RESUMEN
In mammalian skeletal muscle, the propagation of surface membrane depolarization into the interior of the muscle fibre along the transverse (T) tubular network is essential for the synchronized release of calcium from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) in response to the conformational change in the voltage-sensor dihydropyridine receptors. Deficiency in 3-phosphoinositide phosphatase myotubularin (MTM1) has been reported to disrupt T-tubules, resulting in impaired SR calcium release. Here confocal calcium transients recorded in muscle fibres of MTM1-deficient mice were compared with the results from a model where propagation of the depolarization along the T-tubules was modelled mathematically with disruptions in the network assumed to modify the access and transmembrane resistance as well as the capacitance. If, in simulations, T-tubules were assumed to be partially or completely inaccessible to the depolarization and RyRs at these points to be prime for calcium-induced calcium release, all the features of measured SR calcium release could be reproduced. We conclude that the inappropriate propagation of the depolarization into the fibre interior is the initial critical cause of severely impaired SR calcium release in MTM1 deficiency, while the Ca2+ -triggered opening of RyRs provides an alleviating support to the diseased process. KEY POINTS: Myotubular myopathy is a fatal disease due to genetic deficiency in the phosphoinositide phosphatase MTM1. Although the causes are known and corresponding gene therapy strategies are being developed, there is no mechanistic understanding of the disease-associated muscle function failure. Resolving this issue is of primary interest not only for a fundamental understanding of how MTM1 is critical for healthy muscle function, but also for establishing the related cellular mechanisms most primarily or stringently affected by the disease, which are thus of potential interest as therapy targets. The mathematical modelling approach used in the present work proves that the disease-associated alteration of the plasma membrane invagination network is sufficient to explain the dysfunctions of excitation-contraction coupling, providing the first integrated quantitative framework that explains the associated contraction failure.
Asunto(s)
Calcio , Músculo Esquelético , Animales , Ratones , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Calcio de la Dieta , Mamíferos/metabolismo , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMEN
The remodelling of the extracellular matrix plays an important role in skeletal muscle development and regeneration. Syndecan-4 is a cell surface proteoglycan crucial for muscle differentiation. Syndecan-4-/- mice have been reported to be unable to regenerate following muscle damage. To investigate the consequences of the decreased expression of Syndecan-4, we have studied the in vivo and in vitro muscle performance and the excitation-contraction coupling machinery in young and aged Syndecan-4+/- (SDC4) mice. In vivo grip force was decreased significantly as well as the average and maximal speed of voluntary running in SDC4 mice, regardless of their age. The maximal in vitro twitch force was reduced in both EDL and soleus muscles from young and aged SDC4 mice. Ca2+ release from the sarcoplasmic reticulum decreased significantly in the FDB fibres of young SDC4 mice, while its voltage dependence was unchanged regardless of age. These findings were present in muscles from young and aged mice as well. On C2C12 murine skeletal muscle cells, we have also found altered calcium homeostasis upon Syndecan-4 silencing. The decreased expression of Syndecan-4 leads to reduced skeletal muscle performance in mice and altered motility in C2C12 myoblasts via altered calcium homeostasis. The altered muscle force performance develops at an early age and is maintained throughout the life course of the animal until old age.
Asunto(s)
Músculo Esquelético , Sindecano-4 , Animales , Ratones , Calcio/metabolismo , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Sindecano-4/genética , Sindecano-4/metabolismoRESUMEN
The endocannabinoid system (ECS) refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. Cannabinoid receptors (CBRs) are highly expressed in the central nervous system and many peripheral tissues. Evidence suggests that CB1Rs are expressed in human and murine skeletal muscle mainly in the cell membrane, but a subpopulation is present also in the mitochondria. However, very little is known about the latter population. To date, the connection between the function of CB1Rs and the regulation of intracellular Ca2+ signaling has not been investigated yet. Tamoxifen-inducible skeletal muscle-specific conditional CB1 knock-down (skmCB1-KD, hereafter referred to as Cre+/-) mice were used in this study for functional and morphological analysis. After confirming CB1R down-regulation on the mRNA and protein level, we performed in vitro muscle force measurements and found that peak twitch, tetanus, and fatigue were decreased significantly in Cre+/- mice. Resting intracellular calcium concentration, voltage dependence of the calcium transients as well as the activity dependent mitochondrial calcium uptake were essentially unaltered by Cnr1 gene manipulation. Nevertheless, we found striking differences in the ultrastructural architecture of the mitochondrial network of muscle tissue from the Cre+/- mice. Our results suggest a role of CB1Rs in maintaining physiological muscle function and morphology. Targeting ECS could be a potential tool in certain diseases, including muscular dystrophies where increased endocannabinoid levels have already been described.
