RESUMEN
Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin. Prior work has shown that DMD progression can vary, depending on the genetic makeup of the patient. Several modifier alleles have been identified including LTBP4 and SPP1. We previously showed that Spp1 exacerbates the DMD phenotype in the mdx mouse model by promoting fibrosis and by skewing macrophage polarization. Here, we studied the mechanisms involved in Spp1's promotion of fibrosis by using both isolated fibroblasts and genetically modified mice. We found that Spp1 upregulates collagen expression in mdx fibroblasts by enhancing TGFß signaling. Spp1's effects on TGFß signaling are through induction of MMP9 expression. MMP9 is a protease that can release active TGFß ligand from its latent complex. In support for activation of this pathway in our model, we showed that treatment of mdx fibroblasts with MMP9 inhibitor led to accumulation of the TGFß latent complex, decreased levels of active TGFß and reduced collagen expression. Correspondingly, we found reduced active TGFß in Spp1-/-mdxB10 and Mmp9-/-mdxB10 muscles in vivo. Taken together with previous observations of reduced fibrosis in both models, these data suggest that Spp1 acts upstream of TGFß to promote fibrosis in mdx muscles. We found that in the context of constitutively upregulated TGFß signaling (such as in the mdxD2 model), ablation of Spp1 has very little effect on fibrosis. Finally, we performed proof-of-concept studies showing that postnatal pharmacological inhibition of Spp1 reduces fibrosis and improves muscle function in mdx mice.
Asunto(s)
Fibrosis/genética , Distrofia Muscular de Duchenne/metabolismo , Osteopontina/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Colágeno Tipo I/biosíntesis , Modelos Animales de Enfermedad , Distrofina/genética , Distrofina/metabolismo , Matriz Extracelular/metabolismo , Femenino , Fibroblastos/metabolismo , Fibrosis/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Osteopontina/metabolismo , Cultivo Primario de Células , Regeneración/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The spatiotemporal properties of the Ca(2+)-release process in skeletal muscle fibers from normal and mdx fibers were determined using the confocal-spot detection technique. The Ca(2+) indicator OGB-5N was used to record action potential-evoked fluorescence signals at consecutive locations separated by 200 nm along multiple sarcomeres of FDB fibers loaded with 10- and 30-mM EGTA. Three-dimensional reconstructions of fluorescence transients demonstrated the existence of microdomains of increased fluorescence around the Ca(2+)-release sites in both mouse strains. The Ca(2+) microdomains in mdx fibers were regularly spaced along the fiber axis, displaying a distribution similar to that seen in normal fibers. Nevertheless, both preparations differed in that in 10-mM EGTA Ca(2+) microdomains had smaller amplitudes and were wider in mdx fibers than in controls. In addition, Ca(2+)-dependent fluorescence transients recorded at selected locations within the sarcomere of mdx muscle fibers were not only smaller, but also slower than their counterparts in normal fibers. Notably, differences in the spatial features of the Ca(2+) microdomains recorded in mdx and normal fibers, but not in the amplitude and kinetics of the Ca(2+) transients, were eliminated in 30-mM EGTA. Our results consistently demonstrate that Ca(2+)-release flux calculated from release sites in mdx fibers is uniformly impaired with respect to those normal fibers. The Ca(2+)-release reduction is consistent with that previously measured using global detection techniques.
Asunto(s)
Calcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Potenciales de Acción , Animales , Quelantes/farmacología , Ácido Egtácico/farmacología , Indicadores y Reactivos/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Microscopía Confocal , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Sarcómeros/metabolismoRESUMEN
The double knockout mouse for utrophin and dystrophin (utr(-/-)/mdx) has been proposed to be a better model of Duchenne Muscular Dystrophy (DMD) than the mdx mouse because the former displays more similar muscle pathology to that of the DMD patients. In this paper the properties of action potentials (APs) and Ca(2+) transients elicited by single and repetitive stimulation were studied to understand the excitation-contraction (EC) coupling alterations observed in muscle fibers from mdx and utr(-/-)/mdx mice. Based on the comparison of the AP durations with those of fibers from wild-type (WT) mice, fibers from both mdx and utr(-/-)/mdx mice could be divided in two groups: fibers with WT-like APs (group 1) and fibers with significantly longer APs (group 2). Although the proportion of fibers in group 2 was larger in utr(-/-)/mdx (36%) than in mdx mice (27%), the Ca(2+) release elicited by single stimulation was found to be similarly depressed (32-38%) in utr(-/-)/mdx and mdx fibers compared with WT counterparts regardless of the fiber's group. Stimulation at 100 Hz revealed that, with the exception of those from utr(-/-)/mdx mice, group 1 fibers were able to sustain Ca(2+) release for longer than group 2 fibers, which displayed an abrupt limitation even at the onset of the train. The differences in behavior between fibers in groups 1 and 2 became almost unnoticeable at 50 Hz stimulation. In general, fibers from utr(-/-)/mdx mice seem to display more persistent alterations in the EC coupling than those observed in the mdx model.
