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Skeletal muscle suffers atrophy and weakness with aging. Denervation, oxidative stress, and mitochondrial dysfunction are all proposed as contributors to age-associated muscle loss, but connections between these factors have not been established. We examined contractility, mitochondrial function, and intracellular calcium transients (ICTs) in muscles of mice throughout the life span to define their sequential relationships. We performed these same measures and analyzed neuromuscular junction (NMJ) morphology in mice with postnatal deletion of neuronal Sod1 (i-mn-Sod1-/- mice), previously shown to display accelerated age-associated muscle loss and exacerbation of denervation in old age, to test relationships between neuronal redox homeostasis, NMJ degeneration and mitochondrial function. In control mice, the amount and rate of the decrease in mitochondrial NADH during contraction was greater in middle than young age although force was not reduced, suggesting decreased efficiency of NADH utilization prior to the onset of weakness. Declines in both the peak of the ICT and force were observed in old age. Muscles of i-mn-Sod1-/- mice showed degeneration of mitochondrial and calcium handling functions in middle-age and a decline in force generation to a level not different from the old control mice, with maintenance of NMJ morphology. Together, the findings support the conclusion that muscle mitochondrial function decreases during aging and in response to altered neuronal redox status prior to NMJ deterioration or loss of mass and force suggesting mitochondrial defects contribute to sarcopenia independent of denervation.
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Envelhecimento , Cálcio/metabolismo , Mitocôndrias Musculares/patologia , Neurônios/patologia , Estresse Oxidativo , Sarcopenia/patologia , Superóxido Dismutase-1/fisiologia , Animais , Denervação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Contração Muscular , Neurônios/metabolismo , Oxirredução , Sarcopenia/etiologiaRESUMO
BACKGROUND: A persistent atrophy of muscle fibers and an accumulation of fat, collectively referred to as fatty degeneration, commonly occur in patients with chronic rotator cuff tears. The etiology of fatty degeneration and function of the residual rotator cuff musculature have not been well characterized in humans. We hypothesized that muscles from patients with chronic rotator cuff tears have reduced muscle fiber force production, disordered myofibrils, and an accumulation of fat vacuoles. METHODS: The contractility of muscle fibers from biopsy specimens of supraspinatus muscles of 13 patients with chronic full-thickness posterosuperior rotator cuff tears was measured and compared with data from healthy vastus lateralis muscle fibers. Correlations between muscle fiber contractility, American Shoulder and Elbow Surgeons (ASES) scores, and tear size were analyzed. Histology and electron microscopy were also performed. RESULTS: Torn supraspinatus muscles had a 30% reduction in maximum isometric force production and a 29% reduction in normalized force compared with controls. Normalized supraspinatus fiber force positively correlated with ASES score and negatively correlated with tear size. Disordered sarcomeres were noted, along with an accumulation of lipid-laden macrophages in the extracellular matrix surrounding supraspinatus muscle fibers. CONCLUSIONS: Patients with chronic supraspinatus tears have significant reductions in muscle fiber force production. Force production also correlates with ASES scores and tear size. The structural and functional muscle dysfunction of the residual muscle fibers is independent of the additional area taken up by fibrotic tissue. This work may help establish future therapies to restore muscle function after the repair of chronically torn rotator cuff muscles.
