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Duchenne muscular dystrophy (DMD) is a fatal X-linked genetic disease characterized by progressive loss of skeletal muscle. The mechanisms underlying the DMD pathology likely involve the complex interaction between reactive oxygen species (ROS) impaired Ca2+ handling and chronic inflammation, characterized by the presence of immune cells such as neutrophils. Hypochlorous acid (HOCl) is a highly reactive form of ROS produced endogenously via the actions of myeloperoxidase, an enzyme secreted by neutrophils. Myeloperoxidase activity is significantly elevated in dystrophic muscle. This study aimed to determine the effect of HOCl exposure on excitation–contraction coupling and its potential contribution to the dystrophic pathology. Isolated extensor digitorum longus (EDL) muscles and single fibers from C57 (wild type) and mdx (dystrophic) mice were used to investigate the effects of HOCl on whole muscle function, intracellular Ca2+ handling, and myofilament force production. HOCl exposure significantly decreased maximum specific force in isolated EDL muscles by 26% and 49%, respectively, in C57 and mdx mice (P < 0.0001). In single interosseous fibers, HOCl exposure significantly increased resting intracellular Ca2+ concentration by ∼17–19% (P < 0.05) and decreased the amplitude of electrically induced Ca2+ transients by ∼45% and 50%, respectively, in C57 and mdx fibers (C57, P < 0.05; mdx, P < 0.01). These effects of HOCl on resting Ca2+ could be blocked via application of tetracaine (ryanodine receptor blocker) or Gd3+ (stretch-activated channel blocker; C57, P < 0.01; mdx, P < 0.01 for both). The effect of HOCl on Ca2+ transient amplitude was significantly reduced by Gd3+ (C57, P < 0.05; mdx, P < 0.01). In chemically skinned EDL fibers, HOCl exposure decreased maximum Ca2+-activated force by ∼40% in both C57 and mdx fibers (P < 0.001). These results indicate that HOCl potently affects excitation–contraction coupling via impaired Ca2+ handling and myofilament force production. Hence, HOCl potentially links the chronic inflammation, oxidative stress, and impaired Ca2+ handling that underlies the dystrophic pathology.

Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca2+) handling and glucose/glycogen metabolism-related proteins in predominantly slow-twitch, oxidative soleus and fast-twitch, glycolytic extensor digitorum longus (EDL) muscles of 10-month-old wild-type (WT) C57BL/6J and dysferlin-deficient BLAJ male mice. Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy.

The preterm diaphragm is functionally immature compared with its term counterpart. In utero inflammation further exacerbates preterm diaphragm dysfunction. We hypothesized that preterm lambs are more vulnerable to in utero inflammation-induced diaphragm dysfunction compared with term lambs. Pregnant ewes received intra-amniotic (IA) injections of saline or 10 mg lipopolysaccharide (LPS) 2 or 7 days before delivery at 121 days (preterm) or ∼145 days (term) of gestation. Diaphragm contractile function was assessed in vitro. Plasma cytokines, diaphragm myosin heavy chain (MHC) isoforms, and oxidative stress were evaluated. Maximum diaphragm force in preterm control lambs was significantly lower (22%) than in term control lambs ( P < 0.001). Despite similar inflammatory cytokine responses to in utero LPS exposure, diaphragm function in preterm and term lambs was affected differentially. In term lambs, maximum force after a 2-day LPS exposure was significantly lower than in controls (by ~20%, P < 0.05). In preterm lambs, maximum forces after 2-day and 7-day LPS exposures were significantly lower than in controls (by ~30%, P < 0.05). Peak twitch force after LPS exposure was significantly lower in preterm than in controls, but not in term lambs. In term lambs, LPS exposure increased the proportion of MHC-I fibers, increased twitch contraction times, and increased fatigue resistance relative to controls. In preterm diaphragm, the cross-sectional area of embryonic MHC fibers was significantly lower after 7-day versus 2-day LPS exposures. We conclude that preterm lambs are more vulnerable to IA LPS-induced diaphragm dysfunction than term lambs. In utero inflammation exacerbates diaphragm dysfunction and may increase susceptibility to postnatal respiratory failure.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked muscle wasting disease characterised by severe muscle weakness, necrosis, inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a potential therapeutic intervention for DMD boys. We investigated the capacity of NAC to improve dystrophic muscle function in the mdx mouse model of DMD. Young (6 w old) mdx and non-dystrophic C57 mice receiving 2% NAC in drinking water for 6 w were compared with untreated mice. Grip strength and body weight were measured weekly, before the 12 w old mice were anaesthetized and extensor digitorum longus (EDL) muscles excised for functional analysis and tissues sampled for biochemical analyses. Compared to untreated mice, the mean (SD) normalised grip strength was significantly greater in NAC treated mdx [3.13 (0.58) cf. 4.87 (0.78) g bw−1; P