Asunto(s)
Calcio , Endocannabinoides , Receptor Cannabinoide CB1 , Animales , Ratones , Calcio/metabolismo , Músculo Esquelético/metabolismo , Receptor Cannabinoide CB1/genética , Transducción de SeñalRESUMEN
Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum's Ca2+ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca2+ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.
Asunto(s)
Calcio/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Sarcómeros/metabolismo , Animales , Ancirinas/metabolismo , Conectina/metabolismo , Conectina/fisiología , Masculino , Ratones , Ratones Noqueados , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Condicionamiento Físico Animal , Proteínas Serina-Treonina Quinasas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Sarcómeros/fisiología , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.
Asunto(s)
Cartílago Articular/metabolismo , Movimiento Celular , Condrocitos/metabolismo , Canales Iónicos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Osteoartritis de la Rodilla/metabolismo , Células Madre/metabolismo , Transcriptoma , Señalización del Calcio , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Condrocitos/efectos de los fármacos , Condrocitos/patología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Perfilación de la Expresión Génica , Humanos , Canales Iónicos/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Potenciales de la Membrana , Proteínas de Transporte de Membrana/genética , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/patología , Bloqueadores de los Canales de Potasio/farmacología , Células Madre/efectos de los fármacos , Células Madre/patología , Factores de TiempoRESUMEN
Skeletal muscle secrets several bioactive molecules known as myokines. Interleukin-6 (IL-6) has been described as a myokine secreted in response to skeletal muscle injury as well as to macrophage invasion in inflammation. To our knowledge no connection between macrophages and skeletal muscle regarding IL-6 secretion has been described so far. Here we report that co-culturing of C2C12 cells with RAW macrophages enhances IL-6 secretion of the cells cultured together. However, this is not seen in cross-feeding experiments, where culture medium of RAW macrophage culture is used as the culture medium of C2C12 cells or vice versa. Pravastatin, known to induce myopathy, also stimulates IL-6 production in monocultured C2C12 cells and elevates IL-6 concentration in the culture medium of the co-cultures. These results indicate an intricate interaction between skeletal muscle and macrophages in inflammation related to IL-6 production.
Asunto(s)
Interleucina-6 , Pravastatina , Humanos , Inflamación , Macrófagos , Músculo Esquelético , Pravastatina/farmacologíaRESUMEN
Muscular dystrophies are a group of more than 160 different human neuromuscular disorders characterized by a progressive deterioration of muscle mass and strength. The causes, symptoms, age of onset, severity, and progression vary depending on the exact time point of diagnosis and the entity. Congenital myopathies are rare muscle diseases mostly present at birth that result from genetic defects. There are no known cures for congenital myopathies; however, recent advances in gene therapy are promising tools in providing treatment. This review gives an overview of the mouse models used to investigate the most common muscular dystrophies and congenital myopathies with emphasis on their potentials and limitations in respect to human applications.
Asunto(s)
Terapia Genética , Ratones Transgénicos/genética , Distrofias Musculares/genética , Miopatías Estructurales Congénitas/genética , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Distrofias Musculares/patología , Distrofias Musculares/terapia , Miopatías Estructurales Congénitas/patología , Miopatías Estructurales Congénitas/terapiaRESUMEN
Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease.
