RESUMO
Men with chronic heart failure (CHF) have alterations in their skeletal muscle that are partially responsible for a decreased exercise tolerance. The purpose of this study was to investigate whether skeletal muscle alterations in women with CHF are similar to those observed in men and if these alterations are related to exercise intolerance. Twenty-five men and thirteen women with CHF performed a maximal exercise test for evaluation of peak oxygen consumption (VO(2)) and resting left ventricular ejection fraction, after which a biopsy of the vastus lateralis was performed. Twenty-one normal subjects (11 women, 10 men) were also studied. The relationship between muscle markers and peak VO(2) was consistent for CHF men and women. When controlling for gender, analysis showed that oxidative enzymes and capillary density are the best predictors of peak VO(2.) These results indicate that aerobically matched CHF men and women have no differences in skeletal muscle biochemistry and histology. However, when CHF groups were separated by peak exercise capacity of 4.5 metabolic equivalents (METs), CHF men with peak VO(2) >4.5 METs had increased citrate synthase and 3-hydroxyacyl-CoA dehydrogenase compared with CHF men with peak VO(2) <4.5 METs. CHF men with a lower peak VO(2) had increased capillary density compared with men with higher peak VO(2). These observations were not reproduced in CHF women. This suggests that differences may exist in how skeletal muscle adapts to decreasing peak VO(2) in patients with CHF.
Assuntos
Baixo Débito Cardíaco/metabolismo , Baixo Débito Cardíaco/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Caracteres Sexuais , Capilares/patologia , Baixo Débito Cardíaco/fisiopatologia , Doença Crônica , Citrato (si)-Sintase/metabolismo , Enoil-CoA Hidratase/metabolismo , Teste de Esforço , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Consumo de Oxigênio , Resistência Física , Volume SistólicoRESUMO
This study was designed to further characterize peripheral skeletal muscle alterations in patients with chronic obstructive pulmonary disease (COPD) and to evaluate the possible relationship between myosin heavy chain (MyoHC) isoform expression and exercise tolerance in these individuals. MyoHC composition from biopsy of the vastus lateralis muscle was examined in 12 COPD patients (forced expiratory volume in one second (FEV1)=31+/-9% predicted, peak oxygen consumption (V'O2)=15+/-4 mL x kg(-1) x min(-1)) and 10 age-matched normal male subjects (peak V'O2=20+/-5 mL x kg(-1) x min(-1)). The proportion of MyoHC type I was smaller in COPD than in normals (27+/-17% versus 41+/-9%, p<0.05) with an increase in MyoHC type IIa (51+/-15% versus 39+/-9%, p<0.05) and the proportion of MyoHC type IIx being comparable between both groups. A significant relationship was found between peak V'Oo2 mL x kg(-1) x min(-1) and FEV1 % pred (r=0.91, p<0.0001) and the percentage of MyoHC type I (r=0.61, p=0.016). In stepwise multiple regression, only FEV1 % pred was found to be a significant determinant of peak V'O2 (p<0.0001). This variable explained 83% of the total variance of peak V'O2. In summary, this study showed considerable modifications in the phenotypic expression of the myosin heavy chain in the vastus lateralis muscle in patients with chronic obstructive pulmonary disease. An independent effect of myosin heavy chain expression on exercise capacity was not found. These results suggest that chronic inactivity and muscle deconditioning may not be the sole factors explaining peripheral muscle dysfunction in patients with chronic obstructive pulmonary disease.