Background In utero diaphragm development is critically important for postnatal respiratory function and any disturbance to fetal development may lead to diaphragm dysfunction and respiratory complications in the postnatal period. Intrauterine growth restriction (IUGR) has been shown to affect respiratory function in a sex-dependent manner; however, the effect of IUGR on diaphragm function is unknown. Aim This study used a maternal hypoxia-induced mouse model of IUGR to investigate the impact of IUGR on diaphragm function and structure in male and female adult offspring. Materials and methods Pregnant BALB/c mice were housed under hypoxic conditions (10.5% O2 ) from gestational days 11 to 17.5 and then returned to normoxic conditions. Control mice were housed under normoxic conditions throughout pregnancy. At 8 weeks of age, offspring were euthanized and diaphragms isolated for functional assessment in organ bath experiments and for histological analysis. Results IUGR offspring were lighter at birth and remained lighter at 8 weeks of age compared to Controls. While diaphragm force (maximal or twitch) was not affected by treatment or sex, the IUGR group exhibited a longer half-relaxation time after twitch contractions compared to Control. Female offspring had a lower maximum rate of force development and higher fatigue resistance compared to males, independent of IUGR. There was no difference in the diaphragm myofibre cross-sectional area between groups or sexes. Conclusion Sex and IUGR independently affect diaphragm contraction in adult mice without changes in structure. This study demonstrates that IUGR affects diaphragm contractile function in later life and could impair respiratory function if exacerbated under conditions of increased respiratory load.

Key points Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation, oxidative stress and myofibre necrosis. Cysteine precursor antioxidants such as N-acetyl cysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) reduce dystropathology in the mdx mouse model for DMD, and we propose this is via increased synthesis of the amino acid taurine. We compared the capacity of OTC and taurine treatment to increase taurine content of mdx muscle, as well as effects on in vivo and ex vivo muscle function, inflammation and oxidative stress. Both treatments increased taurine in muscles, and improved many aspects of muscle function and reduced inflammation. Taurine treatment also reduced protein thiol oxidation and was overall more effective, as OTC treatment reduced body and muscle weight, suggesting some adverse effects of this drug. These data suggest that increasing dietary taurine is a better candidate for a therapeutic intervention for DMD. Abstract Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease for which there is no widely available cure. Whilst the mechanism of loss of muscle function in DMD and the mdx mouse model are not fully understood, disruptions in intracellular calcium homeostasis, inflammation and oxidative stress are implicated. We have shown that protein thiol oxidation is increased in mdx muscle, and that the indirect thiol antioxidant l-2-oxothiazolidine-4-carboxylate (OTC), which increases cysteine availability, decreases pathology and increases in vivo strength. We propose that the protective effects of OTC are a consequence of conversion of cysteine to taurine, which has itself been shown to be beneficial to mdx pathology. This study compares the efficacy of taurine with OTC in decreasing dystropathology in mdx mice by measuring in vivo and ex vivo contractile function and measurements of inflammation and protein thiol oxidation. Increasing the taurine content of mdx muscle improved both in vivo and ex vivo muscle strength and function, potentially via anti-inflammatory and antioxidant effects of taurine. OTC treatment increased taurine synthesis in the liver and taurine content of mdx muscle, improved muscle function and decreased inflammation. However, OTC was less effective than taurine treatment, with OTC also decreasing body and EDL muscle weights, suggesting that OTC had some detrimental effects. These data support continued research into the use of taurine as a therapeutic intervention for DMD, and suggest that increasing dietary taurine is the better strategy for increasing taurine content and decreasing severity of dystropathology.

Background and objective In utero infection may critically influence diaphragm development and predispose preterm infants to postnatal respiratory failure. We aimed to determine how frequency and gestational age (GA) at time of intra-amniotic (IA) lipopolysaccharide (LPS) exposure affects preterm diaphragm function. Methods Pregnant ewes received IA injections of saline or 10-mg LPS at 7 days or 21 days or weekly injections 21, 14 and 7 days before delivery at 121-day GA. Foetal lambs were killed with pentobarbitone (150 mg/kg; intravenous). Diaphragm contractile function was measured in vitro. Muscle fibre type, activation of protein synthesis and degradation pathways, pro-inflammatory signalling and oxidative stress were evaluated using immunofluorescence staining, RT-qPCR, ELISA, Western blotting and biochemical assay. Results In utero LPS exposure significantly impaired diaphragm contractile function. LPS exposure 7 days before delivery caused maximum specific twitch and tetanic forces 30% lower than controls. When the initial LPS exposure occurred 21 days before delivery maximum specific forces were 40% lower than controls. Earlier LPS exposure also prolonged twitch contraction time, increased fatigue resistance and elevated protein carbonyl content. Despite increased white blood cell counts and interleukin-6 mRNA expression following weekly LPS exposure, there were no significant differences in contractile properties between exposure 21 days before delivery and repeated LPS groups suggesting that frequency of inflammatory exposure does not influence the severity of contractile dysfunction. Conclusions GA at time of initial LPS exposure, rather than frequency of exposure, determines the extent of inflammation-induced diaphragm dysfunction.

Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disease manifested in young boys, for which there is no current cure. We have shown that the amino acid taurine is safe and effective at preventing dystropathology in the mdx mouse model for DMD. This study aimed to establish if treating growing mdx mice with a higher dose of taurine was more effective at improving strength and reducing inflammation and oxidative stress. Mice were treated with a dose of taurine estimated to be 16 g/kg/day, in drinking water from 1-6 weeks of age, after which in vivo and ex vivo muscle strength was assessed, as were measures of inflammation, oxidative stress and taurine metabolism. While the dose did decrease inflammation and protein oxidation in dystrophic muscles, there was no improvement in muscle strength (in contrast with benefits observed with the lower dose) and growth of the young mice was significantly restricted. We present novel data that a high taurine dose increases the cysteine content of both mdx liver and plasma, a possible result of down regulation of the taurine synthesis pathway in the liver (which functions to dispose of excess cysteine, which is toxic). These data caution that a high dose of taurine can have adverse effects and may be less efficacious than lower taurine doses. Therefore, monitoring of taurine dosage needs to be considered in future pre-clinical trials, in anticipation of using taurine as a clinical therapy for growing DMD boys (and other conditions).

Funding provided by the National Health and Medical Research Council (NHMRC) of Australia (1027218). P.N. and K.W. are supported by NHMRC Fellowships (1045824, 1090888). P.W. was supported by the William and Marlene Schrader Postgraduate Scholarship, The University of Western Australia, and C.A. by an NHMRC Preterm Infants CRE top-up scholarship. This study tested the utility of optical coherence tomography (OCT)-based indentation to assess mechanical properties of respiratory tissues in disease. Using OCT-based indentation, the elastic modulus of mouse diaphragm was measured from changes in diaphragm thickness in response to an applied force provided by an indenter. We used a transgenic mouse model of chronic lung disease induced by the overexpression of transforming growth factor-alpha (TGF-α), established by the presence of pleural and peribronchial fibrosis and impaired lung mechanics determined by the forced oscillation technique and plethysmography. Diaphragm elastic modulus assessed by OCT-based indentation was reduced by TGF-α at both left and right lateral locations (p < 0.05). Diaphragm elastic modulus at left and right lateral locations were correlated within mice (r = 0.67, p < 0.01) suggesting that measurements were representative of tissue beyond the indenter field. Co-localised images of diaphragm after TGF-α overexpression revealed a layered fibrotic appearance. Maximum diaphragm force in conventional organ bath studies was also reduced by TGF-α overexpression (p < 0.01). Results show that OCT-based indentation provided clear delineation of diseased diaphragm, and together with organ bath assessment, provides new evidence suggesting that TGF-α overexpression produces impairment in diaphragm function and, therefore, an increase in the work of breathing in chronic lung disease. Publisher PDF

Introduction: Protease-activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles from PAR-1 null and littermate control mice. Methods: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. Results: Soleus muscles from PAR-1 null mice exhibited longer contraction times, a leftward shift in the force–stimulation frequency relationship, and decreased fatiguability compared with controls. PAR-1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR-1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. Conclusions: The absence of PAR-1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991–998, 2014

Dysferlinopathies are a form of muscular dystrophy caused by gene mutations resulting in deficiency of the protein dysferlin. Symptoms manifest later in life in a muscle specific manner, although the pathomechanism is not well understood. This study compared the impact of dysferlin-deficiency on in vivo and ex vivo muscle function, and myofibre type composition in slow (soleus) and fast type (extensor digitorum longus; EDL) muscles using male dysferlin-deficient (dysf-/-) BLAJ mice aged 10 months, compared with wild type (WT) C57Bl/6J mice. There was a striking increase in muscle mass of BLAJ soleus (+25%) (p0.05); however, for the BLAJ EDL, there was an increased proportion of type IIx MHC isoform (+5.5%) and decreased type IIb isoform (-5.5%) (ps

BackgroundPregnant women at a high risk of preterm delivery receive glucocorticoids to accelerate fetal lung maturation and surfactant synthesis. However, the effect of antenatal steroids on the developing diaphragm remains unclear. We hypothesized that maternal betamethasone impairs the fetal diaphragm, and the magnitude of the detrimental effect increases with longer duration of exposure. We aimed to determine how different durations of fetal exposure to maternal betamethasone treatment influence the fetal diaphragm at the functional and molecular levels.MethodsDate-mated merino ewes received intramuscular injections of saline (control) or two doses of betamethasone (5.7 mg) at an interval of 24 h commencing either 2 or 14 days before delivery. Preterm lambs were killed after cesarean delivery at 121-day gestational age. In vitro contractile measurements were performed on the right hemidiaphragm, whereas molecular/cellular analyses used the left costal diaphragm.ResultsDifferent durations of fetal exposure to maternal betamethasone had no consistent effect on the protein metabolic pathway, expression of glucocorticoid receptor and its target genes, cellular oxidative status, or contractile properties of the fetal lamb diaphragm.ConclusionThese data suggest that the potential benefits of betamethasone exposure on preterm respiratory function are not compromised by impaired diaphragm function after low-dose maternal intramuscular glucocorticoid exposure.