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Miofibrilas/ultraestrutura , Manguito Rotador/patologia , Traumatismos dos Tendões/patologia , Tecido Adiposo/patologia , Idoso , Matriz Extracelular/patologia , Matriz Extracelular/ultraestrutura , Feminino , Humanos , Macrófagos/patologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Miofibrilas/patologia , Lesões do Manguito Rotador , Sarcômeros/patologia , Sarcômeros/ultraestruturaRESUMO
BACKGROUND: Rotator cuff tears are one of the most common musculoskeletal complaints and a substantial source of morbidity in elderly patients. Chronic cuff tears are associated with muscle atrophy and an infiltration of fat to the area, a condition known as "fatty degeneration." To improve the treatment of cuff tears in elderly patients, a greater understanding of the changes in the contractile properties of muscle fibers and the molecular regulation of fatty degeneration is essential. METHODS: Using a full-thickness, massive supraspinatus and infraspinatus tear model in elderly rats, we measured fiber contractility and determined changes in fiber type distribution that develop 30 days after tear. We also measured the expression of messenger RNA and micro-RNA transcripts involved in muscle atrophy, lipid accumulation, and matrix synthesis. We hypothesized that a decrease in specific force of muscle fibers, an accumulation of type IIb fibers, and an upregulation in atrophic, fibrogenic, and inflammatory gene expression would occur in torn cuff muscles. RESULTS: Thirty days after the tear, we observed a reduction in muscle fiber force and an induction of RNA molecules that regulate atrophy, fibrosis, lipid accumulation, inflammation, and macrophage recruitment. A marked accumulation of advanced glycation end products and a significant accretion of macrophages in areas of fat accumulation were observed. CONCLUSIONS: The extent of degenerative changes in old rats was greater than that observed in adults. In addition, we identified that the ectopic fat accumulation that occurs in chronic cuff tears does not occur by activation of canonical intramyocellular lipid storage and synthesis pathways.
Assuntos
Envelhecimento/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/metabolismo , Manguito Rotador/metabolismo , Traumatismos dos Tendões/metabolismo , Tecido Adiposo/patologia , Envelhecimento/patologia , Animais , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , MicroRNAs/biossíntese , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/patologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Manguito Rotador/patologia , Lesões do Manguito Rotador , Traumatismos dos Tendões/patologiaRESUMO
Sarcopenia, the progressive loss of muscle mass and function, universally affects older adults and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation. Acylated ghrelin activates its receptor, growth hormone secretagogue receptor 1a (GHSR1a), in the brain by binding to it. Studies have demonstrated that acyl and unacylated ghrelin (UnAG) both have protective effects against acute pathological conditions independent of receptor activation. Here, we investigated the long-term effects of UnAG in age-associated muscle atrophy and contractile dysfunction in mice. Four-month-old and 18-month-old mice were subjected to either UnAG or control treatment for 10 months. UnAG did not affect food consumption or body weight. Gastrocnemius and quadriceps muscle weights were reduced by 20%-30% with age, which was partially protected against by UnAG. Specific force, force per cross-sectional area, measured in isolated extensor digitorum longus muscle was diminished by 30% in old mice; however, UnAG prevented the loss of specific force. UnAG also protected from decreases in mitochondrial respiration and increases in hydrogen peroxide generation of skeletal muscle of old mice. Results of bulk mRNA-seq analysis and our contractile function data show that UnAG reversed neuromuscular junction impairment that occurs with age. Collectively, our data revealed the direct role of UnAG in mitigating sarcopenia in mice, independent of food consumption or body weight, implicating UnAG treatment as a potential therapy against sarcopenia.
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Pulmonary disorders affect 40%-80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragm muscle weakness. Increased intradiaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs) - mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing the effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1-knockout (Thbs1-/-) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGF-ß-related expression signatures and augmentation of a Thy1-expressing subpopulation previously linked to type 2 diabetes. Despite similar weight gain, Thbs1-/- mice were protected from these transcriptomic changes and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1-/- diaphragms maintained normal contractile force and motion after DIO challenge. THBS1 is therefore a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition and a potential therapeutic target in obesity-associated respiratory dysfunction.