Assuntos
Tolerância ao Exercício/fisiologia , Pneumopatias Obstrutivas/metabolismo , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Idoso , Biópsia , Estudos de Casos e Controles , Teste de Esforço , Humanos , Pneumopatias Obstrutivas/fisiopatologia , Masculino , Músculo Esquelético/patologia , Testes de Função RespiratóriaRESUMO
OBJECTIVES: The study was conducted to determine if the capillary density of skeletal muscle is a potential contributor to exercise intolerance in class II-III chronic heart failure (CHF). BACKGROUND: Previous studies suggest that abnormalities in skeletal muscle histology, contractile protein content and enzymology contribute to exercise intolerance in CHF. METHODS: The present study examined skeletal muscle biopsies from 22 male patients with CHF compared with 10 age-matched normal male control patients. Aerobic capacities, myosin heavy chain (MHC) isoforms, enzymes, and capillary density were measured. RESULTS: The patients with CHF demonstrated a reduced peak oxygen consumption when compared to controls (15.0+/-2.5 vs. 19.8+/-5.0 ml x kg(-1) x min(-1), p <0.05). Using cell-specific antibodies to directly assess vascular density, there was a reduction in capillary density in CHF measured as the number of endothelial cells/fiber (1.42+/-0.28 vs. 1.74+/-0.35, p = 0.02). In CHF, capillary density was inversely related to maximal oxygen consumption (r = 0.479, p = 0.02). The MHC IIx isoform was found to be higher in patients with CHF versus normal subjects (28.5+/-13.6 vs. 19.5+/-9.4, p <0.05). CONCLUSIONS: There was a significant reduction in microvascular density in patients with CHF compared with the control group, without major differences in other usual histologic and biochemical aerobic markers. The inverse relationship with peak oxygen consumption seen in the CHF group suggests that a reduction in microvascular density of skeletal muscle may precede other skeletal muscle alterations and play a critical role in the exercise intolerance characteristic of patients with CHF.
Assuntos
Capilares/patologia , Tolerância ao Exercício/fisiologia , Insuficiência Cardíaca/fisiopatologia , Músculo Esquelético/irrigação sanguínea , Biópsia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Contagem de Células , Ecocardiografia , Imagem do Acúmulo Cardíaco de Comporta , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Consumo de Oxigênio , Prognóstico , Volume SistólicoRESUMO
Relaxation rate is an important determinant of axial muscle power production during the oscillatory contractions of undulatory locomotion. Recently, significant differences have been reported in the relaxation rates of rostral versus caudal white muscle fibers of the Atlantic cod Gadus morhua L. The present study investigates the biochemical correlates of this rostral-caudal physiological variation. Using denaturing gel electrophoresis, a series of fresh muscle samples from the dorsal epaxial muscle region was analyzed and several differences were detected. First, a gradual shift occurs in the expression of two troponin T isoforms along the length of the body. Second, rostral muscles were found to contain significantly greater amounts of parvalbumin than caudal muscles. Third, two soluble Ca(2+)-binding proteins, in addition to parvalbumin, were also detected in the rostral muscle samples yet were absent from the caudal samples. This suite of rostral-caudal variations provides a strong biochemical basis for regional differences in the relaxation rates of cod white muscle.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Troponina T/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , PeixesRESUMO
Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.