Introduction: The skeletal muscle weakness associated with many chronic diseases has been attributed to the catabolic effect of pro-inflammatory cytokines. We aimed to determine if local muscle inflammation has direct affects on contractile function and contributes to muscle weakness inde- pendent of muscle atrophy or mechanical injury. Methods: Local muscle inflammation was induced by injecting an algal-derived polysaccharide, carrageenan (10 mg/kg), into the right tibialis anterior muscle in healthy ARC mice. The contralateral muscle was injected with sterile isotonic saline, and the muscles were removed after 24 h for measurement of contractile function and cytokine concentration. Results: Carrageenan significantly reduced maximum specific force, decreased the maximum rate of force development, altered the force-frequency relationship, and increased intramuscular levels of pro-inflammatory cyto- kines and chemokines. Conclusions: These results indicate that carrageenan directly affects contractile function and causes skeletal muscle weakness. Local muscle inflammation may contribute to the weakness observed in inflammatory related disorders. Muscle Nerve 46: 413-420, 2012

The aim of the present study was to examine the temperature dependence of active force in lengthening and shortening muscle. Experiments were done, in vitro, on bundles of intact fibres (fibre length L(0) ~2 mm; sarcomere length ~2.5 μm) isolated from a rat fast muscle (flexor hallucis brevis) and a ramp length change of 5-7% L(0) was applied on the plateau of an isometric tetanic contraction. Ramp lengthening increased and ramp shortening decreased the muscle tension to new approximately steady levels in a velocity-dependent way. The isometric tension and the lower steady tension reached at a given shortening velocity, increased with warming from 10 to 35 °C and the relation between tension and reciprocal absolute temperature was sigmoidal. However, the tension-temperature curve of shortening muscle was sharper and shifted to higher temperature with increased velocity. In contrast, the enhanced steady tension during lengthening at a given velocity was largely temperature-insensitive within the same temperature range; we hypothesize that the tension-temperature curve may be shifted to lower temperatures in lengthening muscle. Consequently, when normalised to the isometric tension at each temperature, the tension during lengthening at a given velocity decreased exponentially with increase of temperature. The residual force enhancement that remains after ramp lengthening showed a similar behaviour and was markedly reduced in warming from 10 to 35 °C. The findings are consistent with the thesis that active force generation in muscle is endothermic and strain-sensitive; during shortening with a faster crossbridge cycle it becomes more pronounced, but during lengthening it becomes depressed as the cycle slows in a velocity-dependent way. The residual force enhancement may be caused by the same process in addition to non-crossbridge mechanism(s).

When an active muscle is stretched, the force increases due to strain of contractile and noncontractile proteins. We examined this force enhancement in rat extensor digitorum longus (EDL) and soleus muscles, which differ in their composition of these proteins, and their susceptibility to damage. Small stretches were applied at different velocities during isometric contractions from which we quantified the velocity-dependent contractile and velocity-independent noncontractile contributions to force enhancement. Whereas the contractile contribution was significantly greater in soleus than EDL, the noncontractile force enhancement was significantly greater in EDL than soleus, and increased ≈6-fold after damaging eccentric contractions. The increased contractile stiffness may be functionally beneficial in slow muscle, as resistance to lengthening is fundamental to maintaining posture. Following stretch-induced muscle damage this capacity is compromised, leading to increased strain of noncontractile proteins that may facilitate the activation of signaling pathways involved in muscle adaptation to injury.

In this study we examined the effects of simulated horizontal and vertical viewpoint jitter on the vection and postural sway induced by radial patterns of optic flow. During each trial, observers were exposed sequentially to 20 s periods of radially expanding flow, radially contracting flow, and static visual scenes. For half the trials, simulated viewpoint jitter was added to the radially expanding/contracting optic flow patterns. In experiment 1, we found that, while this jitter increased the backward postural sway induced by radial expansion, it actually decreased forward postural sway induced by radial contraction. However, in experiment 2 we found that jitter increased both the forward and backward vection induced by radially expanding and contracting flow patterns. We conclude that the processes involved in postural control are more sensitive to the sensory conflicts generated by viewpoint jitter than those involved in the perception of self-motion, and that the observed asymmetries in forward and backward sway are ecological in origin.

We examined the tension change induced by a rapid temperature jump (T-jump) in shortening and lengthening active muscle fibres. Experiments were done on segments of permeabilized single fibres (length (L0) approximately 2 mm, sarcomere length 2.5 microm) from rabbit psoas muscle; [MgATP] was 4.6 mm, pH 7.1, ionic strength 200 mm and temperature approximately 9 degrees C. A fibre was maximally Ca2+-activated in the isometric state and a approximately 3 degrees C, rapid ( 0.05 L0 s(-1), whereas the tension decreased to about < 0.5 x P0 during shortening at 0.1-0.2 L0 s(-1); the unloaded shortening velocity was approximately 1 L0 s(-1) and the curvature of the force-shortening velocity relation was high (a/P0 ratio from Hill's equation of approximately 0.05). In isometric state, a T-jump induced a tension rise of 15-20% to a new steady state; by curve fitting, the tension rise could be resolved into a fast (phase 2b, 40-50 s(-1)) and a slow (phase 3, 5-10 s(-1)) exponential component (as previously reported). During steady lengthening, a T-jump induced a small instantaneous drop in tension, followed by recovery, so that the final tension recorded with and without a T-jump was not significantly different; thus, a T-jump did not lead to a net increase of tension. During steady shortening, the T-jump induced a pronounced tension rise and both its amplitude and the rate (from a single exponential fit) increased with shortening velocity; at 0.1-0.2 L0 s(-1), the extent of fibre shortening during the T-jump tension rise was estimated to be approximately 1.2% L(0) and it was shorter at lower velocities. At a given shortening velocity and over the temperature range of 8-30 degrees C, the rate of T-jump tension rise increased with warming (Q10 approximately 2.7), similar to phase 2b (endothermic force generation) in isometric muscle. Results are discussed in relation to the previous findings in isometric muscle fibres which showed that a T-jump promotes an early step in the crossbridge-ATPase cycle that generates force. In general, the finding that the T-jump effect on active muscle tension is pronounced during shortening, but is depressed/inhibited during lengthening, is consistent with the expectations from the Fenn effect that energy liberation (and acto-myosin ATPase rate) in muscle are increased during shortening and depressed/inhibited during lengthening.

