Baseline comparisons without halothane exposure showed significantly increased mitochondrial mass (CIV, P=0.021) but lower flux control ratios in CI+CII(OXPHOS) and CII(ETS) of MHS mitochondria compared with MHN (P=0.033 and 0.005, respectively) showing that human MHS mitochondria have a functional deficiency. Exposure to halothane triggered a hypermetabolic response in MHS mitochondria, significantly increasing mass-specific oxygen flux in CI(OXPHOS), CI+CII(OXPHOS), CI+CII(ETS), and CII(ETS) (P=0.001-0.012), while the rates in MHN samples were unaltered by halothane exposure. CONCLUSIONS: We present evidence of mitochondrial dysfunction in human MHS skeletal muscle both at baseline and after halothane exposure.


Asunto(s)
Hipertermia Maligna/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Adolescente , Adulto , Anciano , Anestésicos por Inhalación/farmacología , Biopsia , Niño , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/fisiología , Femenino , Predisposición Genética a la Enfermedad , Halotano/farmacología , Humanos , Masculino , Hipertermia Maligna/genética , Hipertermia Maligna/patología , Persona de Mediana Edad , Mitocondrias Musculares/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Adulto Joven
9.
Exp Brain Res ; 235(2): 585-595, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27837258

RESUMEN

Visual capture and the ventriloquism aftereffect resolve spatial disparities of incongruent auditory visual (AV) objects by shifting auditory spatial perception to align with vision. Here, we demonstrated the distinct temporal characteristics of visual capture and the ventriloquism aftereffect in response to brief AV disparities. In a set of experiments, subjects localized either the auditory component of AV targets (A within AV) or a second sound presented at varying delays (1-20 s) after AV exposure (A2 after AV). AV targets were trains of brief presentations (1 or 20), covering a ±30° azimuthal range, and with ±8° (R or L) disparity. We found that the magnitude of visual capture generally reached its peak within a single AV pair and did not dissipate with time, while the ventriloquism aftereffect accumulated with repetitions of AV pairs and dissipated with time. Additionally, the magnitude of the auditory shift induced by each phenomenon was uncorrelated across listeners and visual capture was unaffected by subsequent auditory targets, indicating that visual capture and the ventriloquism aftereffect are separate mechanisms with distinct effects on auditory spatial perception. Our results indicate that visual capture is a 'sample-and-hold' process that binds related objects and stores the combined percept in memory, whereas the ventriloquism aftereffect is a 'leaky integrator' process that accumulates with experience and decays with time to compensate for cross-modal disparities.


Asunto(s)
Localización de Sonidos/fisiología , Disparidad Visual/fisiología , Percepción Visual/fisiología , Estimulación Acústica , Adolescente , Adulto , Análisis de Varianza , Femenino , Humanos , Masculino , Memoria/fisiología , Estimulación Luminosa , Adulto Joven
10.
Biochim Biophys Acta ; 1852(7): 1410-9, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25857619

RESUMEN

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.


Asunto(s)
Interleucina-6/metabolismo , Potenciales de la Membrana , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Estimulación Eléctrica , Interleucina-6/genética , Ratones , Ratones Endogámicos mdx , Fibras Musculares Esqueléticas/fisiología , FN-kappa B/genética
11.
Biol Cybern ; 110(6): 455-471, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27815630

RESUMEN

Vision typically has better spatial accuracy and precision than audition and as a result often captures auditory spatial perception when visual and auditory cues are presented together. One determinant of visual capture is the amount of spatial disparity between auditory and visual cues: when disparity is small, visual capture is likely to occur, and when disparity is large, visual capture is unlikely. Previous experiments have used two methods to probe how visual capture varies with spatial disparity. First, congruence judgment assesses perceived unity between cues by having subjects report whether or not auditory and visual targets came from the same location. Second, auditory localization assesses the graded influence of vision on auditory spatial perception by having subjects point to the remembered location of an auditory target presented with a visual target. Previous research has shown that when both tasks are performed concurrently they produce similar measures of visual capture, but this may not hold when tasks are performed independently. Here, subjects alternated between tasks independently across three sessions. A Bayesian inference model of visual capture was used to estimate perceptual parameters for each session, which were compared across tasks. Results demonstrated that the range of audiovisual disparities over which visual capture was likely to occur was narrower in auditory localization than in congruence judgment, which the model indicates was caused by subjects adjusting their prior expectation that targets originated from the same location in a task-dependent manner.