Assuntos
Cálcio/metabolismo , Troponina C/metabolismo , Animais , Calmodulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculos Psoas/metabolismo , CoelhosRESUMO
The protooncogene c-fos encodes a nuclear protein that acts as a powerful enhancer of gene transcription, and shares the same general patterns of reactivity to stimulation as ornithine decarboxylase, an essential enzyme in cell replication and differentiation; c-fos enhances expression of proteins whose genes possess the AP-1 consensus sequence, which include ornithine decarboxylase. In the current study, we examined the regulation of c-fos during early postnatal development of the rat central nervous system. Regional profiles of basal c-fos mRNA paralleled the ontogenetic profiles of cell replication and differentiation; in the earliest developing region (midbrain + brainstem) c-fos was low by birth and did not change over the first 2 postnatal weeks; in the forebrain, where replication and differentiation peak somewhat later, c-fos levels were higher during the first postnatal week than in the second; in the latest developing region (cerebellum), basal c-fos expression was low at birth and increased markedly during the second postnatal week, coincidentally with the increase in neuronal cell replication/differentiation. The resemblance of these patterns to those of ornithine decarboxylase, which is regulated in part by beta adrenergic input, led us to evaluate the potential role of beta receptors in the ontogenetic control of c-fos. In the forebrain of 2-day-old rats, intracisternal administration of isoproterenol, a beta adrenergic agonist, produced > 20-fold stimulation of c-fos within 10 min of drug administration. The induction was mediated through the beta receptor, as confirmed by studies with selective adrenergic blocking agents, and could be reproduced by administration of cAMP analogs.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica , Genes fos , Receptores Adrenérgicos beta/fisiologia , Fatores Etários , Animais , Encéfalo/crescimento & desenvolvimento , AMP Cíclico/fisiologia , Feminino , Isoproterenol/farmacologia , Gravidez , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Cardiac myosin heavy chain expression undergoes a perinatal transition from predominance of beta-MHC to alpha-MHC. In the current study, we tested the effects of glucocorticoids in this early transition period, by treating pregnant rats with dexamethasone on gestational days 17, 18 and 19, using doses below (0.05 mg/kg), at (0.2 mg/kg) or above (0.8 mg/kg) the threshold for growth retardation. Cardiac MHC isoforms were resolved with a denaturing SDS-PAGE system, followed by quantitative densitometry. In normal animals alpha-MHC was only 10% of the total on gestational day 18 but rose to 35% by postnatal day 1, and to 95% by the end of the first month postpartum. During the early phase of this transition, the lowest dose of dexamethasone significantly promoted alpha-MHC expression without inhibiting body or heart growth; regression analysis indicated a 40% increase in the slope of MHC isoform transition with respect to tissue weight. In contrast, the higher, growth-retarding doses of dexamethasone either failed to enhance alpha-MHC expression or caused biphasic changes, with inhibition at ages corresponding to the onset of weight deficits; regression analysis indicated that the effects of the higher doses on MHC could all be accounted for by changes in tissue weight. Glucocorticoid levels rise substantially in the period surrounding parturition, and serve to program the development and coupling of adenylate cyclase to membrane receptors; because adenylate cyclase has been shown to elicit the beta-MHC to alpha-MHC transition in vitro, our results suggest that glucocorticoids, along with thyroid hormone and beta-adrenergic stimulation, influence the ontogenetic program of MHC isoform transition.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dexametasona/farmacologia , Miocárdio/metabolismo , Miosinas/biossíntese , Efeitos Tardios da Exposição Pré-Natal , Análise de Variância , Animais , Animais Recém-Nascidos , Peso Corporal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Feto/metabolismo , Coração/efeitos dos fármacos , Coração/embriologia , Coração/crescimento & desenvolvimento , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/embriologia , Ventrículos do Coração/metabolismo , Troca Materno-Fetal , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-Dawley , Análise de RegressãoRESUMO
The myofilament protein compositions of muscle fibres from the transverse muscle mass of the tentacles and the transverse muscle mass of the arms of the loliginid squid Sepioteuthis lessoniana were compared. These two muscle masses are distinct types, differing in their ultrastructural and behavioural properties. The transverse muscle of the tentacles consists of specialized muscle fibres that exhibit cross-striation and unusually short sarcomeres and thick filaments. The transverse muscle of the arms consists of obliquely striated muscle fibres that are typical of cephalopod skeletal muscle in general. The specialization of the tentacle muscle results in a high shortening speed and reflects its role in creating rapid elongation of the tentacles during prey capture. Comparison of samples of myofilament preparations of the two muscle fibre types using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping of myosin heavy chains from the two muscle fibre types, however, showed little evidence of differences in contractile protein isoforms. Thus, specialization for high shortening speed appears to have occurred primarily through changes in the dimensions and arrangement of the myofilament lattice, rather than through changes in biochemistry. The thick filament core protein paramyosin was tentatively identified in the squid muscle fibres. This protein was less abundant in the short thick filament cross-striated tentacle muscle cells than in the obliquely striated arm cells.