Introduction: Transforming growth factor alpha (TGFα) is increased in the lung tissue of patients with chronic respiratory disease. Animal models show that deficiency in the early growth response (Egr-1) gene accelerates the disease process. Aim: To examine the effects of TGFα on lung and diaphragm mechanics. Method: Doxycycline produced conditional expression of TGFα in the airways of transgenic mice (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGFα(+/-)) heterozygous for the Egr-1 gene. Four-week old mice were fed doxycycline in chow (Dox) or a control diet (Control) for three weeks. Lung mechanics were assessed in anaesthetised and mechanically ventilated mice by the forced oscillation technique. Separate groups of mice were euthanized and the diaphragm removed for ex vivo study. Specific diaphragmatic force was measured in an organ bath and transverse elastance measured by optical elastography. Results: Fibrosis was identified in the lung pleura, peribronchial space and diaphragm with associated thickening of the central tendon. Lung elastance ( P =0.02), tissue damping ( P =0.01) and airway resistance ( P =0.02) at functional residual capacity were increased in the Dox group (n=12) compared with Control (n=7). Specific diaphragmatic force fell with increasing collagen within the muscle layer (r=-0.77, P =0.001, n=16). Transverse diaphragm elastance was reduced ( P =0.04) in the Dox group (n=10) compared with control (n=8) suggesting fibrosis related dissociation of skeletal fibres. Conclusion: Expression of TGFα impairs respiratory function characterised by increased lung loading and compromised diaphragm mechanics. Findings support a role of TGFα in chronic lung disease.

Lengthening of active muscle is an essential feature of animal locomotion, but the molecular processes occurring are incompletely understood. We therefore examined and modelled tension responses to ramp stretches (5% fibre length, L0) over a wide range of velocities (0.1–10 L0 s−1) of tetanized intact rat muscle fibre bundles (L0∼2 mm) with a resting sarcomere length of 2.5 μm at 20°C. Tension rose to a peak during stretch and decayed afterwards to a level which was higher than the prestretch tetanic tension. This residual force enhancement was insensitive to velocity. The tension rise during stretch showed an early transition (often appearing as an inflection) at ∼1 ms. Both the stretch (L1) and the tension rise at this transition increased in proportion to velocity. A second transition, marked by a reduction in slope, occurred at a stretch of ∼18 nm per half-sarcomere; the rise in tension at this transition increased with velocity towards a plateau. Based on analyses of the velocity dependence of the tension and modelling, we propose that the initial steep increase in tension arises from increasing strain of all attached crossbridges and that the first transition reflects the tension loss due to the original post-stroke heads executing a reverse power stroke. Modelling indicates that the reduction in slope at the second transition occurs when the last of the heads that were attached at the start of the ramp become detached. Thereafter, the crossbridge cycle is largely truncated, with prepower stroke crossbridges rapidly detaching at high strain and attaching at low strain, the tension being borne mainly by the prestroke heads. Analysis of the tension decay after the ramp and the velocity dependence of the peak tension suggest that a non-crossbridge component increasingly develops tension throughout the stretch; this decays only slowly, reaching at 500 ms after the ramp ∼20% of its peak value. This is supported by the finding that, in the presence of 10 μmN-benzyl-p-toluene sulphonamide (a myosin inhibitor), while isometric tension is reduced to ∼15%, and the crossbridge contribution to stretch-induced tension rise is reduced to 30–40%, the peak non-crossbridge contribution and the residual force enhancement remain high. We propose that the residual force enhancement is due to changes upon activation in parallel elastic elements, specifically that titin stiffens and C-protein–actin interactions may be recruited.

We studied, by experiment and by kinetic modelling, the characteristics of the force increase on heating (endothermic force) in muscle. Experiments were done on maximally Ca2+-activated, permeabilized, single fibres (length ∼2 mm; sarcomere length, 2.5 μm) from rabbit psoas muscle; [MgATP] was 4.6 mm, pH 7.1 and ionic strength was 200 mm. A small-amplitude (∼3°C) rapid laser temperature-jump (0.2 ms T-jump) at 8–9°C induced a tension rise to a new steady state and it consisted of two (fast and slow) exponential components. The T-jump-induced tension rise became slower as [MgADP] was increased, with half-maximal effect at 0.5 mm[MgADP]; the pre- and post-T-jump tension increased ∼20% with 4 mm added [MgADP]. As determined by the tension change to small, rapid length steps (

We have examined the effects of N-benzyl-p-toluene sulphonamide (BTS), a potent and specific inhibitor of fast muscle myosin-II, using small bundles of intact fibres or single fibres from rat foot muscle. BTS decreased tetanic tension reversibly in a concentration-dependent manner with half-maximal inhibition at approximately approximately 2 microM at 20 degrees C. The inhibition of tension with 10 microM BTS was marked at the three temperatures examined (10, 20 and 30 degrees C), but greatest at 10 degrees C. BTS decreased active muscle stiffness to a lesser extent than tetanic tension indicating that not all of the tension inhibition was due to a reduced number of attached cross-bridges. BTS-induced inhibition of active tension was not accompanied by any change in the free myoplasmic Ca2+ transients. The potency and specificity of BTS make it a very suitable myosin inhibitor for intact mammalian fast muscle and should be a useful tool for the examination of outstanding questions in muscle contraction.

