Blebbistatin: use as inhibitor of muscle contraction

Blebbistatin (BLEB) is a recently discovered compound that inhibits myosin-II ATPase activity. In this study, we tested BLEB in intact and skinned isolated rat cardiac trabeculae, rat intact myocytes, and single rabbit psoas myofibrils. BLEB (10 μM) reduced twitch force and cell shortening that was reversed by exposure to light. BLEB treatment of skinned trabeculae in the dark (1 hr) reduced Ca²⁺-activated force . Rapid (<5 ms) BLEB application in Ca²⁺-activated rabbit myofibrils reduced force proportional to [BLEB]. Two-photon Indo1-AM ratio-metric confocal line-scan microscopy revealed no impact of BLEB on the cytosolic Ca²⁺ transient. BLEB reduced contractile force in skinned trabeculae without affecting tension-dependent myofilament ATPase activity. We conclude that BLEB specifically uncouples cardiac myofilament activation from Ca²⁺ activation without affecting EC coupling or cross-bridge cycling parameters. This agent could be useful to uncouple myofilament contractility from electrical events that lead to sarcoplasmic reticulum Ca²⁺ release in the cardiac myocyte (uncoupling agent) However, the compound is very sensitive to light, a property that limits its application to mechanistic physiological studies.     

Effect of contraction on lymphatic,venous, and tissue electrolytes and metabolites in rabbit skeletal muscle

Summary The effect of muscle contraction on lymphatic and plasma [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], [P_i], [lactate] ([Lac^–]); [creatine] ([Cr]), ideal osmolality (OSM), and [protein] was evaluated in femoral venous blood and lymph specimens sampled from the calf muscles of rabbits before, in the course of, and after contractions. In addition, total [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Cl^–], and [H₂O] were analyzed in the muscle tissue. To facilitate lymph sampling both hind limbs were passively flexed and extended, in imitation of natural running movements, by an electrically driven crank. The muscles of one side also performed superimposed rhythmic isotonic contractions. Before contractions, lymphatic [K⁺], [Na⁺], [Ca²⁺], [Mg²⁺], [Lac^–], [Cr], and OSM did not significantly differ from corresponding femoral venous concentrations, [Cl^–], and [P_i] were significantly higher, [protein] significantly lower in the lymph than in the plasma. During contractions lymphatic [K⁺], OSM, [Lac^–], and [P_i] were raised significantly more in the lymph compared with the plasma concentrations. [Na⁺], [Cl^–], [Ca²⁺], and [Mg²⁺] showed only small changes in the course of contractions and thereafter, and they were altered in a similar way in the lymph and plasma. It was suggested that lymphatic and interstitial concentrations were in equilibrium. Comparing inactive with active muscles, the latter lost K⁺ but gained Na⁺, Cl^–, and H₂O, whereas minimal changes occurred in the [Ca²⁺] and [Mg²⁺]. The changes were discussed in connection with the hypothesis that electrolyte shifts might be involved in the activation of the muscular non-proprioceptive interstitial nerve endings which appear to play a role in reflexogenic cardiovascular and respiratory control.A preliminary report of this work has been given elsewhere [33]Supported by Deutsche Forschungsgemeinschaft     

Shared and distinct mechanisms of skeletal muscle atrophy: A narrative review

Maintenance of skeletal muscle mass and function is an incredibly nuanced balance of anabolism and catabolism that can become distorted within different pathological conditions. In this paper we intend to discuss the distinct intracellular signaling events that regulate muscle protein atrophy for a given clinical occurrence. Aside from the common outcome of muscle deterioration, several conditions have at least one or more distinct mechanisms that creates unique intracellular environments that facilitate muscle loss. The subtle individuality to each of these given pathologies can provide both researchers and clinicians with specific targets of interest to further identify and increase the efficacy of medical treatments and interventions.     