Assuntos
Decapodiformes/fisiologia , Contração Muscular/fisiologia , Proteínas Musculares/análise , Músculos/fisiologia , Actinina/análise , Actinas/análise , Animais , Brometo de Cianogênio , Eletroforese em Gel de Poliacrilamida , Músculos/química , Miosinas/análise , Mapeamento de Peptídeos , Serina Endopeptidases/metabolismoRESUMO
Cardiac myosin heavy chain (MHC) expression undergoes an ontogenetic transition from beta to alpha MHC isoforms. Although thyroid hormone plays a role in this change, the timing of the events suggests the participation of other factors. Using a new, denaturing SDS-PAGE procedure that cleanly resolves the beta and alpha heavy chains, we have assessed the role of beta-adrenergic stimulation on this transition in fetal and neonatal rat hearts. In control animals at embryonic day 20, less than 15% of the MHC was the alpha-form, and the proportion increased to approximately 35% by postnatal day 1 and to 80% by postnatal day 8. Although catecholamine levels rise abruptly at birth, and cyclic AMP levels increase the expression of alpha-MHC in vitro, neither premature beta-adrenergic stimulation (maternal treatment with terbutaline on embryonic days 17, 18 and 19) nor continuous prenatal blockade of beta-receptors (maternal propranolol infusions from embryonic day 7 onward) influenced the developmental profile. Because beta-receptors in fetal and neonatal heart are functionally linked to adenylate cyclase, and cyclic AMP has been shown to promote the expression of alpha-MHC, the lack of effect of terbutaline or propranolol suggests that activation of adenylate cyclase through fetal cardiac beta-receptors is not sufficient to mediate the switchover without participation of other factors, such as thyroid or steroid hormones, or hypoxia.
Assuntos
Coração Fetal/efeitos dos fármacos , Isoenzimas/metabolismo , Miocárdio/enzimologia , Miosinas/metabolismo , Simpatomiméticos/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Animais Recém-Nascidos , Feminino , Ventrículos do Coração , Isoenzimas/química , Miosinas/química , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Ratos , Ratos Sprague-DawleyRESUMO
When Ca2+ binds to troponin C (TnC), all 26 troponin-tropomyosin (Tn-Tm) complexes of a regulatory strand change in concert from the inactive to the active configuration. To see if the complexes respond similarly when they are activated by rigor crossbridges in the absence of Ca2+, we determined the slope (ns) of the bell-shaped pS/tension (pS = -log [MgATP], where S = MgATP2-) relationship between pS 5, where the tension is maximal, and pS 2.3, where fibers are fully relaxed. In control skinned rabbit psoas fibers the ns value is greater than 4; it progressively decreases with TnC extraction. This decrease in ns with TnC extraction is analogous to the decrease in the slope (Hill coefficient) of the pCa/tension (pCa = -log [Ca2+]) relationship with extraction. Complete TnC extraction reduces the maximum substrate-induced tension by only 25%; in contrast, it reduces the maximum Ca2+ induced tension to zero. The effects of TnC extraction on the slope of the pS/tension curve are explained by the assumptions that (1) extracted Tn-Tm complexes no longer change in concert with their neighbors but change independently of them, and (2) co-operative signals cannot cross extracted Tn-Tm complexes. The ns value, therefore, like the nH, is a direct function of the number of contiguous, intact, Tn-Tm complexes in a stretch of a regulatory strand. To describe qualitatively the bi-phasic pS/tension relationship, the mono-phasic pCa/tension relationship, and the effects of TnC extraction on them, we introduce a version of the concerted-transition formalism which includes two activating ligands, Ca2+ and rigor crossbridges.
