Lengthening Contractions Research Articles

Reactive oxygen species generation is not different during isometric and lengthening contractions of mouse muscle

Skeletal muscles can be injured by lengthening contractions, when the muscles are stretched while activated. Lengthening contractions produce structural damage that leads to the degeneration and regeneration of damaged muscle fibers by mechanisms that have not been fully elucidated. Reactive oxygen species (ROS) generated at the time of injury may initiate degenerative or regenerative processes. In the present study we hypothesized that lengthening contractions that damage the muscle would generate more ROS than isometric contractions that do not cause damage. To test our hypothesis, we subjected muscles of mice to lengthening contractions or isometric contractions and simultaneously monitored intracellular ROS generation with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-DCFH), which is oxidized by ROS to form the fluorescent product CM-DCF. We found that CM-DCF fluorescence was not different during or shortly after lengthening contractions compared with isometric controls, regardless of the amount of stretch and damage that occurred during the lengthening contractions. The only exception was that after severe stretches, the increase in CM-DCF fluorescence was impaired. We conclude that lengthening contractions that damage the muscle do not generate more ROS than isometric contractions that do not cause damage. The implication is that ROS generated at the time of injury are not the initiating signals for subsequent degenerative or regenerative processes.

TRPV1 and TRPV4 Play Pivotal Roles in Delayed Onset Muscle Soreness

Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes tenderness and movement related pain after some delay (delayed-onset muscle soreness, DOMS). We previously demonstrated that nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) are up-regulated in exercised muscle through up-regulation of cyclooxygenase (COX)-2, and they sensitized nociceptors resulting in mechanical hyperalgesia. There is also a study showing that transient receptor potential (TRP) ion channels are involved in DOMS. Here we examined whether and how TRPV1 and/or TRPV4 are involved in DOMS. We firstly evaluated a method to measure the mechanical withdrawal threshold of the deep tissues in wild-type (WT) mice with a modified Randall-Selitto apparatus. WT, TRPV1−/− and TRPV4−/− mice were then subjected to LC. Another group of mice received injection of murine NGF-2.5S or GDNF to the lateral gastrocnemius (LGC) muscle. Before and after these treatments the mechanical withdrawal threshold of LGC was evaluated. The change in expression of NGF, GDNF and COX-2 mRNA in the muscle was examined using real-time RT-PCR. In WT mice, mechanical hyperalgesia was observed 6–24 h after LC and 1–24 h after NGF and GDNF injection. LC induced mechanical hyperalgesia neither in TRPV1−/− nor in TRPV4−/− mice. NGF injection induced mechanical hyperalgesia in WT and TRPV4−/− mice but not in TRPV1−/− mice. GDNF injection induced mechanical hyperalgesia in WT but neither in TRPV1−/− nor in TRPV4−/− mice. Expression of NGF and COX-2 mRNA was significantly increased 3 h after LC in all genotypes. However, GDNF mRNA did not increase in TRPV4−/− mice. These results suggest that TRPV1 contributes to DOMS downstream (possibly at nociceptors) of NGF and GDNF, while TRPV4 is located downstream of GDNF and possibly also in the process of GDNF up-regulation.

Targeted inhibition of TGF-β results in an initial improvement but long-term deficit in force production after contraction-induced skeletal muscle injury

Transforming growth factor-β (TGF-β) is a proinflammatory cytokine that regulates the response of many tissues following injury. Previous studies in our lab have shown that treating muscles with TGF-β results in a dramatic accumulation of type I collagen, substantial fiber atrophy, and a marked decrease in force production. Because TGF-β promotes atrophy and fibrosis, our objective was to investigate whether the inhibition of TGF-β after injury would enhance the recovery of muscle following injury. We hypothesized that inhibiting TGF-β after contraction-induced injury would improve the functional recovery of muscles by preventing muscle fiber atrophy and weakness, and by limiting the accumulation of fibrotic scar tissue. To test this hypothesis, we induced an injury using a series of in situ lengthening contractions to extensor digitorum longus muscles of mice treated with either a bioneutralizing antibody against TGF-β or a sham antibody. Compared with controls, muscles from mice receiving TGF-β inhibitor showed a greater recovery in force 3 days and 7 days after injury but had a decrease in force compared with controls at the 21-day time point. The early enhancement in force in the TGF-β inhibitor group was associated with an initial improvement in tissue morphology, but, at 21 days, while the control group was fully recovered, the TGF-β inhibitor group displayed an irregular extracellular matrix and an increase in atrogin-1 gene expression. These results indicate that the inhibition of TGF-β promotes the early recovery of muscle function but is detrimental overall to full muscle recovery following moderate to severe muscle injuries.

Enhanced force production in old age is not a far stretch: an investigation of residual force enhancement and muscle architecture

In older adults, isometric force production is enhanced following a voluntary lengthening contraction when compared with isometric force produced at the same muscle length without a prior lengthening contraction. This phenomenon is termed residual force enhancement (RFE), and appears to be related to the age-related maintenance of eccentric (ECC) strength. However, it is unknown whether age-related changes in muscle architecture contribute to greater RFE at short and long muscle lengths in old age. Neuromuscular properties of the knee extensors were assessed on a HUMAC NORM dynamometer. Torque was examined in young (26 ± 3 year, n = 11) and old men (77 ± 6 year, n = 11) during brief maximal voluntary isometric contractions (MVC) at 80° and 120° (180° representing full knee extension) and then compared with torque during a steady-state phase at the same joint angle following a maximal voluntary lengthening contraction at 30°/sec over a 60° joint excursion; either from 140 to 80° (long), or from 180 to 120° (short). Ultrasound images were obtained from the vastus lateralis during the isometric phase for each condition. When comparing the ECC torque with the MVC isometric torque, old men had 17% greater ECC:MVC ratios than young men, confirming an age-related maintenance of ECC strength. The extent of RFE was greater at long versus short but independent of age. At rest, old had shorter (∼18%) and less pennated (∼22%) fascicles. However, changes in fascicle length and pennation during contraction did not contribute to RFE in either group. Thus, age-related changes in muscle architecture may not contribute to RFE.