Asunto(s)
Potenciales de Acción/fisiología , Canales de Calcio Tipo L/genética , Acoplamiento Excitación-Contracción/genética , Fibras Musculares de Contracción Rápida/citología , Fibras Musculares de Contracción Lenta/citología , Debilidad Muscular/genética , Músculo Esquelético/embriología , Empalme Alternativo/genética , Animales , Calcio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Debilidad Muscular/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
Very recently, the diamide insecticide chlorantraniliprole was shown to induce Ca2+-release from sarcoplasmic reticulum (SR) vesicles isolated from mammalian skeletal muscle through the activation of the SR Ca2+ channel ryanodine receptor. As this result raises severe concerns about the safety of this chemical, we aimed to learn more about its action. To this end, single-channel analysis was performed, which showed that chlorantraniliprole induced high-activity bursts of channel opening that accounts for the Ca2+-releasing action described before.
Asunto(s)
Insecticidas , Canal Liberador de Calcio Receptor de Rianodina , ortoaminobenzoatos , Animales , Calcio , Diamida , Insecticidas/farmacología , Músculo Esquelético , Rianodina , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Retículo Sarcoplasmático , ortoaminobenzoatos/farmacologíaRESUMEN
Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the Ank1 gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca2+ handling, cytosolic Ca2+ levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca2+ content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca2+ transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca2+ release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca2+ release, likely as a consequence of a decreased Ca2+ store due to the reduction of the SR volume in sAnk1 KO muscle fibers.
Asunto(s)
Ancirinas/metabolismo , Calcio/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Ancirinas/genética , Masculino , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/metabolismo , Retículo Sarcoplasmático/genéticaRESUMEN
Aspergillus fumigatus is an opportunistic pathogen, the leading cause of invasive and disseminated aspergillosis in systemic immunocompromised patients, and an important cause of mortality. The aim of the present study was to adapt a pulmonary aspergillosis murine model, to determine pathodynamical parameters quantitatively, and to follow the progression of fungal infection in vivo. The nasal inoculation of Aspergillus conidia in mice previously subjected to immunosuppression with cyclophosphamide (CP) turned out to be a more suitable model than that of immunosuppressed with hydrocortisone (HC). The following parameters were found to correlate quantitatively with the progress of the infection: (i) survival rate, (ii) weight loss of mice, (iii) infected focal plaque size, (iv) hyphal density, (v) hyphal length distribution of A. fumigatus, and the (vi) the histopathological status and scores. These parameters will be essential elements for the development of antifungal drugs and therapies, and important for the investigation of the pathogenicity in different strains of A. fumigatus.
Asunto(s)
Aspergillus fumigatus/crecimiento & desarrollo , Modelos Animales de Enfermedad , Hifa/crecimiento & desarrollo , Aspergilosis Pulmonar Invasiva/microbiología , Aspergilosis Pulmonar Invasiva/patología , Animales , Peso Corporal , Recuento de Colonia Microbiana , Histocitoquímica , Huésped Inmunocomprometido , Ratones , Índice de Severidad de la Enfermedad , Análisis de SupervivenciaRESUMEN
Store-operated Ca2+ entry (SOCE) is a Ca2+-entry process activated by the depletion of intracellular stores and has an important role in many cell types. In skeletal muscle, however, its role during physiological muscle activation has been controversial. To address this question, sarcoplasmic reticulum (SR) calcium release in a mouse strain with a naturally occurring mutation in the myostatin gene (Compact (Cmpt)) leading to a hypermuscular yet reduced muscle-force phenotype was compared to that in wild-type mice. To elicit Ca2+ release from the SR of flexor digitorum brevis (FDB) fibers, either a ryanodine receptor agonist (4-chloro-meta-cresol) or depolarizing pulses were used. In muscles from Cmpt mice, endogenous protein levels of STIM1 and Orai1 were reduced, and consequently, SOCE after 4-chloro-meta-cresol-induced store depletion was suppressed. Although the voltage dependence of SR calcium release was not statistically different between wild-type and Cmpt fibers, the amount of releasable calcium was significantly reduced in the latter, indicating a smaller SR content. To assess the immediate role of SOCE in replenishing the SR calcium store, the evolution of intracellular calcium concentration during a train of long-lasting depolarizations to a maximally activating voltage was monitored. Cmpt mice exhibited a faster decline in calcium release, suggesting a compromised ability to refill the SR. A simple model that incorporates a reduced SOCE as an important partner in regulating immediate calcium influx through the surface membrane readily accounts for the steady-state reduction in SR calcium content and its more pronounced decline after calcium release.