Asunto(s)
Percepción Auditiva , Modelos Biológicos , Animales , Teorema de Bayes , Humanos , Juicio , Localización de Sonidos , Percepción Espacial , Percepción Visual
13.
J Biol Chem ; 289(27): 19180-90, 2014 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-24847052

RESUMEN

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca(2+) dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca(2+) depending on cellular activity. Resting intracellular calcium ([Ca(2+)]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 [Ca(2+)]r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca(2+) or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca(2+)]r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca(2+)]r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca(2+)]r in MHS muscle fibers and decreases the amplitude of [Ca(2+)]r rise triggered by halothane, but had no effect on [Ca(2+)]r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca(2+) transient elicited by high [K(+)]e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca(2+)]r and the Ca(2+) 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 Ca(2+) transients associated with K(+)-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.


Asunto(s)
Calcio/metabolismo , Hipertermia Maligna/metabolismo , Músculo Esquelético/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Halotano/farmacología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Hipertermia Maligna/patología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Niacinamida/análogos & derivados , Niacinamida/farmacología , Potasio/metabolismo , Sodio/metabolismo , Intercambiador de Sodio-Calcio/genética , Porcinos , Tiourea/análogos & derivados , Tiourea/farmacología
14.
Stem Cells ; 32(9): 2492-501, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24916688

RESUMEN

Certain lower organisms achieve organ regeneration by reverting differentiated cells into tissue-specific progenitors that re-enter embryonic programs. During muscle regeneration in the urodele amphibian, postmitotic multinucleated skeletal myofibers transform into mononucleated proliferating cells upon injury, and a transcription factor-msx1 plays a role in their reprograming. Whether this powerful regeneration strategy can be leveraged in mammals remains unknown, as it has not been demonstrated that the dedifferentiated progenitor cells arising from muscle cells overexpressing Msx1 are lineage-specific and possess the same potent regenerative capability as their amphibian counterparts. Here, we show that ectopic expression of Msx1 reprograms postmitotic, multinucleated, primary mouse myotubes to become proliferating mononuclear cells. These dedifferentiated cells reactivate genes expressed by embryonic muscle progenitor cells and generate only muscle tissue in vivo both in an ectopic location and inside existing muscle. More importantly, distinct from adult muscle satellite cells, these cells appear both to fuse with existing fibers and to regenerate myofibers in a robust and time-dependent manner. Upon transplantation into a degenerating muscle, these dedifferentiated cells generated a large number of myofibers that increased over time and replenished almost half of the cross-sectional area of the muscle in only 12 weeks. Our study demonstrates that mammals can harness a muscle regeneration strategy used by lower organisms when the same molecular pathway is activated.


Asunto(s)
Reprogramación Celular/fisiología , Leucocitos Mononucleares/citología , Fibras Musculares Esqueléticas/citología , Regeneración/fisiología , Células Madre/citología , Animales , Técnicas de Cultivo de Célula , Ciclo Celular/fisiología , Desdiferenciación Celular/fisiología , Leucocitos Mononucleares/metabolismo , Factor de Transcripción MSX1/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Trasplante de Células Madre , Células Madre/metabolismo
15.
Proc Natl Acad Sci U S A ; 109(20): 7923-8, 2012 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-22547813

RESUMEN

Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca(2+) release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca(V)1.1, the principal subunit of the L-type Ca(2+) channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca(2+) entry. The Ca(V)1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Ca(V) channels [Carpenter D, et al. (2009) BMC Med Genet 10:104-115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (Ca(V)1.1 null) myotubes. Unlike previously described MH-linked mutations in Ca(V)1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca(2+) release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Ca(V)1.1 R174W-expressing myotubes, resting myoplasmic Ca(2+) levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Ca(V)1.1. Our results indicate that Ca(V)1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca(2+) leak from the SR, and that perturbation of Ca(V)1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.