We examined the tension responses induced by rapid temperature-jumps (T-jumps) applied at different times during twitch and tetanic contractions in small intact fibre bundles (5-10 fibres) isolated from a fast foot muscle (flexor hallucis brevis) of the rat. A rapid T-jump of 2-4 degrees C was induced by a 0.2 ms infrared (lambda = 1.32 microm) laser pulse applied to the fibre bundle immersed in a 50 microl trough of physiological saline, the temperature of which was clamped at different steady temperatures ranging from 10 to 30 degrees C. In a tetanic contraction, the tension increased to the same steady level when a standard T-jump was applied at different intervals after the onset of stimulation; thus, with maximal activation, an enhanced force generation by T-jump leads to a new steady state. In a twitch contraction, a T-jump induced a large, potentiation of tension when it was applied during the rising phase. Whereas the twitch relaxation subsequent to a T-jump was faster in all cases, the amplitude of the twitch tension potentiation decreased as the T-jump was delayed with respect to the stimulus, and there was no increase of tension when a T-jump was placed on the relaxation phase of the twitch. The increase of tension induced by a T-jump applied on the rising phase resulted in peak tension that was greater than the tension in control twitches at the steady post-T-jump temperature; therefore tension was higher than that expected on the basis of steady state temperature dependence of twitch tension. Whether these effects on a twitch contraction arise from differential fibre-heating by a T-jump that leads to shortening and development of sarcomere length disorder etc remain unclear. However, the findings may be interpreted as indicating that twitch tension increment by a T-jump occurs when excitation (the action potential) leading to calcium release and thin filament activation occur at the low temperature, whereas the crossbridge force-generation processes (and Ca2+-uptake) proceed at the higher temperature.

In many muscle pathologies, impairment of skeletal muscle function is closely linked to changes in the mechanical properties of the muscle constituents. Optical coherence micro-elastography (OCME) uses optical coherence tomography (OCT) imaging of tissue under a quasi-static, compressive mechanical load to map variations in tissue mechanical properties on the micro-scale. We present the first study of OCME on skeletal muscle tissue. We show that this technique can resolve features of muscle tissue including fibers, fascicles and tendon, and can also detect necrotic lesions in skeletal muscle from the mdx mouse model of Duchenne muscular dystrophy. In many instances, OCME provides better or additional contrast complementary to that provided by OCT. These results suggest that OCME could provide new understanding and opportunity for assessment of skeletal muscle pathologies.

Protease-activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles from PAR-1 null and littermate control mice.Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry.Soleus muscles from PAR-1 null mice exhibited longer contraction times, a leftward shift in the force-stimulation frequency relationship, and decreased fatiguability compared with controls. PAR-1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR-1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls.The absence of PAR-1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991-998, 2014.

Elastography images tissue under a load to map variations in mechanical properties. We present our recent progress in three-dimensional compression optical coherence elastography and demonstrate mechanical contrast in skeletal muscle tissue.

PINNIGER, G. J., J. R. STEELE, and H. GROELLER. Does fatigue induced by repeated dynamic efforts affect hamstring muscle function? Med. Sci. Sports Exerc.,Vol. 32, No. 3, pp. 647‐ 653, 2000. Purpose: The purpose of this study was to determine the effects of hamstring fatigue induced by repeated maximal efforts on hamstring muscle function during maximal sprint running. Methods: Twelve subjects performed three maximal 40-m sprints during which time high-speed film of the subjects’ sprint action and EMG of five lower extremity muscles were recorded (nonfatigued condition, NFC). Subjects then performed specific and general hamstring fatigue tasks followed by three final 40-m sprints (fatigued condition, FC) during which time high-speed film and EMG of the same muscles were again recorded. Results: Statistical analysis of the kinematic data indicated the following significant ( P , 0.05) changes in the subjects’ running action from the NFC to the FC: decreased hip and knee flexion at maximum knee extension in the swing phase of the sprint cycle, decreased leg angular velocity immediately before foot-ground contact (FGC), and decreased angular displacement of the trunk, thigh, and leg segments during the late swing phase. Statistical analysis of the EMG data indicated a significant increase in the duration of hamstring activity and earlier cessation of rectus femoris activity during the swing phase of the sprint stride. Conclusions: It was concluded that these changes in the kinematic and EMG parameters of sprint running primarily served as protective

During normal daily activities, muscles are required to lengthen as frequently as they shorten to produce movement. Lengthening muscle actions are associated with high forces and low energy consumption, but can often result in muscle injury. These unique features are not explained adequately by current (cross-bridge) theories of muscle contraction. Using specific myosin inhibitors and different temperatures the author has examined the molecular mechanisms of stretch-induced force enhancement. The results, which suggest that lengthening force arises from the strain of both cross-bridge and non-cross-bridge components of the sarcomere, help to refine understanding of the molecular mechanisms of muscle contraction.