Assuntos
Citoesqueleto de Actina/fisiologia , Citoesqueleto/fisiologia , Contração Muscular , Músculos/fisiologia , Troponina/fisiologia , Animais , Cálcio/metabolismo , Músculos/ultraestrutura , Coelhos , Troponina/metabolismo , Troponina CRESUMO
Changes in contractile protein expression during myogenesis are usually categorized as developmentally programmed or neuronally dependent. Studies on aneurogenic chick embryos indicated that the neuronally dependent phase begins at about Embryonic Day 15, immediately prior to the fetal transition in myosin and C-protein expression. The prime candidate for the neuronal event that induces the fetal transition is the conversion to the adult form of singly innervated neuromuscular junctions (NMJs), which occurs contemporaneously with the fetal transition. Using curare to inhibit the conversion to focal innervation, we find that the fetal transition proceeds unimpaired, demonstrating that there is no causal link between the fetal transition and the conversion to focal innervation. Furthermore, because the doses of curare used inhibit motor activity by more than 80%, the fetal transition can occur in the absence of normal levels of motor activity. These observations show that the fetal transition in ovo is not induced by either a specific change in innervation or use. Rather, the dependence on innervation seems to be a consequence of the need for muscle activity to prevent atrophy, and the fetal transition appears to have characteristics more like the preprogrammed contractile protein transitions that precede it.
Assuntos
Proteínas de Transporte/fisiologia , Neurônios Motores/embriologia , Músculos/embriologia , Miosinas/fisiologia , Junção Neuromuscular/embriologia , Acetilcolinesterase/metabolismo , Animais , Western Blotting , Embrião de Galinha , Curare/farmacologiaRESUMO
The properties of extraocular muscles (EOMs) are quite different from those of the trunk and limb. Here we show that there is a novel pattern of troponin T (TnT) expression in EOMs which most likely contributes to the fine control of ocular movement and may reflect their innervation by cranial motoneurons. Three regions of the muscle were analysed to distinguish the TnT isoforms present in the fast singly-innervated fibres from those in the multiply-innervated fibres. More than 95% of the TnT in the singly-innervated fibres is TnT3f, which exhibits the most graded response to changes in calcium concentration during activation (Schachat et al., J. molec. Biol. 198, 551-4). In multiply-innervated fibres, which exhibit tonic contractures, the slow troponin T TnT2s is expressed. While neither TnT3f nor TnT2s is unique to EOM, this pattern is unusual in two respects: first, both TnT3f and TnT2s are minor components of the trunk and limb musculature, and second, most muscles express several fast and both slow TnT species. Although EOM occupies a highly specialized physiological niche, its unusual physiology is not reflected in the presence of new TnT isoforms but in the expression of a different ratio of the known species of TnT.
Assuntos
Músculos Oculomotores/metabolismo , Troponina/metabolismo , Animais , Miosinas/metabolismo , Músculos Oculomotores/inervação , Músculos Oculomotores/fisiologia , Coelhos , Troponina TRESUMO
The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT2f and alpha 2 Tm exhibit steeper pCa/tension relations than those in which either TnT1f or TnT3f and alpha beta Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the Tn-Tm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo.
Assuntos
Músculos/fisiologia , Tropomiosina/metabolismo , Troponina/metabolismo , Animais , Cálcio/metabolismo , Proteínas Contráteis/análise , Contração Muscular , Polimorfismo Genético , Coelhos , Troponina TRESUMO
Ca2+ binding to troponin C (TnC), a subunit of the thin filament regulatory strand, activates vertebrate skeletal muscle contraction. Tension, however, increases with Ca2+ too abruptly to be the result of binding to sites on individual TnCs. Because extraction of one TnC on average per regulatory strand dramatically reduces the slope of the tension/Ca2+ relationship, we proposed that all 26 troponin-tropomyosin complexes of the regulatory strand form a co-operative system. This study of permeabilized (chemically skinned) rabbit psoas fibers analyzes the extraction time-course, the distribution of extraction sites on regulatory strands and the effects of extraction on the co-operativity of the tension/Ca2+ relationship. Two components of TnC are resolved in the time-course of extraction: a "rapidly extracting" component that can be selectively removed without affecting tension or co-operativity, and a "slow extracting" component whose loss reduces tension and co-operativity. Extraction of [14C]TnC shows that the slowly extracting component is lost randomly, so that, after removal of 5% of the TnC, most extracted strands have lost one TnC. Extraction interrupts the transmission of co-operativity by dividing the regulatory strand into smaller, independent co-operative systems; it reduces tension by preventing Ca2+ activation of TnC-depleted regulatory units. Co-operativity of the tension/Ca2+ relationship is modeled with the concerted-transition formalism for intact systems of 26 regulatory units, and for the smaller systems in extracted fibers.