Asunto(s)
Calcio/metabolismo , Caveolina 1/genética , Acoplamiento Excitación-Contracción/fisiología , Predisposición Genética a la Enfermedad/genética , Hipertermia Maligna/genética , Músculo Esquelético/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/genética , Análisis de Varianza , Anestésicos/farmacología , Temperatura Corporal , Cafeína/farmacología , ADN Complementario/genética , Acoplamiento Excitación-Contracción/genética , Fluorescencia , Genes Dominantes/genética , Humanos , Microelectrodos , Mutación Missense/genética , Retículo Sarcoplasmático/metabolismo
16.
J Neurosci ; 33(38): 15044-9, 2013 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-24048834

RESUMEN

The medial nucleus of the trapezoid body (MNTB) in the superior olivary complex (SOC) is an inhibitory hub considered critical for binaural sound localization. We show that genetic ablation of MNTB neurons in mice only subtly affects this ability by prolonging the minimum time required to detect shifts in sound location. Furthermore, glycinergic innervation of the SOC is maintained without an MNTB, consistent with the existence of parallel inhibitory inputs. These findings redefine the role of MNTB in sound localization and suggest that the inhibitory network is more complex than previously thought.


Asunto(s)
Glicina/metabolismo , Inhibición Neural/fisiología , Núcleo Olivar/citología , Núcleo Olivar/fisiología , Localización de Sonidos/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Estimulación Acústica , Animales , Animales Recién Nacidos , Vías Auditivas/fisiología , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Antagonistas de Aminoácidos Excitadores/farmacología , Lateralidad Funcional , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Proteínas de Homeodominio/genética , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibición Neural/efectos de los fármacos , Inhibición Neural/genética , Técnicas de Placa-Clamp , Localización de Sonidos/efectos de los fármacos , Estricnina/farmacología , Valina/análogos & derivados , Valina/farmacología
17.
FASEB J ; 27(3): 991-1000, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23159934

RESUMEN

Malignant hyperthermia (MH) susceptibility has been attributed to a leaky sarcoplasmic reticulum (SR) caused by missense mutations in RYR1 or CACNA1S, and the MH crisis has been attributed solely to massive self-sustaining release of Ca(2+) from SR stores elicited by triggering agents. Here, we show in muscle cells from MH-RyR1(R163C) knock-in mice that increased passive SR Ca(2+) leak causes an enlarged basal influx of sarcolemmal Ca(2+) that results in chronically elevated myoplasmic free Ca(2+) concentration ([Ca(2+)]i) at rest. We discovered that Gd(+3) and GsMTx-4 were more effective than BTP2 or expression of the dominant-negative Orai1(E190Q) in reducing both Ca(2+) entry and [Ca(2+)]i, implicating a non-STIM1/Orai1 SOCE pathway in resetting resting [Ca(2+)]i. Indeed, two nonselective cationic channels, TRPC3 and TRPC6, are overexpressed, and [Na]i is chronically elevated in MH-RyR1(R163C) muscle cells. [Ca(2+)]i and [Na(+)]i are persistently elevated in vivo and further increased by halothane in MH-RyR1(R163C/WT) muscle. These increases are markedly attenuated by local perfusion of Gd(+3) or GsMTx-4 and completely suppressed by dantrolene. These results contribute a new paradigm for understanding MH pathophysiology by demonstrating that nonselective sarcolemmal cation channel activity plays a critical role in causing myoplasmic Ca(2+) and Na(+) overload both at rest and during the MH crisis.-Eltit, J. M., Ding, X., Pessah, I. N., Allen, P. D., Lopez, J. R. Nonspecific sarcolemmal cation channels are critical for the pathogenesis of malignant hyperthermia.