Modulation of troponin C affinity for the thin filament by different cross-bridge states in skinned skeletal muscle fibers

In vertebrate skeletal muscle, the C-domain of troponin C (TnC) serves as an anchor; the N-domain regulates the position of troponin-tropomyosin on the thin filament after changes in intracellular Ca2+. Another type of thin-filament regulation is provided by cross-bridges. In this study, we use skinned fibers reconstituted with chicken recombinant TnC (rTnC) to examine TnC-thin filament affinity when cross-bridges containing different ligands are formed. Dissociation and equilibrium binding of apo-TnC (i.e., lacking divalent cations) under different conditions were monitored by a standard test for maximum tension (P (o)). After 10 min in low-Mg2+ relaxing solution, rTnC dissociation (i.e., tension loss) was 80% vs only 45% in rigor. In rigor, adding myosin subfragment 1 (S1) reduced dissociation approximately twofold, whereas stretching to reduce filament overlap increased dissociation to nearly the value for relaxed fibers. Dissociation of rTnC after addition of Pi or MgADP to form A.M.Pi or A.M.ADP cross-bridges was significantly greater than with rigor (A.M) bridges. The increase in P (o) during equilibration with different concentrations of rTnC showed that the affinity for rTnC binding to the thin filament increased progressively with stronger cross-bridges: rTnC concentrations for half-maximal reconstitution (K (0.5)) were 8.1, 3.7, 2.9, and 1.1 microM for A + M.ADP.Pi, A.M.Pi, A.M, and A.M + S1. Cross-bridges containing MgADP(-) (A.M.ADP) were also less effective than rigor bridges in promoting rTnC binding. We conclude that cross-bridge state and number both modulate TnC affinity for the thin filament and that the TnC C-domain is a central element in this pathway.     

A coupled electromechanical model for the excitation-dependent contraction of skeletal muscle

This work deals with the development and implementation of an electromechanical skeletal muscle model. To this end, a recently published hyperelastic constitutive muscle model with transversely isotropic characteristics, see Ehret et al. (2011), has been weakly coupled with Ohm’s law describing the electric current. In contrast to the traditional way of active muscle modelling, this model is rooted on a non-additive decomposition of the active and passive components. The performance of the proposed modelling approach is demonstrated by the use of three-dimensional illustrative boundary-value problems that include electromechanical analysis on tissue strips. Further, simulations on the biceps brachii muscle document the applicability of the model to realistic muscle geometries.     

The age-related loss of skeletal muscle mass and function: Measurement and physiology of muscle fibre atrophy and muscle fibre loss in humans

Age-related loss of skeletal muscle mass and function, sarcopenia, is associated with physical frailty and increased risk of morbidity (chronic diseases), in addition to all-cause mortality. The loss of muscle mass occurs incipiently from middle-age (∼1%/year), and in severe instances can lead to a loss of ∼50% by the 8–9th decade of life. This review will focus on muscle deterioration with ageing and highlight the two underpinning mechanisms regulating declines in muscle mass and function: muscle fibre atrophy and muscle fibre loss (hypoplasia) – and their measurement. The mechanisms of muscle fibre atrophy in humans relate to imbalances in muscle protein synthesis (MPS) and breakdown (MPB); however, since there is limited evidence for basal alterations in muscle protein turnover, it would appear that “anabolic resistance” to fundamental environmental cues regulating diurnal muscle homeostasis (namely physical activity and nutrition), underlie age-related catabolic perturbations in muscle proteostasis. While the ‘upstream’ drivers of the desensitization of aged muscle to anabolic stimuli are poorly defined, they most likely relate to impaired efficiency of the conversion of nutritional/exercise stimuli into signalling impacting mRNA translation and proteolysis. Additionally, loss of muscle fibres has been shown in cadaveric studies using anatomical fibre counts, and from iEMG studies demonstrating motor unit loss, albeit with few molecular investigations of this in humans. We suggest that defining countermeasures against sarcopenia requires improved understandings of the co-ordinated regulation of muscle fibre atrophy and fibre loss, which are likely to be inextricably linked.     