Assuntos
Músculos/metabolismo , Tropomiosina/metabolismo , Troponina/metabolismo , Regulação Alostérica , Animais , Cálcio/metabolismo , Cinética , Substâncias Macromoleculares , Contração Muscular , Coelhos , Troponina CRESUMO
The distribution of troponin T (TnT) species in typed single muscle fibres was analysed using one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and a monoclonal antibody specific for fast TnT. Fibres taken from erector spinae (Es), plantaris (Plt), diaphragm (Dia) and soleus (Sol) muscles of adult rabbits were pretyped as fast-twitch-glycolytic (FG), fast-twitch-oxidative-glycolytic (FOG), slow-twitch-oxidative (SO) or promiscuous (P) using a combination of histochemical staining and PAGE. Although none of the four size classes of TnT was either muscle or fibre type specific, their pattern of expression differed in each muscle and between the fibre types. FG fibres expressed TnT1f or TnT2f as predominant species, depending on the muscle; TnT3f and TnT4f were minor components. In contrast, all size classes of TnT were expressed in varying proportions in FOG fibres from Es and Plt, while those from Dia resembled FG fibres, expressing TnT1f as their major species. P fibres from Es, Plt, and Sol exhibited a distinctive pattern of fast TnT expression, TnT3f being the predominant species. Dia differed from the other muscles as TnT1f was the dominant fast TnT species in its P fibres as it is in the Dia fast fibres. Quantitative analysis of one- and two-dimensional gels revealed that the P fibres could be divided into two classes, those that exhibited discoordinate expression of fast and slow TnTs, myosin light chains and myosin heavy chains and those in which their expression was coordinate. In addition low levels of TnT4f were detected in SO fibres and of slow TnT in fast fibres.
Assuntos
Músculos/metabolismo , Troponina/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Histocitoquímica , Imunoquímica , Miosinas/metabolismo , Coelhos , Troponina TRESUMO
The extent and nature of fast troponin T (TnT) heterogeneity has been assessed in rabbit skeletal muscle. Previous studies identified two major fast TnT species (TnT1f and TnT2f), in the fast white muscle erector spinae, differing in their N-terminal cyanogen bromide (CNBr) fragments. Here a monoclonal antibody that recognizes a conserved region of TnT was used to characterize two additional TnT species (TnT3f and TnT4f) in the epaxial and limb musculature and a minor species (TnTcf) in craniofacial muscles. A combination of CNBr peptide mapping, immunoblotting and specific labelling of the N-terminus shows that these TnT species also differ in their N-terminal region. This observation is consistent with cDNA studies that predicted the N-terminal region is hypervariable. One additional species, a variant of TnT2f present in the tongue, was identified by two-dimensional gel electrophoresis. The limited number of TnT variants indicates that the full potential for heterogeneity inferred from the cDNA studies is not realized. This conclusion is supported by immunoblot analysis with a monoclonal antibody that recognizes an epitope in the hypervariable N-terminal region which is present in all variants of TnT1f and TnT2f but absent from the lower molecular weight species TnT3f and TnT4f.