Asunto(s)
Calcio/metabolismo , Fibras Musculares Esqueléticas/citología , Retículo Sarcoplasmático/metabolismo , Animales , Fenómenos Electrofisiológicos , Masculino , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiología , Mutación , Miostatina/genéticaRESUMEN
KEY POINTS: Dynamin 2 is a ubiquitously expressed protein involved in membrane trafficking processes. Mutations in the gene encoding dynamin 2 are responsible for a congenital myopathy associated with centrally located nuclei in the muscle fibres. Using muscle fibres from a mouse model of the most common mutation responsible for this disease in humans, we tested whether altered Ca2+ signalling and excitation-contraction coupling contribute to muscle weakness. The plasma membrane network that carries the electrical excitation is moderately perturbed in the diseased muscle fibres. The excitation-activated Ca2+ input fluxes across both the plasma membrane and the membrane of the sarcoplasmic reticulum are defective in the diseased fibres, which probably contributes to muscle weakness in patients. ABSTRACT: Mutations in the gene encoding dynamin 2 (DNM2) are responsible for autosomal dominant centronuclear myopathy (AD-CNM). We studied the functional properties of Ca2+ signalling and excitation-contraction (EC) coupling in muscle fibres isolated from a knock-in (KI) mouse model of the disease, using confocal imaging and the voltage clamp technique. The transverse-tubule network organization appeared to be unaltered in the diseased fibres, although its density was reduced by â¼10% compared to that in control fibres. The density of Ca2+ current through CaV1.1 channels and the rate of voltage-activated sarcoplasmic reticulum Ca2+ release were reduced by â¼60% and 30%, respectively, in KI vs. control fibres. In addition, Ca2+ release in the KI fibres reached its peak value 10-50 ms later than in control ones. Activation of Ca2+ transients along the longitudinal axis of the fibres was more heterogeneous in the KI than in the control fibres, with the difference being exacerbated at intermediate membrane voltages. KI fibres exhibited spontaneous Ca2+ release events that were almost absent from control fibres. Overall, the results of the present study demonstrate that Ca2+ signalling and EC coupling exhibit a number of dysfunctions likely contributing to muscle weakness in DNM2-related AD-CNM.
Asunto(s)
Dinamina II/genética , Acoplamiento Excitación-Contracción , Fibras Musculares Esqueléticas/metabolismo , Miopatías Estructurales Congénitas/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Células Cultivadas , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/fisiología , Mutación Missense , Miopatías Estructurales Congénitas/genética , Miopatías Estructurales Congénitas/fisiopatologíaRESUMEN
KEY POINTS: Marijuana was found to cause muscle weakness, although the exact regulatory role of its receptors (CB1 cannabinoid receptor; CB1R) in the excitation-contraction coupling (ECC) of mammalian skeletal muscle remains unknown. We found that CB1R activation or its knockout did not affect muscle force directly, whereas its activation decreased the Ca2+ -sensitivity of the contractile apparatus and made the muscle fibres more prone to fatigue. We demonstrate that CB1Rs are not connected to the inositol 1,4,5-trisphosphate pathway either in myotubes or in adult muscle fibres. By contrast, CB1Rs constitutively inhibit sarcoplasmic Ca2+ release and sarcoplasmic reticulum Ca2+ ATPase during ECC in a Gi/o protein-mediated way in adult skeletal muscle fibres but not in myotubes. These results help with our understanding of the physiological effects and pathological consequences of CB1R activation in skeletal muscle and may be useful in the development of new cannabinoid drugs. ABSTRACT: Marijuana was found to cause muscle weakness, although it is unknown whether it affects the muscles directly or modulates only the motor control of the central nervous system. Although the presence of CB1 cannabinoid receptors (CB1R), which are responsible for the psychoactive effects of the drug in the brain, have recently been demonstrated in skeletal muscle, it is unclear how CB1R-mediated signalling affects the contraction and Ca²âº homeostasis of mammalian skeletal muscle. In the present study, we demonstrate that in vitro CB1R activation increased muscle fatigability and decreased the Ca2+ -sensitivity of the contractile apparatus, whereas it did not alter the amplitude of single twitch contractions. In myotubes, CB1R agonists neither evoked, nor influenced inositol 1,4,5-trisphosphate (IP3 )-mediated Ca2+ transients, nor did they alter excitation-contraction coupling. By contrast, in isolated muscle fibres of wild-type mice, although CB1R agonists did not evoke IP3 -mediated Ca2+ transients too, they significantly reduced the amplitude of the depolarization-evoked transients in a pertussis-toxin sensitive manner, indicating a Gi/o protein-dependent mechanism. Concurrently, on skeletal muscle fibres isolated from CB1R-knockout animals, depolarization-evoked Ca2+ transients, as well qas Ca2+ release flux via ryanodine receptors (RyRs), and the total amount of released Ca2+ was significantly greater than that from wild-type mice. Our results show that CB1R-mediated signalling exerts both a constitutive and an agonist-mediated inhibition on the Ca2+ transients via RyR, regulates the activity of the sarcoplasmic reticulum Ca2+ ATPase and enhances muscle fatigability, which might decrease exercise performance, thus playing a role in myopathies, and therefore should be considered during the development of new cannabinoid drugs.