The association of objectively measured physical activity and sedentary behavior with skeletal muscle strength and muscle power in older adults: A systematic review and meta-analysis

BackgroundEngaging in physical activity (PA) and avoiding sedentary behavior (SB) are important for healthy ageing with benefits including the mitigation of disability and mortality. Whether benefits extend to key determinants of disability and mortality, namely muscle strength and muscle power, is unclear.AimsThis systematic review aimed to describe the association of objective measures of PA and SB with measures of skeletal muscle strength and muscle power in community-dwelling older adults.MethodsSix databases were searched from their inception to June 21^st, 2020 for articles reporting associations between objectively measured PA and SB and upper body or lower body muscle strength or muscle power in community dwelling adults aged 60 years and older. An overview of associations was visualized by effect direction heat maps, standardized effect sizes were estimated with albatross plots and summarized in box plots. Articles reporting adjusted standardized regression coefficients (β) were included in meta-analyses.ResultsA total of 112 articles were included representing 43,796 individuals (range: 21 to 3726 per article) with a mean or median age from 61.0 to 88.0 years (mean 56.4 % female). Higher PA measures and lower SB were associated with better upper body muscle strength (hand grip strength), upper body muscle power (arm curl), lower body muscle strength, and lower body muscle power (chair stand test). Median standardized effect sizes were consistently larger for measures of PA and SB with lower compared to upper body muscle strength and muscle power. The meta-analyses of adjusted β coefficients confirmed the associations between total PA (TPA), moderate-to-vigorous PA (MVPA) and light PA (LPA) with hand grip strength (β = 0.041, β = 0.057, and β = 0.070, respectively, all p ≤ 0.001), and TPA and MVPA with chair stand test (β = 0.199 and β = 0.211, respectively, all p ≤ 0.001).ConclusionsHigher PA and lower SB are associated with greater skeletal muscle strength and muscle power, particularly with the chair stand test.     

Null mutations causing depletion of the type 1 ryanodine receptor (RYR1) are commonly associated with recessive structural congenital myopathies with cores

Mutations of the ryanodine receptor cause dominant and recessive forms of congenital myopathies with cores. Quantitative defects of RYR1 have been reported in families presenting with recessive forms of the disease and epigenic regulation has been recently proposed to explain potential maternal monoallelic silencing of the RYR1 gene. We investigated nine families presenting with a recessive form of the disease and showing a quantitative defect of RYR1 expression. Genetic analysis allowed the identification of a mutation on both alleles of the RYR1 gene for all patients, 15 being novel variants. We evidenced for all patients an alteration of the expression of the RYR1 gene caused by amorphic mutations responsible either for mRNA or protein instability. In seven families the variant present on the second allele was a missense mutation. In the remaining two families the second variant led to a hypomorphic expression of the RYR1 gene and was associated with a severe neonatal phenotype, pointing out the minimal amount of RYR1 needed for skeletal muscle function. Noticeably, a novel additional exon 3b was characterized in the most severely affected cases. This study showed that all cases presenting with a quantitative defect of RYR1 expression in our panel of patients affected by recessive core myopathies were caused by the presence of one recessive null allele and that variability of the phenotype depended on the nature of the mutation present on the second allele. Our study also indicated that presence of a second mutation must be investigated in sporadic cases or in dominant cases presenting with a familial clinical variability.     

Hormonal regulation of skeletal muscle hypertrophy in rats: the testosterone to cortisol ratio