Assuntos
Variação Genética , Troponina/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Fenômenos Químicos , Química , Epitopos , Imunoquímica , Fragmentos de Peptídeos , Mapeamento de Peptídeos , Coelhos , Troponina/metabolismo , Troponina TRESUMO
The pathologic changes in myotonic dystrophy (DM) skeletal muscle biopsies have been analyzed at both the histochemical and molecular level. A histochemical stain for pretyping single fibers in conjunction with sodium dodecyl sulphate-polyacrylamide gel electrophoresis allowed biochemical differences to be pinpointed in specific histochemical fiber types. These biochemical differences can be related to histochemical changes in fiber type observed in cross-section of the DM biopsies. Such changes included specific fiber type atrophy, hypertrophy, and disproportion. The pathogenesis of DM appears to be characterized by a large increase in the number of promiscuous fibers, that is, those fibers that express both fast and slow myosins. This promiscuity, which is rare in control muscle (less than 2%), is also prevalent at high levels in some family members at risk for DM. The observed promiscuity, although probably not a primary effect of DM, appears to be linked to the histochemical changes in fiber type observed in the DM biopsies.
Assuntos
Músculos/análise , Miosinas/análise , Distrofia Miotônica/metabolismo , Adolescente , Adulto , Biópsia , Eletroforese em Gel de Poliacrilamida , Feminino , Histocitoquímica , Humanos , Masculino , Músculos/metabolismo , Músculos/patologia , Distrofia Miotônica/patologia , Dodecilsulfato de SódioRESUMO
We have examined the intermediate filament (IF) protein content of vascular smooth muscle (SM) cells from several arteries and veins in rabbits and quantitated the changes which occur in SM cell expression of these proteins in response to cholesterol feeding. Cells from control rabbit arteries expressed 30% of their IF protein as desmin, while veins expressed 50% as desmin. During development of diet-induced atherosclerosis, morphological changes in arterial SM cells in the intima correlate with changes in IF expression. There is a significant increase in total IF protein content, vimentin increased differentially in thoracic aorta and desmin in pulmonary artery. In abdominal aorta both increase equally. Cholesterol feeding also resulted in changes in the expression of subspecies of desmin, vimentin, and actin in the thoracic arch. Although cholesterol feeding did not produce obvious morphological changes in the veins examined, venous SM IF protein expression was also altered. In the vena cava of cholesterol-fed rabbits there was an increase in vimentin expression without the parallel increase in desmin that occurred in the arterial system. These studies show that cholesterol feeding of rabbits induces measurable changes in the amounts of IF proteins in both arterial atherosclerotic lesions and venous SM cells.
Assuntos
Arteriosclerose/metabolismo , Hipercolesterolemia/metabolismo , Proteínas de Filamentos Intermediários/análise , Músculo Liso Vascular/análise , Actinas/análise , Animais , Aorta Abdominal/análise , Aorta Torácica/análise , Arteriosclerose/etiologia , Arteriosclerose/patologia , Colesterol na Dieta/administração & dosagem , Desmina/análise , Hipercolesterolemia/patologia , Filamentos Intermediários/análise , Filamentos Intermediários/ultraestrutura , Masculino , Músculo Liso Vascular/ultraestrutura , Artéria Pulmonar/análise , Coelhos , Veias Cavas/análise , Vimentina/análiseRESUMO
Two species of alpha-actinin from rabbit fast skeletal muscles were identified with a monospecific antisera. Designated alpha-actinin1f and alpha-actinin2f, their distribution in muscles does not correlate with histochemically defined fast fiber type. Rather, the presence of each correlates with Z-line width and with the expression of different thin filament Ca2+-regulatory complexes. alpha-Actinin1f is expressed with troponin T 1f-alpha beta tropomyosin, and alpha-actinin2f with troponin T 2f-alpha 2 tropomyosin. CNBr peptide maps show that the fast alpha-actinin species differ in primary structure. In contrast, the slow alpha-actinin is indistinguishable from alpha-actinin1f. Further evidence for the similarity of alpha-actinin1f and slow alpha-actinin comes from electron microscopic studies which show that fibers that express these species exhibit thick Z-lines. So, unlike other contractile proteins, the multiple forms of alpha-actinin do not reflect the distinction between fast- and slow-twitch muscles.