RESUMEN
Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADP-stimulated mitochondrial respiration in situ using saponin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADP-stimulated oxygen consumption and the dissociation constant (K(m)) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant role in mitochondrial positioning and respiratory function in cardiac and skeletal muscle.
Asunto(s)
Respiración de la Célula/fisiología , Desmina/genética , Filamentos Intermedios/metabolismo , Mitocondrias/metabolismo , Miocardio/metabolismo , Adenosina Difosfato/farmacología , Animales , Cardiomiopatías/metabolismo , Desmina/metabolismo , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Filamentos Intermedios/ultraestructura , Ratones , Ratones Noqueados , Microscopía Electrónica , Mitocondrias/ultraestructura , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Miocardio/citologíaRESUMEN
Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystem disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed severe disruption of muscle architecture and degeneration. Structural abnormalities included loss of lateral alignment of myofibrils and abnormal mitochondrial organization. The consequences of these abnormalities were most severe in the heart, which exhibited progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. The present data demonstrate the essential role of desmin in the maintenance of myofibril, myofiber, and whole muscle tissue structural and functional integrity, and show that the absence of desmin leads to muscle degeneration.
Asunto(s)
Desmina/fisiología , Músculo Esquelético/ultraestructura , Músculo Liso/ultraestructura , Miocardio/ultraestructura , Animales , Desmina/genética , Marcación de Gen , Proteínas de Filamentos Intermediarios/biosíntesis , Ratones , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Sarcómeros/metabolismoRESUMEN
The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.
Asunto(s)
Fusión Celular , Desmina/biosíntesis , Expresión Génica/efectos de los fármacos , Músculos/fisiología , Proteína MioD/biosíntesis , Miogenina/biosíntesis , ARN sin Sentido/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Secuencia de Consenso , ADN Complementario/metabolismo , Desmina/antagonistas & inhibidores , Secuencias Hélice-Asa-Hélice , Ratones , Datos de Secuencia Molecular , Músculos/citología , Músculos/metabolismo , Proteína MioD/antagonistas & inhibidores , Proteína MioD/metabolismo , Miogenina/antagonistas & inhibidores , Miogenina/metabolismo , ARN Mensajero/biosíntesis , Homología de Secuencia de Aminoácido , TransfecciónAsunto(s)
Citoesqueleto/fisiología , Desmina/fisiología , Músculo Esquelético/fisiología , Animales , Citoesqueleto/química , Desmina/química , Desmina/genética , Desarrollo Embrionario y Fetal , Femenino , Humanos , Filamentos Intermedios/química , Filamentos Intermedios/fisiología , Ratones , Ratones Noqueados , Microscopía Electrónica , Modelos Biológicos , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/ultraestructura , Músculo Liso/crecimiento & desarrollo , Músculo Liso/fisiología , Músculo Liso/ultraestructura , Miocardio/química , Miocardio/ultraestructura , Miofibrillas/química , Miofibrillas/fisiología , Embarazo , Regeneración/fisiología , Cicatrización de Heridas/fisiologíaRESUMEN
Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earliest known myogenic markers in both skeletal muscle and heart. Its expression precedes that of all known muscle proteins including the members of the MyoD family of myogenic helix-loop-helix (mHLH) regulators with the exception of myf5. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. In vitro studies using both antisense RNA and homologous recombination techniques in embryonic stem (ES) cells demonstrated that desmin plays a crucial role during myogenesis, as inhibition of desmin expression blocked myoblast fusion and myotube formation. Both in C2C12 cells and differentiating embryoid bodies, the absence of desmin interferes with the normal myogenic program, as manifested by the inhibition of the mHLH transcription regulators. To investigate the function of desmin in all muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, a considerable number of these mice are viable and fertile, potentially due to compensation by vimentin, nestin or synemin. However, desmin null mice demonstrate a multisystem disorder involving cardiac, skeletal and smooth muscle, beginning early in their postnatal life. Histological and electron microscopic analysis in both heart and skeletal muscle tissues reveals severe disruption of muscle architecture and degeneration. Structural abnormalities include loss of lateral alignment of myofibrils, perturbation of myofibril anchorage to the sarcolemma, abnormal mitochondrial number and organization, and loss of nuclear shape and positioning. Loose cell adhesion and increased intercellular space are prominent defects. The consequences of these abnormalities are most severe in the heart, which exhibits progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. There is a direct correlation between severity of damage and muscle usage, possibly due to increased susceptibility to normal mechanical damage and/or to repair deficiency in the absence of desmin. In conclusion, the studies so far have demonstrated that though desmin is absolutely necessary for muscle differentiation in vitro, muscle development can take place in vivo in the absence of this intermediate filament protein. However, desmin seems to play an essential role in the maintenance of myofibril, myofiber and whole muscle tissue structural and functional integrity.