This study determined the influence that the catabolic hormone, corticosterone (C), and the anabolic hormone, testosterone (T), had in regulating skeletal muscle hypertrophy using the rat hind limb ablation model. Specifically, the ratio of T : C (TCR) was manipulated via hormone implants and injections and concentrations measured to evaluate the relative contribution of each hormone to skeletal muscle protein balance. Skeletal muscle growth was measured 16 days after gastrocnemius muscle ablation. Elevations in plasma concentrations of C (via daily C injections, 50 mg · kg^–1 body mass) resulted in TCR of 0.007 that was less than the control group TCR of 0.249. In this C-injected group, whole body and skeletal muscle atrophy was elicited-this being greater in the fast-twitch plantaris muscle than in the slow-twitch soleus muscle. The overloaded leg resisted the C-induced atrophy. Castration of animals (TCR 0.024) resulted in less whole body and skeletal muscle growth. However, elevations in plasma concentrations of T (two groups, with TCR of 1.35 and 1.64) did not result in significantly greater muscle growth. Furthermore, T was also ineffective in antagonizing the C-induced atrophy in a group that received both T implants and C injections. This group had a TCR of 0.175 that was similar to the control group ratio of 0.249 that received no manipulations. We concluded that glucocorticoids were able to induce pronounced atrophy, but at the same time overloaded muscles were able to over-ride the glucocorticoid signal. Plasma concentrations of C were a better predictor of muscle growth/atropy than T and/or the TCR. In addition, it is suggested that the volume of contractile activity of the muscle is perhaps an important determinant of C-induced atrophy, because less atrophy occurs in the more active slow twitch muscles.     

Changes in the action potential and contractile properties of skeletal muscle in human's with repetitive stimulation after long-term dry immersion

This paper compares the effects of 7-daydry immersion and intermittent muscle contraction on electrical and mechanical failure during muscle fatigue in the human triceps surae muscle electrically stimulated at 50 impulses·s⁻¹ via its motor nerve. Intermittent contractions of 60-s duration were separated by 1-s intervals for identical duration of tension development. The 60-s intermittent contractions decreased tetanic force to 57% (P<0.05) of initial values, but force reduction was not significantly different in the two fatigue tests: the fatigue index was 36.2 (SEM 5.4)% versus 38.6 (SEM 2.8)%, respectively (P>0.05). Whilst identical force reduction was present in the two fatigue tests, it would appear that concomitant electrical failure was considerably different. This electromechanical dissociation would suggest that a slowing of conduction along nerve and muscle membranes did not explain the observed mechanical failure. It is suggested that intracellular processes played major role in contractile failure during intermittent contractions after muscle disuse.     

Invertebrate muscles: thin and thick filament structure; molecular basis of contraction and its regulation, catch and asynchronous muscle

This is the second in a series of canonical reviews on invertebrate muscle. We cover here thin and thick filament structure, the molecular basis of force generation and its regulation, and two special properties of some invertebrate muscle, catch and asynchronous muscle. Invertebrate thin filaments resemble vertebrate thin filaments, although helix structure and tropomyosin arrangement show small differences. Invertebrate thick filaments, alternatively, are very different from vertebrate striated thick filaments and show great variation within invertebrates. Part of this diversity stems from variation in paramyosin content, which is greatly increased in very large diameter invertebrate thick filaments. Other of it arises from relatively small changes in filament backbone structure, which results in filaments with grossly similar myosin head placements (rotating crowns of heads every 14.5nm) but large changes in detail (distances between heads in azimuthal registration varying from three to thousands of crowns). The lever arm basis of force generation is common to both vertebrates and invertebrates, and in some invertebrates this process is understood on the near atomic level. Invertebrate actomyosin is both thin (tropomyosin:troponin) and thick (primarily via direct Ca(++) binding to myosin) filament regulated, and most invertebrate muscles are dually regulated. These mechanisms are well understood on the molecular level, but the behavioral utility of dual regulation is less so. The phosphorylation state of the thick filament associated giant protein, twitchin, has been recently shown to be the molecular basis of catch. The molecular basis of the stretch activation underlying asynchronous muscle activity, however, remains unresolved.     