Asunto(s)
Distrofina/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Fibras Musculares Esqueléticas/fisiología , Utrofina/metabolismo , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Distrofina/genética , Electrofisiología , Acoplamiento Excitación-Contracción/genética , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Utrofina/genéticaRESUMEN
Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC(4)(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC(4)(5). The peak DeltaF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC(4)(5) exhibit DeltaF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.
Asunto(s)
Microtúbulos/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Potenciales de Acción/fisiología , Algoritmos , Animales , Membrana Celular/metabolismo , Interpretación Estadística de Datos , Electrofisiología , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/citología , Plásmidos , Potenciometría , Prenilación , TransfecciónRESUMEN
BACKGROUND: Sarcospan (SSPN) is a transmembrane protein that interacts with the sarcoglycans (SGs) to form a tight subcomplex within the dystrophin-glycoprotein complex that spans the sarcolemma and interacts with laminin in the extracellular matrix. Overexpression of SSPN ameliorates Duchenne muscular dystrophy in murine models. METHODS: Standard cloning approaches were used to identify nanospan, and nanospan-specific polyclonal antibodies were generated and validated. Biochemical isolation of skeletal muscle membranes and two-photon laser scanning microscopy were used to analyze nanospan localization in muscle from multiple murine models. Duchenne muscular dystrophy biopsies were analyzed by immunoblot analysis of protein lysates as well as indirect immunofluorescence analysis of muscle cryosections. RESULTS: Nanospan is an alternatively spliced isoform of sarcospan. While SSPN has four transmembrane domains and is a core component of the sarcolemmal dystrophin-glycoprotein complex, nanospan is a type II transmembrane protein that does not associate with the dystrophin-glycoprotein complex. We demonstrate that nanospan is enriched in the sarcoplasmic reticulum (SR) fractions and is not present in the T-tubules. SR fractions contain membranes from three distinct structural regions: a region flanking the T-tubules (triadic SR), a SR region across the Z-line (ZSR), and a longitudinal SR region across the M-line (LSR). Analysis of isolated murine muscles reveals that nanospan is mostly associated with the ZSR and triadic SR, and only minimally with the LSR. Furthermore, nanospan is absent from the SR of δ-SG-null (Sgcd-/-) skeletal muscle, a murine model for limb girdle muscular dystrophy 2F. Analysis of skeletal muscle biopsies from Duchenne muscular dystrophy patients reveals that nanospan is preferentially expressed in type I (slow) fibers in both control and Duchenne samples. Furthermore, nanospan is significantly reduced in Duchenne biopsies. CONCLUSIONS: Alternative splicing of proteins from the SG-SSPN complex produces δ-SG3, microspan, and nanospan that localize to the ZSR and the triadic SR, where they may play a role in regulating resting calcium levels as supported by previous studies (Estrada et al., Biochem Biophys Res Commun 340:865-71, 2006). Thus, alternative splicing of SSPN mRNA generates three protein isoforms (SSPN, microspan, and nanospan) that differ in the number of transmembrane domains affecting subcellular membrane association into distinct protein complexes.