Assuntos
Diafragma , Camundongos Knockout , Obesidade , Trombospondina 1 , Animais , Trombospondina 1/metabolismo , Trombospondina 1/genética , Diafragma/fisiopatologia , Diafragma/metabolismo , Diafragma/patologia , Obesidade/metabolismo , Camundongos , Contração Muscular/fisiologia , Masculino , Adipogenia , Adipócitos/metabolismo , Adipócitos/patologia , Dieta Hiperlipídica/efeitos adversos , Fibroblastos/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Diferenciação CelularRESUMO
Pulmonary disorders impact 40-80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragmatic weakness. Increased intra-diaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs)-mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1 knockout ( Thbs1 -/- ) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGFß-related expression signatures, and augmentation of a Thy1 -expressing sub-population previously linked to type 2 diabetes. Despite similar weight gain, Thbs1 -/- mice were protected from these transcriptomic changes, and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1 -/- diaphragms maintained normal contractile force and motion after DIO challenge. These findings establish THBS1 as a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition, and potential therapeutic target in obesity-associated respiratory dysfunction.
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Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.
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Distrofia Muscular Animal , Distrofia Muscular de Duchenne , Camundongos , Animais , Cães , Distrofia Muscular de Duchenne/metabolismo , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofina/genética , Contração Muscular/fisiologia , Modelos Animais de Doenças , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismoRESUMO
Tension and regional average sarcomere length (L(s)) behavior were examined during repeated stretches of single, permeabilized, relaxed muscle fibers isolated from the soleus muscles of rats. We tested the hypothesis that during stretches of single permeabilized fibers, the global fiber strain is distributed non-uniformly along the length of a relaxed fiber in a repeatable pattern. Each fiber was subjected to eight constant-velocity stretch and release cycles with a strain of 32% and strain rate of 54% s(-1). Stretch-release cycles were separated by a 4.5 min interval. Throughout each stretch-release cycle, sarcomere lengths were measured using a laser diffraction technique in which 20 contiguous sectors along the entire length of a fiber segment were scanned within 2 ms. The results revealed that: (1) the imposed length change was not distributed uniformly along the fiber, (2) the first stretch-release cycle differed from subsequent cycles in passive tension and in the distribution of global fiber strain, and (3) a characteristic "signature" for the L(s) response emerged after cycle 3. The findings support the conclusions that longitudinal heterogeneity exists in the passive stiffness of individual muscle fibers and that preconditioning of fibers with stretch-release cycles produces a stable pattern of sarcomere strains.
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Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Sarcômeros/fisiologia , Animais , Masculino , Músculo Esquelético/lesões , Ratos , Relaxamento , Entorses e Distensões , Estresse MecânicoRESUMO
Aging is accompanied by loss of muscle mass and force, known as sarcopenia. Muscle atrophy, weakness, and neuromuscular junction (NMJ) degeneration reminiscent of normal muscle aging are observed early in adulthood for mice deficient in Cu, Zn-superoxide dismutase (SOD, Sod1-/-). Muscles of Sod1-/- mice also display impaired mitochondrial ATP production and increased mitochondrial reactive oxygen species (ROS) generation implicating oxidative stress in sarcopenia. Restoration of CuZnSOD specifically in neurons of Sod1-/- mice (SynTgSod1-/-) prevents muscle atrophy and loss of force, but whether muscle mitochondrial function is preserved is not known. To establish links among CuZnSOD expression, mitochondrial function, and sarcopenia, we examined contractile properties, mitochondrial function and ROS production, intracellular calcium transients (ICT), and NMJ morphology in lumbrical muscles of 7-9 month wild type (WT), Sod1-/-, and SynTgSod1-/- mice. Compared with WT values, mitochondrial ROS production was increased 2.9-fold under basal conditions and 2.2-fold with addition of glutamate and malate in Sod1-/- muscle fibers while oxygen consumption was not significantly altered. In addition, NADH recovery was blunted following contraction and the peak of the ICT was decreased by 25%. Mitochondrial function, ROS generation and calcium handling were restored to WT values in SynTgSod1-/- mice, despite continued lack of CuZnSOD in muscle. NMJ denervation and fragmentation were also fully rescued in SynTgSod1-/- mice suggesting that muscle mitochondrial and calcium handling defects in Sod1-/- mice are secondary to neuronal oxidative stress and its effects on the NMJ rather than the lack of muscle CuZnSOD. We conclude that intact neuronal function and innervation are key to maintaining excitation-contraction coupling and muscle mitochondrial function.