Diaphragmatic contractility is reduced in preterm lambs after lipopolysaccharide (LPS) exposure in utero. The mechanism of impaired fetal diaphragm contractility after LPS exposure is unknown. We hypothesise that in utero exposure to LPS induces a deficiency of mitochondrial complex activity and oxidative damage in the fetal diaphragm. To test this hypothesis, we used a well-established preterm ovine model of chorioamnionitis: Pregnant ewes received intra-amniotic (IA) saline or 10 mg LPS, at 2 d or 7 d prior to surgical delivery at 121 d GA (term = 150 d). The fetus was killed humanely immediately after delivery for tissue sampling. Mitochondrial fractions were prepared from the isolated diaphragm and mitochondrial electron transfer chain activities were evaluated using enzymatic assays. Oxidative stress was investigated by quantifying mitochondrial oxidative protein levels and determining antioxidant gene and protein (catalase, superoxide dismutase 2 and glutathione peroxidase 1) expression. The activity of the erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signalling pathway was examined by quantifying the Nrf2 protein content of cell lysate and nuclear extract. A 2 d LPS exposure in utero significantly decreased electron transfer chain complex II and IV activity (p

Preterm birth is associated with inflammation of the fetal membranes (chorioamnionitis). We aimed to establish how chorioamnionitis affects the contractile function and phenotype of the preterm diaphragm. Pregnant ewes received intra-amniotic injections of saline or 10 mg LPS, 2 days or 7 days before delivery at 121 days of gestation (term = 150 d). Diaphragm strips were dissected for the assessment of contractile function after terminal anesthesia. The inflammatory cytokine response, myosin heavy chain (MHC) fibers, proteolytic pathways, and intracellular molecular signaling were analyzed using quantitative PCR, ELISA, immunofluorescence staining, biochemical assays, and Western blotting. Diaphragm peak twitch force and maximal tetanic force were approximately 30% lower than control values in the 2-day and 7-day LPS groups. Activation of the NF-κB pathway, an inflammatory response, and increased proteasome activity were observed in the 2-day LPS group relative to the control or 7-day LPS group. No inflammatory response was evident after a 7-day LPS exposure. Seven-day LPS exposure markedly decreased p70S6K phosphorylation, but no effect on other signaling pathways was evident. The proportion of MHC IIa fibers was lower than that for control samples in the 7-day LPS group. MHC I fiber proportions did not differ between groups. These results demonstrate that intrauterine LPS impairs preterm diaphragmatic contractility after 2-day and 7-day exposures. Diaphragm dysfunction, resulting from 2-day LPS exposure, was associated with a transient activation of proinflammatory signaling, with subsequent increased atrophic gene expression and enhanced proteasome activity. Persistently impaired contractility for the 7-day LPS exposure was associated with the down-regulation of a key component of the protein synthetic signaling pathway and a reduction in the proportions of MHC IIa fibers.

Duchenne and Becker muscular dystrophy severity depends upon the nature and location of the DMD gene lesion and generally correlates with the dystrophin open reading frame. However, there are striking exceptions where an in-frame genomic deletion leads to severe pathology or protein-truncating mutations (nonsense or frame-shifting indels) manifest as mild disease. Exceptions to the dystrophin reading frame rule are usually resolved after molecular diagnosis on muscle RNA. We report a moderate/severe Becker muscular dystrophy patient with an in-frame genomic deletion of DMD exon 5. This mutation has been reported by others as resulting in Duchenne or Intermediate muscular dystrophy, and the loss of this in-frame exon in one patient led to multiple splicing events, including omission of exon 6, that disrupts the open reading frame and is consistent with a severe phenotype. The patient described has a deletion of dystrophin exon 5 that does not compromise recognition of exon 6, and although the deletion does not disrupt the reading frame, his clinical presentation is more severe than would be expected for classical Becker muscular dystrophy. We suggest that the dystrophin isoform lacking the actin-binding sequence encoded by exon 5 is compromised, reflected by the phenotype resulting from induction of this dystrophin isoform in mouse muscle in vivo. Hence, exon skipping to address DMD-causing mutations within DMD exon 5 may not yield an isoform that confers marked clinical benefit. Additional studies will be required to determine whether multi-exon skipping strategies could yield more functional dystrophin isoforms, since some BMD patients with larger in-frame deletions in this region have been reported with mild phenotypes.

Rationale The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue. Methods Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content. Results Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio. Conclusion These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure. Pediatr Pulmonol. 2013; 48:640–648. © 2013 Wiley Periodicals, Inc.

The skeletal muscle weakness associated with many chronic diseases has been attributed to the catabolic effect of pro-inflammatory cytokines. We aimed to determine if local muscle inflammation has direct affects on contractile function and contributes to muscle weakness independent of muscle atrophy or mechanical injury.Local muscle inflammation was induced by injecting an algal-derived polysaccharide, carrageenan (10 mg/kg), into the right tibialis anterior muscle in healthy ARC mice. The contralateral muscle was injected with sterile isotonic saline, and the muscles were removed after 24 h for measurement of contractile function and cytokine concentration.Carrageenan significantly reduced maximum specific force, decreased the maximum rate of force development, altered the force-frequency relationship, and increased intramuscular levels of pro-inflammatory cytokines and chemokines.These results indicate that carrageenan directly affects contractile function and causes skeletal muscle weakness. Local muscle inflammation may contribute to the weakness observed in inflammatory related disorders.