Asunto(s)
Empalme Alternativo , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Sarcoglicanopatías/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Proteínas Portadoras/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Sarcoglicanopatías/genética , Sarcoglicanopatías/patología , Sarcoglicanos/genética , Retículo Sarcoplasmático/ultraestructuraRESUMEN
In the degenerative disease Duchenne muscular dystrophy, inflammatory cells enter muscles in response to repetitive muscle damage. Immune factors are required for muscle regeneration, but chronic inflammation creates a profibrotic milieu that exacerbates disease progression. Osteopontin (OPN) is an immunomodulator highly expressed in dystrophic muscles. Ablation of OPN correlates with reduced fibrosis and improved muscle strength as well as reduced natural killer T (NKT) cell counts. Here, we demonstrate that the improved dystrophic phenotype observed with OPN ablation does not result from reductions in NKT cells. OPN ablation skews macrophage polarization toward a pro-regenerative phenotype by reducing M1 and M2a and increasing M2c subsets. These changes are associated with increased expression of pro-regenerative factors insulin-like growth factor 1, leukemia inhibitory factor, and urokinase-type plasminogen activator. Furthermore, altered macrophage polarization correlated with increases in muscle weight and muscle fiber diameter, resulting in long-term improvements in muscle strength and function in mdx mice. These findings suggest that OPN ablation promotes muscle repair via macrophage secretion of pro-myogenic growth factors.
Asunto(s)
Macrófagos/metabolismo , Distrofia Muscular Animal/patología , Osteopontina/fisiología , Animales , Polaridad Celular , Macrófagos/citología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculos/metabolismo , Músculos/patología , Músculos/fisiología , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/fisiología , Osteopontina/genética , Osteopontina/metabolismo , Fenotipo , RegeneraciónRESUMEN
A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver, skeletal muscle, and even the brain. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP, calpain, FKBP12, beta2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and alpha-actinin; and membrane proteins, i.e. alpha1s-DHPR, RyR1, cardiac Na/Ca(2+) exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical and functional evidence. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions.
Asunto(s)
ADN/administración & dosificación , Electroporación/métodos , Músculo Esquelético/fisiología , Transfección/métodos , Animales , ADN/genética , Ratones , RatasRESUMEN
The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.
Asunto(s)
Clonación Molecular/métodos , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Animales , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Miembro Posterior , Extremidad Inferior , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Proteínas Musculares/biosíntesis , Proteínas Musculares/química , Proteínas Musculares/genéticaRESUMEN
Ca(2)(+) transients elicited by action potentials were measured using MagFluo-4, at 20-22 degrees C, in intact muscle fibres enzymatically dissociated from mice of different ages (7, 10, 15 and 42 days). The rise time of the transient (time from 10 to 90% of the peak) was 2.4 and 1.1 ms in fibres of 7- and 42-day-old mice, respectively. The decay of the transient was described by a double exponential function, with time constants of 1.8 and 16.4 ms in adult, and of 4.6 and 105 ms in 7-day-old animals. The fractional recovery of the transient peak amplitude after 10 ms, F(2(10))/F(1), determined using twin pulses, was 0.53 for adult fibres and ranged between 0.03 and 0.60 in fibres of 7-day-old animals This large variance may indicate differences in the extent of inactivation of Ca(2)(+) release, possibly related to the difference in ryanodine receptor composition between young and old fibres. At the 7 and 10 day stages, fibres responded to Ca(2)(+)-free solutions with a larger decrease in the transient peak amplitude (25% versus 11% in adult fibres), possibly indicating a contribution of Ca(2)(+) influx to the Ca(2)(+) transient in younger animals. Cyclopiazonic acid (1 mum), an inhibitor of the sarcoplasmic reticulum (SR) Ca(2)(+)-ATPase, abolished the Ca(2)(+) transient decay in fibres of 7- and 10-day-old animals and significantly reduced its rate in older animals. Analysis of the transients with a Ca(2)(+) removal model showed that the results are consistent with a larger relative contribution of the SR Ca(2)(+) pump and a lower expression of myoplasmic Ca(2)(+) buffers in fibres of young versus old animals.
Asunto(s)
Señalización del Calcio/fisiología , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Calcio/farmacología , Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Técnicas In Vitro , Indoles/farmacología , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacosRESUMEN
Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation-contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca2+ release by measuring Ca2+ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca2+ release elicited by voltage pulses was reduced by approximately 67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca2+ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca2+ may be altered in the mdx mouse.