Assuntos
Cálcio , Músculo Esquelético , Animais , Cálcio/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias , Músculo Esquelético/metabolismo , Neurônios/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
BACKGROUND: Excess reactive oxygen species (ROS) and muscle weakness occur in parallel in multiple pathological conditions. However, the causative role of skeletal muscle mitochondrial ROS (mtROS) on neuromuscular junction (NMJ) morphology and function and muscle weakness has not been directly investigated. METHODS: We generated mice lacking skeletal muscle-specific manganese-superoxide dismutase (mSod2KO) to increase mtROS using a cre-Lox approach driven by human skeletal actin. We determined primary functional parameters of skeletal muscle mitochondrial function (respiration, ROS, and calcium retention capacity) using permeabilized muscle fibres and isolated muscle mitochondria. We assessed contractile properties of isolated skeletal muscle using in situ and in vitro preparations and whole lumbrical muscles to elucidate the mechanisms of contractile dysfunction. RESULTS: The mSod2KO mice, contrary to our prediction, exhibit a 10-15% increase in muscle mass associated with an ~50% increase in central nuclei and ~35% increase in branched fibres (P < 0.05). Despite the increase in muscle mass of gastrocnemius and quadriceps, in situ sciatic nerve-stimulated isometric maximum-specific force (N/cm2 ), force per cross-sectional area, is impaired by ~60% and associated with increased NMJ fragmentation and size by ~40% (P < 0.05). Intrinsic alterations of components of the contractile machinery show elevated markers of oxidative stress, for example, lipid peroxidation is increased by ~100%, oxidized glutathione is elevated by ~50%, and oxidative modifications of myofibrillar proteins are increased by ~30% (P < 0.05). We also find an approximate 20% decrease in the intracellular calcium transient that is associated with specific force deficit. Excess superoxide generation from the mitochondrial complexes causes a deficiency of succinate dehydrogenase and reduced complex-II-mediated respiration and adenosine triphosphate generation rates leading to severe exercise intolerance (~10 min vs. ~2 h in wild type, P < 0.05). CONCLUSIONS: Increased skeletal muscle mtROS is sufficient to elicit NMJ disruption and contractile abnormalities, but not muscle atrophy, suggesting new roles for mitochondrial oxidative stress in maintenance of muscle mass through increased fibre branching.
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A laser-diffraction technique was developed that rapidly reports the lengths of sarcomeres (L(s)) in serially connected sectors of permeabilized single fibers. The apparatus translates a laser beam along the entire length of a fiber segment within 2 ms, with brief stops at each of 20 contiguous sectors. We tested the hypothesis that during lengthening contractions, when maximally activated fibers are stretched, sectors that contain the longer sarcomeres undergo greater increases in L(s) than those containing shorter sarcomeres. Fibers (n = 16) were obtained from the soleus muscles of adult male rats and the middle portions (length = 1.05 +/- 0.11 mm; mean +/- SD) were investigated. Single stretches of strain 27% and a strain rate of 54% s(-1) were initiated at maximum isometric stress and resulted in a 19 +/- 9% loss in isometric stress. The data on L(s) revealed that 1), the stretch was not distributed uniformly among the sectors, and 2), during the stretch, sectors at long L(s) before the stretch elongated more than those at short lengths. The findings support the hypothesis that during stretches of maximally activated skeletal muscles, sarcomeres at longer lengths are more susceptible to damage by excessive strain.