Asunto(s)
Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Músculo Esquelético/fisiología , Receptor Cannabinoide CB1/fisiología , Retículo Sarcoplasmático/metabolismo , Animales , Benzoxazinas/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Fatiga Muscular/fisiología , Músculo Esquelético/efectos de los fármacos , Naftalenos/farmacología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Myostatin, a member of the transforming growth factor ß family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (Mstn(Cmpt-dl1Abc) or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa-force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca(2+) concentration ([Ca(2+)]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nM, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nM, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mM s(-1)) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mM s(-1)) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.
Asunto(s)
Señalización del Calcio/genética , Hipertonía Muscular/genética , Mutación , Miostatina/genética , Animales , Señalización del Calcio/fisiología , Potenciales Evocados , Acoplamiento Excitación-Contracción/genética , Acoplamiento Excitación-Contracción/fisiología , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Fibras Musculares Esqueléticas/fisiología , Hipertonía Muscular/fisiopatología , Miostatina/fisiologíaRESUMEN
Skeletal muscle excitationcontraction (EC) coupling is altered in several models of phosphatidylinositol phosphate (PtdInsP) phosphatase deficiency and ryanodine receptor activity measured in vitro was reported to be affected by certain PtdInsPs, thus prompting investigation of the physiological role of PtdInsPs in EC coupling. We measured intracellular Ca2+ transients in voltage-clamped mouse muscle fibres microinjected with a solution containing a PtdInsP substrate (PtdIns(3,5)P2 or PtdIns(3)P) or product (PtdIns(5)P or PtdIns) of the myotubularin phosphatase MTM1. No significant change was observed in the presence of either PtdIns(5)P or PtdIns but peak SR Ca2+ release was depressed by ~30% and 50% in fibres injected with PtdIns(3,5)P2 and PtdIns(3)P, respectively, with no concurrent alteration in the membrane current signals associated with the DHPR function as well as in the voltage dependence of Ca2+ release inactivation. In permeabilized muscle fibres, the frequency of spontaneous Ca2+ release events was depressed in the presence of the three tested phosphorylated forms of PtdInsP with PtdIns(3,5)P2 being the most effective, leading to an almost complete disappearance of Ca2+ release events. Results support the possibility that pathological accumulation of MTM1 substrates may acutely depress ryanodine receptor-mediated Ca2+ release. Overexpression of a mCherry-tagged form of MTM1 in muscle fibres revealed a striated pattern consistent with the triadic area. Ca2+ release remained although unaffected by MTM1 overexpression and was also unaffected by the PtdIns-3-kinase inhibitor LY2940002, suggesting that the 3-phosphorylated PtdIns lipids active on voltage-activated Ca2+ release are inherently maintained at a low level, inefficient on Ca2+ release in normal conditions.