Asunto(s)
Canales de Calcio/metabolismo , Hipertermia Maligna/metabolismo , Células Musculares/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo L , Cationes Bivalentes/metabolismo , Cationes Monovalentes/metabolismo , Hipertermia Maligna/genética , Hipertermia Maligna/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Células Musculares/patología , Mutación Missense , Proteína ORAI1 , Canal Liberador de Calcio Receptor de Rianodina/genética , Sarcolema/genética , Sarcolema/patología , Sodio/metabolismo , Molécula de Interacción Estromal 1
18.
Ear Hear ; 35(6): e282-90, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25127320

RESUMEN

OBJECTIVES: During the earlier years of the HIV/AIDS epidemic, initial reports described sensorineural hearing loss in up to 49% of individuals with HIV/AIDS. During those years, patients commonly progressed to advanced stages of HIV disease and frequently had neurological complications. However, the abnormalities on pure-tone audiometry and brainstem-evoked responses outlined in small studies were not always consistently correlated with advanced stages of HIV/AIDS. Moreover, these studies could not exclude the confounding effect of concurrent opportunistic infections and syphilis. Additional reports also have indicated that some antiretroviral medications may be ototoxic; thus, it has been difficult to make conclusions regarding the cause of changes in hearing function in HIV-infected patients. More recently, accelerated aging has been suggested as a potential explanation for the disproportionate increase in complications of aging described in many HIV-infected patients; hence, accelerated aging-associated hearing loss may also be playing a role in these patients. DESIGN: We conducted a large cross-sectional analysis of hearing function in over 300 patients with HIV-1 infection and in 137 HIV-uninfected controls. HIV-infected participants and HIV-uninfected controls underwent a 2-hr battery of hearing tests including the Hearing Handicap Inventory, standard audiometric pure-tone air and bone conduction testing, tympanometric testing, and speech reception and discrimination testing. RESULTS: Three-way analysis of variance (ANOVA) and logistic regression analysis of 278 eligible HIV-infected subjects stratified by disease stage in early HIV disease (n = 127) and late HIV disease (n = 148) and 120 eligible HIV-uninfected controls revealed no statistically significant differences among the three study groups in either overall 4-frequency pure-tone average (4-PTA) or hearing loss prevalence in either ear. Three-way ANOVA showed significant differences in word recognition scores in the right ear among groups, a significant group effect on tympanogram static admittance in both ears and a significant group effect on tympanic gradient in the right ear. There was significantly larger admittance and gradient in controls as compared to the HIV-infected group at late stage of disease. Hearing loss in the HIV-infected groups was associated with increased age and was similar to that described in the literature for the general population. Three-way ANOVA analysis also indicated significantly greater pure-tone thresholds (worse hearing) at low frequencies in HIV patients in the late stage of disease compared with HIV-uninfected controls. This difference was also found by semi-parametric mixed effects models. CONCLUSIONS: Despite reports of "premature" or "accelerated" aging in HIV-infected subjects, we found no evidence of hearing loss occurring at an earlier age in HIV-infected patients compared to HIV-uninfected controls. Similar to what is described in the general population, the probability of hearing loss increased with age in the HIV-infected subjects and was more common in patients over 60 years of age. Interestingly, HIV-infected subjects had worse hearing at lower frequencies and have significant differences in tympanometry compared to HIV-uninfected controls; these findings deserve further study.