Assuntos
Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Sarcômeros/fisiologia , Sarcômeros/ultraestrutura , Animais , Células Cultivadas , Masculino , Ratos , Ratos Endogâmicos F344 , Estatística como AssuntoRESUMO
BACKGROUND: We have previously shown that the deletion of the superoxide scavenger, CuZn superoxide dismutase, in mice (Sod1-/- mice) results in increased oxidative stress and an accelerated loss of skeletal muscle mass and force that mirror the changes seen in old control mice. The goal of this study is to define the effect of oxidative stress and ageing on muscle weakness and the Excitation Contraction (EC) coupling machinery in age-matched adult (8-10 months) wild-type (WT) and Sod1-/- mice in comparison with old (25-28 months) WT mice. METHODS: In vitro contractile assays were used to measure muscle contractile parameters. The activity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump was measured using an NADH-linked enzyme assay. Immunoblotting and immunofluorescence techniques were used to measure protein expression, and real-time reverse transcription PCR was used to measure gene expression. RESULTS: The specific force generated by the extensor digitorum longus muscle was reduced in the Sod1-/- and old WT mice compared with young WT mice along with significant prolongation of time to peak force, increased half relaxation time, and disruption of intracellular calcium handling. The maximal activity of the SERCA calcium uptake pump was significantly reduced in gastrocnemius muscle from both old WT (≈14%) and adult Sod1-/- (≈33%) mice compared with young WT mice along with increased expression of sarcolipin, a known inhibitor of SERCA activity. Protein levels of the voltage sensor and calcium uptake channel proteins dihydropyridine receptor α1 and SERCA2 were significantly elevated (≈45% and ≈57%, respectively), while the ratio of calstabin, a channel stabilizing protein, to ryanodine receptor was significantly reduced (≈21%) in Sod1-/- mice compared with young WT mice. The changes in calcium handling were accompanied by substantially elevated levels of global protein carbonylation and lipid peroxidation. CONCLUSIONS: Our data suggest that the muscle weakness in Sod1-/- and old WT mice is in part driven by reactive oxygen species-mediated EC uncoupling and supports a role for reduced SERCA pump activity in compromised muscle function. The novel quantitative mechanistic data provided here can lead to potential therapeutic interventions of SERCA dysfunction for sarcopenia and muscle diseases.
Assuntos
Acoplamento Excitação-Contração , Debilidade Muscular/etiologia , Debilidade Muscular/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Animais , Biomarcadores , Peso Corporal , Cálcio/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Espaço Intracelular/metabolismo , Camundongos , Camundongos Knockout , Debilidade Muscular/fisiopatologia , Músculo Esquelético/fisiopatologia , Processamento de Proteína Pós-Traducional , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Superóxido Dismutase-1/metabolismoRESUMO
Skeletal muscle contraction results from molecular interactions of myosin "crossbridges" with adjacent actin filament binding sites. The binding of myosin to actin can be "weak" or "strong," and only strong binding states contribute to force production. During active shortening, the number of strongly bound crossbridges declines with increasing shortening velocity. Forcibly stretching a muscle that is actively shortening at high velocity results in no apparent negative consequences, whereas stretch of an isometrically (fixed-length) contracting muscle causes ultrastructural damage and a decline in force-generating capability. Our working hypothesis is that stretch-induced damage is uniquely attributable to the population of crossbridges that are strongly bound. We tested the hypothesis that stretch-induced force deficits decline as the prevailing shortening velocity is increased. Experiments were performed on permeabilized segments of individual skeletal muscle fibers obtained from human subjects. Fibers were maximally activated and allowed either to generate maximum isometric force (Fo), or to shorten at velocities that resulted in force maintenance of ≈50% Fo or ≈2% Fo For each test condition, a rapid stretch equivalent to 0.1 × optimal fiber length was applied. Relative to prestretch Fo, force deficits resulting from stretches applied during force maintenance of 100, ≈50, and ≈2% Fo were 23.2 ± 8.6, 7.8 ± 4.2, and 0.3 ± 3.3%, respectively (means ± SD, n = 20). We conclude that stretch-induced damage declines with increasing shortening velocity, consistent with the working hypothesis that the fraction of strongly bound crossbridges is a causative factor in the susceptibility of skeletal muscle to stretch-induced damage.NEW & NOTEWORTHY Force deficits caused by stretch of contracting muscle are most severe when the stretch is applied during an isometric contraction, but prevented if the muscle is shortening at high velocity when the stretch occurs. This study indicates that velocity-controlled modulation of the number of strongly bound crossbridges is the basis for the observed relationship between stretch-induced muscle damage and prevailing shortening velocity.
