Gavin J. Pinniger

Crossbridge and non-crossbridge contributions to tension in lengthening rat muscle: force-induced reversal of the power stroke

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.

On the ascent: the soleus operating length is conserved to the ascending limb of the force–length curve across gait mechanics in humans

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 explored 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 tested whether modulation of operating lengths occurred between walking and running, two gaits that require different levels of force production and different muscle–tendon mechanics, and examined 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 (walk, 0.70–0.94 L0; run, 0.65–0.99 L0). Furthermore, the mean normalized muscle length at the time of the peak activation of the muscle 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-dependent differences in muscle–tendon dynamics, and appear to be preferentially selected for stable force production compared with 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<0.05) so that the normalized length changes during walking and running remain independent of L0. The correlation between L0 and absolute length change was not explained on the basis of muscle moment arms or joint excursion, suggesting that regulation of muscle strain may occur via tendon stretch.

Blockade of TNF in vivo using cV1q antibody reduces contractile dysfunction of skeletal muscle in response to eccentric exercise in dystrophic mdx and normal mice

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.

Effects of simulated viewpoint jitter on visually induced postural sway

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.

Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy

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.

Endothermic force generation, temperature-jump experiments and effects of increased [MgADP] in rabbit psoas muscle fibres

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 (<1.4%L0 complete in <0.5 ms), the increase of force by [MgADP] was not associated with a concomitant increase of stiffness; the quick tension recovery after length steps (Huxley–Simmons phase 2) was slower with added MgADP. In steady‐state experiments, the tension was larger at higher temperatures and the plot of tension versus reciprocal absolute temperature was sigmoidal, with a half‐maximal tension at 10–12°C; the relation with added 4 mm MgADP was shifted upwards on the tension axis and towards lower temperatures. The potentiation of tension with 4 mm added MgADP was 20–25% at low temperatures (∼5–10°C), but ∼10% at the physiological temperatures (∼30°C). The shortening velocity was decreased with increased [MgADP] at low and high temperatures. The sigmoidal relation between tension and reciprocal temperature, and the basic effects of increased [MgADP] on endothermic force, can be qualitatively simulated using a five‐step kinetic scheme for the crossbridge/A‐MATPase cycle where the force generating conformational change occurs in a reversible step before the release of inorganic phosphate (Pi), it is temperature sensitive (Q10 of ∼4) and the release of MgADP occurs by a subsequent, slower, two‐step mechanism. Modelling shows that the sigmoidal relation between force and reciprocal temperature arises from conversion of preforce‐generating (A‐M.ADP.Pi) states to force‐bearing (A‐M.ADP) states as the temperature is raised. A tension response to a simulated T‐jump consists of three (one fast and two slow) components, but, by combining the two slow components, they could be reduced to two; their relative amplitudes vary with temperature. The model can qualitatively simulate features of the tension responses induced by large‐T‐jumps from low starting temperatures, and those induced by small‐T‐jumps from different starting temperatures and, also, the interactive effects of Pi and temperature on force in muscle fibres.

Three-dimensional optical coherence micro-elastography of skeletal muscle tissue

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.

Lipopolysaccharide-induced weakness in the preterm diaphragm is associated with mitochondrial electron transport chain dysfunction and oxidative stress.

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<0.05). A 7 d LPS exposure inhibited superoxide dismutase 2 and catalase expression at gene and protein levels, and Nrf2 pathway activity (p<0.05) compared with control and 2 d LPS groups, respectively. Diaphragm mitochondria accumulated oxidised protein after a 7 d LPS exposure. We conclude that intrauterine exposure to LPS induces mitochondrial oxidative stress and electron chain dysfunction in the fetal diaphragm, that is further exacerbated by impairment of the antioxidant signalling pathway and decreased antioxidant activity.

Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

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) ∼2 mm, sarcomere length 2.5 μm) from rabbit psoas muscle; [MgATP] was 4.6 mm, pH 7.1, ionic strength 200 mm and temperature ∼9°C. A fibre was maximally Ca2+‐activated in the isometric state and a ∼3°C, rapid (< 0.2 ms), laser T‐jump applied when the tension was approximately steady in the isometric state, or during ramp shortening or ramp lengthening at a limited range of velocities (0–0.2 L0 s−1). The tension increased to 2‐ to 3 ×P0 (isometric force) during ramp lengthening at velocities > 0.05 L0 s−1, whereas the tension decreased to about < 0.5 ×P0 during shortening at 0.1–0.2 L0 s−1; the unloaded shortening velocity was ∼1 L0 s−1 and the curvature of the force–shortening velocity relation was high (a/P0 ratio from Hills equation of ∼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 ∼1.2%L0 and it was shorter at lower velocities. At a given shortening velocity and over the temperature range of 8–30°C, the rate of T‐jump tension rise increased with warming (Q10≈ 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.

The effects of the myosin-II inhibitor N-benzyl-p-toluene sulphonamide on fatigue in mouse single intact toe muscle fibres

Aim:  This study determined whether fatigue in skeletal muscle is primarily due to the repeated elevations of myoplasmic free calcium concentration ([Ca2+]i) or to metabolite accumulation.