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Potenciales de Acción , Calcio/metabolismo , Acoplamiento Excitación-Contracción , Fibras Musculares Esqueléticas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Animales , Canales de Calcio/metabolismo , Ratones , Fibras Musculares Esqueléticas/fisiología , Proteínas Tirosina Fosfatasas no Receptoras/genéticaRESUMEN
The antifungal protein of Penicillium chrysogenum (PAF) inhibits the growth of important pathogenic filamentous fungi, including members of the Aspergillus family and some dermatophytes. Furthermore, PAF was proven to have no toxic effects on mammalian cells in vitro. To prove that PAF could be safely used in therapy, experiments were carried out to investigate its in vivo effects. Adult mice were inoculated with PAF intranasally in different concentrations, up to 2700 µg·kg⻹ daily, for 2 weeks. Even at the highest concentration--a concentration highly toxic in vitro for all affected molds used, animals neither died due to the treatment nor were any side effects observed. Histological examinations did not find pathological reactions in the liver, in the kidney, and in the lungs. Mass spectrometry confirmed that a measurable amount of PAF was accumulated in the lungs after the treatment. Lung tissue extracts from PAF treated mice exerted significant antifungal activity. Small-animal positron emission tomography revealed that neither the application of physiological saline nor that of PAF induced any inflammation while the positive control lipopolysaccharide did. The effect of the drug on the skin was examined in an irritative dermatitis model where the change in the thickness of the ears following PAF application was found to be the same as in control and significantly less than when treated with phorbol-12-myristate-13-acetate used as positive control. Since no toxic effects of PAF were found in intranasal application, our result is the first step for introducing PAF as potential antifungal drug in therapy.
Asunto(s)
Antifúngicos/administración & dosificación , Proteínas Fúngicas/administración & dosificación , Penicillium chrysogenum/metabolismo , Administración por Inhalación , Animales , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Antifúngicos/toxicidad , Relación Dosis-Respuesta a Droga , Femenino , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/toxicidad , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Pulmón/diagnóstico por imagen , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Tomografía de Emisión de Positrones , Medición de Riesgo , Piel/efectos de los fármacos , Factores de Tiempo , Pruebas de ToxicidadRESUMEN
In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²âº released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Proteínas Musculares/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Línea Celular , Células HEK293 , Humanos , Datos de Secuencia Molecular , Contracción Muscular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/aislamiento & purificación , Músculo Esquelético/metabolismo , NAD/metabolismo , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Distribución TisularRESUMEN
Septin7 as a unique member of the GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be essential in the formation of hetero-oligomeric septin complexes. As a cytoskeletal component, Septin7 is involved in many important cellular processes. However, its contribution in striated muscle physiology is poorly described. In skeletal muscle, a highly orchestrated process of migration is crucial in the development of functional fibers and in regeneration. Here, we describe the pronounced appearance of Septin7 filaments and a continuous change of Septin7 protein architecture during the migration of myogenic cells. In Septin7 knockdown C2C12 cultures, the basic parameters of migration are significantly different, and the intracellular calcium concentration change in migrating cells are lower compared to that of scrambled cultures. Using a plant cytokinin, forchlorfenuron, to dampen septin dynamics, the altered behavior of the migrating cells is described, where Septin7-depleted cells are more resistant to the treatment. These results indicate the functional relevance of Septin7 in the migration of myoblasts, implying its contribution to muscle myogenesis and regeneration.
Asunto(s)
Músculo Esquelético , Septinas , Línea Celular , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Septinas/metabolismo , Animales , RatonesRESUMEN
Basic leucine zipper (bZIP) transcription factors are crucial components of differentiation, cellular homeostasis and the environmental stress defense of eukaryotes. In this work, we further studied the consequence of gene deletion and overexpression of two bZIP transcription factors, NapA and RsmA, on superoxide production, mitochondrial morphology and hyphal diameter of Aspergillus nidulans. We have found that reactive oxygen species production was influenced by both gene deletion and overexpression of napA under tert-butylhydroperoxide (tBOOH) elicited oxidative stress. Furthermore, gene expression of napA negatively correlated with mitochondrial volumetric ratio as well as sterigmatocystin production of A. nidulans. High rsmA expression was accompanied with elevated relative superoxide ratio in the second hyphal compartment. A negative correlation between the expression of rsmA and catalase enzyme activity or mitochondrial volumetric ratio was also confirmed by statistical analysis. Hyphal diameter was independent on either rsmA and napA expression as well as 0.2 mM tBOOH treatment.