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Elasticidade/fisiologia , Contração Isométrica/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Actinas/metabolismo , Adulto , Humanos , Masculino , Fenômenos Mecânicos , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Adulto JovemRESUMO
Permeabilized individual skeletal muscle fibers offer the opportunity to evaluate contractile behavior in a system that is greatly simplified, yet physiologically relevant. Here we describe the steps required to prepare, permeabilize and preserve small samples of skeletal muscle. We then detail the procedures used to isolate individual fiber segments and attach them to an experimental apparatus for the purpose of controlling activation and measuring force generation. We also describe our technique for estimating the cross-sectional area of fiber segments. The area measurement is necessary for normalizing the absolute force to obtain specific force, a measure of the intrinsic force-generating capability of the contractile system.
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Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Animais , Força Muscular , Músculo Esquelético/fisiologiaRESUMO
Compromised mitochondrial respiratory function is associated with advancing age. Damage due to an increase in reactive oxygen species (ROS) with age is thought to contribute to the mitochondrial deficits. The coenzyme nicotinamide adenine dinucleotide in its reduced (NADH) and oxidized (NAD(+)) forms plays an essential role in the cyclic sequence of reactions that result in the regeneration of ATP by oxidative phosphorylation in mitochondria. Monitoring mitochondrial NADH/NAD(+) redox status during recovery from an episode of high energy demand thus allows assessment of mitochondrial function. NADH fluoresces when excited with ultraviolet light in the UV-A band and NAD(+) does not, allowing NADH/NAD(+) to be monitored in real time using fluorescence microscopy. Our goal was to assess mitochondrial function by monitoring the NADH fluorescence response following a brief period of high energy demand in muscle from adult and old wild-type mice. This was accomplished by isolating whole lumbrical muscles from the hind paws of 7- and 28-month-old mice and making simultaneous measurements of force and NADH fluorescence responses during and after a 5 s maximum isometric contraction. All muscles exhibited fluorescence oscillations that were qualitatively similar and consisted of a brief transient increase followed by a longer transient period of reduced fluorescence and, finally, an increase that included an overshoot before recovering to resting level. Compared with the adult mice, muscles from the 28 mo mice exhibited a delayed peak during the first fluorescence transient and an attenuated recovery following the second transient. These findings indicate an impaired mitochondrial capacity to maintain NADH/NAD(+) redox homeostasis during contractile activity in skeletal muscles of old mice.
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A progressive loss of skeletal muscle mass and force generating capacity occurs with aging. Mice are commonly used in the study of aging-associated changes in muscle size and strength, with most models of aging demonstrating 15-35% reductions in muscle mass, cross-sectional area (CSA), maximum isometric force production (Po) and specific force (sPo), which is Po/CSA. The lumbrical muscle of the mouse forepaw is exceptionally small, with corresponding short diffusion distances that make it ideal for in vitro pharmacological studies and measurements of contractile properties. However, the aging-associated changes in lumbrical function have not previously been reported. To address this, we tested the hypothesis that compared to adult (12month old) mice, the forepaw lumbrical muscles of old (30month old) mice exhibit aging-related declines in size and force production similar to those observed in larger limb muscles. We found that the forepaw lumbricals were composed exclusively of fibers with type II myosin heavy chain isoforms, and that the muscles accumulated connective tissue with aging. There were no differences in the number of fibers per whole-muscle cross-section or in muscle fiber CSA. The whole muscle CSA in old mice was increased by 17%, but the total CSA of all muscle fibers in a whole-muscle cross-section was not different. No difference in Po was observed, and while sPo normalized to total muscle CSA was decreased in old mice by 22%, normalizing Po by the total muscle fiber CSA resulted in no difference in sPo. Combined, these results indicate that forepaw lumbrical muscles from 30month old mice are largely protected from the aging-associated declines in size and force production that are typically observed in larger limb muscles.