Summary The region over which skeletal muscles operate on their force-length (F-L) relationship is fundamental to the mechanics, control and economy of movement. Yet, surprisingly little experimental data exist on normalized length operating ranges of muscle during human gait, or how they are modulated when mechanical demands (such as force output) change. Here we explore the soleus muscle (SOL) operating lengths experimentally in a group of healthy young adults by combining subject-specific F-L relationships with in vivo muscle imaging during gait. We test whether modulation of operating lengths occurs between walking and running, two gaits that require different levels of force production and different muscle-tendon mechanics, and examine the relationship between optimal fascicle lengths (L0) and normalized operating lengths during these gaits. We found that the mean active muscle lengths reside predominantly on the ascending limbs of the F-L relationship in both gaits (0.70 - 0.94 L0, walk; 0.65 - 0.99 L0, run). Furthermore, the mean normalized muscle length at the time of the muscle's peak activation was the same between the two gaits (0.88 L0). The active operating lengths were conserved, despite a fundamentally different fascicle strain pattern between walking (stretch-shorten cycle) and running (near continuous shortening). Taken together, these findings indicate that the SOL operating length is highly conserved despite gait-dependant differences in muscle-tendon dynamics, and appear to be preferentially selected for stable force production compared to optimal force output (although length-dependent force capacity is high when maximal forces are expected to occur). Individuals with shorter L0 undergo smaller absolute muscle excursions (p

This study evaluated the contribution of the pro-inflammatory cytokine, tumour necrosis factor (TNF) to the severity of exercise-induced muscle damage and subsequent myofibre necrosis in mdx mice. Adult mdx and non-dystrophic C57 mice were treated with the mouse-specific TNF antibody cV1q before undergoing a damaging eccentric contraction protocol performed in vivo on a custom built mouse dynamometer. Muscle damage was quantified by (i) contractile dysfunction (initial torque deficit) immediately after the protocol, (ii) subsequent myofibre necrosis 48 h later. Blockade of TNF using cV1q significantly reduced contractile dysfunction in mdx and C57 mice compared with mice injected with the negative control antibody (cVaM) and un-treated mice. Furthermore, cV1q treatment significantly reduced myofibre necrosis in mdx mice. This in vivo evidence that cV1q reduces the TNF-mediated adverse response to exercise-induced muscle damage supports the use of targeted anti-TNF treatments to reduce the severity of the functional deficit and dystropathology in DMD.

Analysis of tension responses to ramp length changes in muscle can provide important information about the crossbridge cycle. During a ramp length change, the force response of an active muscle shows an early change in slope (the P1 transition) followed by a later, gradual change in slope (the P2 transition). Modeling shows that the first transition reflects the tension change associated with the crossbridge power stroke in shortening and with its reversal in lengthening; the reduction in slope at the second transition occurs when most of the crossbridges (myosin heads) that were attached at the start of the ramp become detached; the steady tension during shortening is borne mainly by post-stroke heads whereas tension during lengthening is borne mostly by pre-stroke heads. After the P2 transition, the tension reaches a steady level in the model whereas in the experiments the tension continues to increase during lengthening or to decrease during shortening; this tension change is seen at a wide range of sarcomere lengths and even when active force is reduced by a myosin inhibitor. It appears that some non-crossbridge components in muscle fibers stiffen upon activation and contribute to the continued tension rise during lengthening; release of such tension leads to tension decline during shortening. Thus, non-crossbridge visco-elasticity in sarcomeres may also contribute to energy storage and release during in situ muscle function.

Duchenne muscular dystrophy (DMD) is a genetic disorder in which muscle weakness and fragility contribute to ongoing muscle degeneration. Although exercise-induced muscle damage is associated with adaptation that protects normal muscle from further damage, exploiting this process to protect dystrophic muscle has been avoided for fear of inducing excessive muscle degeneration. However, muscle-specific over-expression of the class 1:Ea isoform of insulin-like growth factor-1 (IGF-1) reduces myofibre necrosis in dystrophic mdx mice (a model for DMD) and, therefore, may enhance the adaptation process in response to eccentric exercise. To test this hypothesis, we evaluated the effect of transgenic class 1:Ea IGF-1 over-expression on the susceptibility to muscle damage and subsequent adaptation in 12-week-old dystrophic mdx and non-dystrophic control mice. Experiments were conducted in vivo using a custom-built isokinetic mouse dynamometer to measure the deficit in joint torque (indicating muscle damage) after 20 maximal lengthening (eccentric) contractions. Adaptation to this damaging exercise was evaluated by repeating the protocol 7 days after the initial exercise. The over-expression of IGF-1 significantly increased the normalised joint torque in non-dystrophic mice and appeared to ameliorate the muscle weakness in dystrophic mice. All mice displayed a marked reduction in the susceptibility to muscle damage on day 7; however, this adaptation was unaffected by IGF-1, showing that IGF-1 does not protect the dystrophic muscles of adult mdx mice against damage resulting from maximal lengthening contractions.