Asunto(s)
Infecciones por VIH/epidemiología , Pérdida Auditiva Sensorineural/epidemiología , Pruebas de Impedancia Acústica , Síndrome de Inmunodeficiencia Adquirida/epidemiología , Adulto , Factores de Edad , Anciano , Audiometría de Tonos Puros , Estudios de Casos y Controles , Estudios Transversales , Femenino , VIH-1 , Pérdida Auditiva/epidemiología , Pérdida Auditiva/fisiopatología , Pérdida Auditiva Sensorineural/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Discriminación del Habla , Prueba del Umbral de Recepción del Habla , Adulto Joven
19.
Proc Natl Acad Sci U S A ; 108(17): 7046-51, 2011 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-21482776

RESUMEN

The skeletal muscle dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) are known to engage a form of conformation coupling essential for muscle contraction in response to depolarization, referred to as excitation-contraction coupling. Here we use WT and Ca(V)1.1 null (dysgenic) myotubes to provide evidence for an unexplored RyR1-DHPR interaction that regulates the transition of the RyR1 between gating and leak states. Using double-barreled Ca(2+)-selective microelectrodes, we demonstrate that the lack of Ca(V)1.1 expression was associated with an increased myoplasmic resting [Ca(2+)] ([Ca(2+)](rest)), increased resting sarcolemmal Ca(2+) entry, and decreased sarcoplasmic reticulum (SR) Ca(2+) loading. Pharmacological control of the RyR1 leak state, using bastadin 5, reverted the three parameters to WT levels. The fact that Ca(2+) sparks are not more frequent in dysgenic than in WT myotubes adds support to the hypothesis that the leak state is a conformation distinct from gating RyR1s. We conclude from these data that this orthograde DHPR-to-RyR1 signal inhibits the transition of gated RyR1s into the leak state. Further, it suggests that the DHPR-uncoupled RyR1 population in WT muscle has a higher propensity to be in the leak conformation. RyR1 leak functions are to keep [Ca(2+)](rest) and the SR Ca(2+) content in the physiological range and thus maintain normal intracellular Ca(2+) homeostasis.


Asunto(s)
Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Animales , Canales de Calcio Tipo L/genética , Señalización del Calcio/efectos de los fármacos , Éteres Difenilos Halogenados/farmacología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Ratones , Ratones Mutantes , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/citología , Canal Liberador de Calcio Receptor de Rianodina/genética , Sarcolema/genética
20.
Cells ; 13(3)2024 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-38334656

RESUMEN

Background: Postoperative cognitive dysfunction (POCD) is a common disorder after general anesthesia in elderly patients, the precise mechanisms of which remain unclear. Methods: We investigated the effect of isoflurane with or without dantrolene pretreatment on intracellular calcium concentration ([Ca2+]i), reactive oxygen species (ROS) production, cellular lactate dehydrogenase (LDH) leak, calpain activity, and cognitive function using the Morris water maze test of young (3 months), middle-aged (12-13 months), and aged (24-25 months) C57BL6/J mice. Results: Aged cortical and hippocampal neurons showed chronically elevated [Ca2+]i compared to young neurons. Furthermore, aged hippocampal neurons exhibited higher ROS production, increased LDH leak, and elevated calpain activity. Exposure to isoflurane exacerbated these markers in aged neurons, contributing to increased cognitive deficits in aged mice. Dantrolene pretreatment reduced [Ca2+]i for all age groups and prevented or significantly mitigated the effects of isoflurane on [Ca2+]i, ROS production, LDH leak, and calpain activity in aged neurons. Dantrolene also normalized or improved age-associated cognitive deficits and mitigated the cognitive deficits caused by isoflurane. Conclusions: These findings suggest that isoflurane-induced cytotoxicity and cognitive decline in aging are linked to disruptions in neuronal intracellular processes, highlighting the reduction of [Ca2+]i as a potential therapeutic intervention.


Asunto(s)
Anestesia , Anestésicos por Inhalación , Disfunción Cognitiva , Isoflurano , Fármacos Neuroprotectores , Ratones , Humanos , Animales , Persona de Mediana Edad , Anciano , Isoflurano/efectos adversos , Anestésicos por Inhalación/toxicidad , Fármacos Neuroprotectores/uso terapéutico , Calpaína , Especies Reactivas de Oxígeno/efectos adversos , Dantroleno/farmacología , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/inducido químicamente , Ratones Endogámicos C57BL , Neuronas
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