Asunto(s)
Desmina/fisiología , Músculos/embriología , Proteínas del Tejido Nervioso , Animales , Células Cultivadas , Regulación hacia Abajo , Corazón/embriología , Proteínas de Filamentos Intermediarios/metabolismo , Ratones , Miofibrillas/ultraestructura , Nestina , Vimentina/metabolismoRESUMEN
The functional role of the skeletal muscle intermediate filament system was investigated by measuring the magnitude of muscle force loss after cyclic eccentric contraction (EC) in normal and desmin null mouse extensor digitorum longus muscles. Isometric stress generated was significantly greater in wild-type (313 +/- 8 kPa) compared with knockout muscles (276 +/- 13 kPa) before EC (P < 0.05), but 1 h after 10 ECs, both muscle types generated identical levels of stress ( approximately 250 kPa), suggesting less injury to the knockout. Differences in injury susceptibility were not explained by the different absolute stress levels imposed on wild-type versus knockout muscles (determined by testing older muscles) or by differences in fiber length or mechanical energy absorbed. Morphometric analysis of longitudinal electron micrographs indicated that Z disks from knockout muscles were more staggered (0.36 +/- 0. 03 microm) compared with wild-type muscles (0.22 +/- 0.03 microm), which may indicate that the knockout cytoskeleton is more compliant. These data demonstrate that lack of the intermediate filament system decreases isometric stress production and that the desmin knockout muscle is less vulnerable to mechanical injury.
Asunto(s)
Desmina/deficiencia , Músculo Esquelético/fisiopatología , Factores de Edad , Análisis de Varianza , Animales , Desmina/genética , Metabolismo Energético/genética , Técnicas In Vitro , Filamentos Intermedios/genética , Filamentos Intermedios/fisiología , Contracción Isométrica/genética , Ratones , Ratones Noqueados , Contracción Muscular/genética , Músculo Esquelético/fisiología , Periodicidad , Estrés MecánicoRESUMEN
A differentiating system based on embryonic stem (ES) cell-derived embryoid bodies (EBs) which recapitulates the in vivo cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation.
Asunto(s)
Citoesqueleto/fisiología , Desmina/fisiología , Músculo Esquelético/embriología , Músculo Liso/embriología , Células Madre/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Corazón/embriología , Ratones , Mutación , Recombinación Genética , Transducción de SeñalRESUMEN
The serial sarcomere number of skeletal muscle changes in response to chronic length perturbation. The role of the intermediate filament desmin in regulating these changes was investigated by comparing the architectural adaptations of the tibialis anterior, extensor digitorum longus (EDL) and soleus from wild-type mice with those of homozygous desmin knockout mice after hindlimb immobilization. After 28 days, serial sarcomere number increased significantly in the lengthened wild-type tibialis anterior (by approximately 9%) and EDL (by approximately 17%). Surprisingly, muscles from desmin knockout mice also experienced significant serial remodeling, with the serial sarcomere number of the tibialis anterior increasing by approximately 10% and that of the EDL by approximately 27%. A consistent result was observed in the shortened soleus: a significant decrease in sarcomere number was observed in the muscles from both wild-type (approximately 26%) and knockout (approximately 12%) mice. Thus, although desmin is not essential for sarcomerogenesis or sarcomere subtraction in mouse hindlimb muscles, the results do suggest subtle differences in the nature of sarcomere number adaptation. We speculate that desmin may play a role in regulating the optimal arrangement of sarcomeres within the muscle or in sensing the magnitude of the immobilization effect itself.
Asunto(s)
Desmina/fisiología , Suspensión Trasera/fisiología , Músculo Esquelético/fisiología , Sarcómeros/fisiología , Animales , Desmina/genética , Desmina/metabolismo , Ratones , Ratones Noqueados , Músculo Esquelético/ultraestructura , Sarcómeros/ultraestructuraRESUMEN
Desmin is the muscle-specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. There have been increasing reports of human cardiomyopathies associated with abnormal accumulation and aggregation of desmin filaments. Recently identified desmin mutations in humans suffering from skeletal muscle myopathy and cardiomyopathy suggest that these diseases might arise as a consequence of impaired function of desmin filaments. Previous generation of desmin null mice in our laboratory demonstrated that the absence of desmin results in myocyte ultrastructural defects and myocyte cell death leading to fibrosis and calcification of the myocardium. However, the effects that these defects have on cardiac function were not addressed. To further our understanding of desmin function in vivo, and in order to address the direct involvement of desmin in cardiomyopathy, we investigated the effect of the absence of desmin on myocardial mass, myocyte size and shape, changes in gene expression and cardiac systolic and diastolic function in mice. Morphometric characterization of isolated cardiomyocytes demonstrated a 24% increase in cell volume in the desmin null mice, solely due to an increase in transverse section area, suggesting for the first time that mice lacking the intermediate filament protein desmin develop concentric cardiomyocyte hypertrophy. This type of hypertrophy was accompanied by induction of embryonic gene expression and later by ventricular dilatation, and compromised systolic function. These results demonstrate that desmin is essential for normal cardiac function, and they suggest that the absence of an intact desmin filament system, rather than accumulation of the protein, may be responsible for the pathology seen in some of the desmin associated cardiomyopathies.