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Envelhecimento/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Miosinas de Músculo Esquelético/fisiologia , Fatores Etários , Animais , Constituição Corporal/fisiologia , Tecido Conjuntivo/fisiologia , Contração Isométrica/fisiologia , Camundongos , Modelos Animais , Cadeias Pesadas de Miosina , Isoformas de ProteínasRESUMO
Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.
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Fibras Musculares Esqueléticas/fisiologia , Animais , Feminino , Humanos , Contração Isométrica/fisiologia , Masculino , Camundongos , Fibras Musculares Esqueléticas/citologia , Permeabilidade , Ratos , Sarcômeros/fisiologiaRESUMO
Advanced age is associated with increases in muscle passive stiffness, but the contributors to the changes remain unclear. Our purpose was to determine the relative contributions of muscle fibers and extracellular matrix (ECM) to muscle passive stiffness in both adult and old animals. Passive mechanical properties were determined for isolated individual muscle fibers and bundles of muscle fibers that included their associated ECM, obtained from tibialis anterior muscles of adult (8-12 mo old) and old (28-30 mo old) mice. Maximum tangent moduli of individual muscle fibers from adult and old muscles were not different at any sarcomere length tested. In contrast, the moduli of bundles of fibers from old mice was more than twofold greater than that of fiber bundles from adult muscles at sarcomere lengths >2.5 µm. Because ECM mechanical behavior is determined by the composition and arrangement of its molecular constituents, we also examined the effect of aging on ECM collagen characteristics. With aging, muscle ECM hydroxyproline content increased twofold and advanced glycation end-product protein adducts increased threefold, whereas collagen fibril orientation and total ECM area were not different between muscles from adult and old mice. Taken together, these findings indicate that the ECM of tibialis anterior muscles from old mice has a higher modulus than the ECM of adult muscles, likely driven by an accumulation of densely packed extensively crosslinked collagen.
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Matriz Extracelular/fisiologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiologia , Envelhecimento/fisiologia , Animais , Colágeno/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Hidroxiprolina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/fisiologia , Sarcômeros/metabolismo , Sarcômeros/ultraestruturaRESUMO
Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.
Assuntos
Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Larva , Peixe-ZebraRESUMO
Mice deficient in Cu,Zn superoxide dismutase (Sod1 (-/-) mice) demonstrate elevated oxidative stress associated with rapid age-related declines in muscle mass and force. The decline in mass for muscles of Sod1 (-/-) mice is explained by a loss of muscle fibers, but the mechanism underlying the weakness is not clear. We hypothesized that the reduced maximum isometric force (F o) normalized by cross-sectional area (specific F o) for whole muscles of Sod1 (-/-) compared with wild-type (WT) mice is due to decreased specific F o of individual fibers. Force generation was measured for permeabilized fibers from muscles of Sod1 (-/-) and WT mice at 8 and 20 months of age. WT mice were also studied at 28 months to determine whether any deficits observed for fibers from Sod1 (-/-) mice were similar to those observed in old WT mice. No effects of genotype were observed for F o or specific F o at either 8 or 20 months, and no age-associated decrease in specific F o was observed for fibers from Sod1 (-/-) mice, whereas specific F o for fibers of WT mice decreased by 20 % by 28 months. Oxidative stress has also been associated with decreased maximum velocity of shortening (V max), and we found a 10 % lower V max for fibers from Sod1 (-/-) compared with WT mice at 20 months. We conclude that the low specific F o of muscles of Sod1 (-/-) mice is not explained by damage to contractile proteins. Moreover, the properties of fibers of Sod1 (-/-) mice do not recapitulate those observed with aging in WT animals.