Pathology of skeletal muscle: Principles of reaction patterns and histochemistry and experience with 195 biopsies

Summary Technique and interpretation of muscle biopsies require the coodinated team work between referring clinician, surgeon, and pathologist so that optimal management of a patient with neuromuscular disease may be attained. Investigation of muscle diseases has advanced so much with an interdisciplinary approach that not only can accurate diagnosis be offered for the benefit of the patient in certain instances, but also genetic counselling provided and pre-natal diagnosis established. The principal reaction patterns and pathogenetic mechanisms of skeletal muscle as a contractile and metabolically active tissue are described; the diagnostic usefulness of enzyme histochemistry and the basic principles of the motor unit are discussed and illustrated. For the practicing pathologist, adequate tissue preparation of a muscle biopsy specimen requires interest and a willingness to dedicate time, effort and funds. While the paraffin-embedded section is still very valuable, enzyme histochemistry provides certain highly diagnostic information not otherwise obtainable. Likewise, there must be an interest in electron microscopy and appreciation for its value in depicting ultrastructural abnormalities when certain reaction patterns are apparent on light microscopic sections, enzyme histochemical stains, or Epon-embedded thick sections. Finally, concurrent sural nerve biopsy also requires optimal processing and interpretation.Presented in part at the Canadian Association of Neuropathologists' annual meeting, September 29–October 1, 1977, Vancouver, Canada, and at the Virginia Society of Histology Technicians' Spring Workshop, March 29–31, 1979, Charlottesville, Virginia     

The expression pattern of contractile and intermediate filament proteins in developing skeletal muscle and rhabdomyosarcoma of childhood: Diagnostic and prognostic utility

In order to investigate whether rhabdomyosarcoma (RMS) can be related to equivalent stages of skeletal muscle development, muscle tissue of 21 human foetuses and 112 primary RMSs were characterized immunohistochemically using antibodies directed against vimentin, desmin, muscle-specific actin (HHF35), sarcomeric actin (sr-actin), smooth muscle actin (sm-actin), and troponin-T. During fetal skeletal muscle development, all myotubes/fibres of the first and second generations expressed desmin, HHF35, and sr-actin. Vimentin was almost exclusively present in immature primary and secondary myotubes/fibres. Troponin-T was expressed in immature myotubes/fibres of the first and second generations as well as mature fibres of the second generation. Sm-actin was never expressed. Vimentin was expressed in 96 per cent of primary and 98 per cent of relapsed RMS; HHF35 in 96 and 98 per cent, respectively; desmin in 95 and 100 per cent; troponin-T in 82 and 75 per cent; sr-actin in 71 and 86 per cent; and sm-actin in 13 and 17 per cent. The proportion of RMS cells reacting with vimentin, HHF35, and desmin was consistently higher than those expressing sr-actin and troponin-T. Neither the shape nor size of neoplastic RMS cells nor the histopathological types were related to the expression pattern of the investigated markers. RMS with aberrant expression of two or more markers predicted a worse prognosis than RMS in which at most one marker was aberrantly expressed (25 per cent and 54 per cent 10-year survival, P=0·01). These results demonstrate that HHF35, desmin, sr-actin, and troponin-T have the potential to confirm the commitment of the tumours to the myogenic pathway which supports the diagnosis of RMS. However, it was impossible to relate RMS to equivalent stages of skeletal muscle development. Aberrant marker expression by RMS cells correlated significantly with patients' survival.     

Intensity of X-ray reflections from skeletal muscle thin filaments partially occupied with myosin heads: effect of cooperative binding

For quantitative analysis of contractile proteins of muscle by means of X-ray diffraction, it is important to know how the intensities of individual reflections are related to the number of diffracting objects, i.e., the amount of constituent contractile protein in the muscle cell. Here we diffused various amounts of exogenous myosin subfragment-1 (S1) into overstretched skinned skeletal muscle fibers, either in the presence or absence of Ca²⁺ , and derived the relationship between the S1 content and the intensities of reflections arising from the S1. In theory, the intensities should be proportional to the square of the S1 content (square law). However, the intensity--content relation deviated systematically from the square law as the S1 content was lowered, and it was better described as a linear function at the lower end of the S1 contents (<20% of saturation level). Model calculations show that the way of deviation is explained by the cooperative manner of S1 binding to the regulated thin filament. This revised version was published online in July 2006 with corrections to the Cover Date.