Peak power is reduced following lengthening contractions despite a maintenance of shortening velocity

Following repetitive lengthening contractions, power (the product of torque and velocity) is impaired during shortening contractions. However, the relative contribution of each component to power loss and the underlying factors are unclear. We investigated neuromuscular properties of the dorsiflexors in 8 males (27 ± 3 years) and 8 females (26 ± 4 years) for a potential sex-related difference before, during, and after 150 unaccustomed maximal lengthening actions. Velocity-dependent power was determined from shortening contractions at 8 levels (1 N · m to 70% of maximum voluntary isometric contraction (MVC)) before, after, and throughout recovery assessed at 0-30 min, 24 h, and 48 h. Immediately following task termination, both sexes displayed similar impairments of 30%, 4%, and 10% in MVC torque, shortening velocity, and overall peak power, respectively (P < 0.05). Peak rate of isometric torque development (RTD) was reduced by 10% in males, but females exhibited a 35% reduction (P < 0.05). Rate of torque development for the MVC remained depressed in both sexes throughout the 30 min recovery period; however, the RTD returned to normal by 24 h in males but did not recover by 48 h in females. Power was reduced preferentially at higher loads (i.e., 60% MVC), with a greater loss in females (65%) than males (45%). For lower loads (<20% MVC), power was impaired minimally (4%-8%; P < 0.05) and recovered within 30 min in both groups. The reduction in maximal angular velocity persisted until 30 min of recovery, and peak power did not recover until 24 h for both sexes. Unaccustomed lengthening contractions decreased power preferentially at higher loads, whereas peak power was reduced minimally owing to maintenance of maximal shortening velocity.

Upregulated glial cell line‐derived neurotrophic factor through cyclooxygenase‐2 activation in the muscle is required for mechanical hyperalgesia after exercise in rats

Unaccustomed strenuous exercise that includes lengthening contraction (LC) often causes delayed onset muscle soreness (DOMS), characterised as muscular mechanical hyperalgesia. Previously we reported that a bradykinin-like substance released from the muscle during exercise plays a pivotal role in triggering the process of muscular mechanical hyperalgesia by upregulating nerve growth factor (NGF) in exercised muscle of rats. We show here that cyclooxygenase (COX)-2 and glial cell line-derived neurotrophic factor (GDNF) are also involved in DOMS. COX-2 inhibitors but not COX-1 inhibitors given orally before LC completely suppressed the development of DOMS, but when given 2 days after LC they failed to reverse the mechanical hyperalgesia. COX-2 mRNA and protein in exercised muscle increased six- to 13-fold in mRNA and 1.7-2-fold in protein 0-12 h after LC. COX-2 inhibitors did not suppress NGF upregulation after LC. Instead, we found GDNF mRNA was upregulated seven- to eight-fold in the exercised muscle 12 h-1 day after LC and blocked by pretreatment of COX-2 inhibitors. In situ hybridisation studies revealed that both COX-2 and GDNF mRNA signals increased at the periphery of skeletal muscle cells 12 h after LC. The accumulation of COX-2 mRNA signals was also observed in small blood vessels. Intramuscular injection of anti-GDNF antibody 2 days after LC partly reversed DOMS. Based on these findings, we conclude that GDNF upregulation through COX-2 activation is essential to mechanical hyperalgesia after exercise.

G-21 Free Communication/Poster - Lengthening Muscle Contractions

G-21 Free Communication/Poster - Lengthening Muscle Contractions

Effect of a work‐matched bout of lengthening or shortening contractions on satellite cell proliferation in human muscle

PurposeTo determine the extent of satellite cell (SC) activity following a work‐matched bout of lengthening (LC) or shortening (SHC) contractions.MethodsParticipants (aged 22.6 ± 1.5 years) were assigned to either LC (n=6) or SHC (n=6). A control muscle biopsy was taken from the non‐exercised leg prior to exercise. Subjects performed a bout of unilateral LC or SHC of the knee extensors. A biopsy was obtained from the exercised leg 24 h after exercise. Muscle soreness and strength were assessed pre‐, immediately post‐exercise, and every 24 h for 4 d. Immunohistochemistry was used to quantify total and fiber‐type specific SC content from exercised and control legs in LC and SHC. Repeated‐measures ANOVA (p&lt;0.05) was used to analyze data.ResultsParticipants performed 40.8 ± 2.9 KJ of work, with no differences between groups (p=0.65). LC caused greater soreness than SHC (p&lt;0.01). LC resulted in prolonged strength loss that recovered 4 days post‐exercise (p&lt;0.01). SHC caused immediate but not prolonged strength loss. SC per muscle fiber increased following LC (0.106 ± 0.031 and 0.126 ± 0.048 pre‐ and post‐exercise, respectively; p=0.028), while SHC caused no change in SC number (0.095 ± 0.025 and 0.101 ± 0.032 pre‐ and post‐exercise, respectively). There was no fiber type‐specific SC response.ConclusionWe confirm SC activation following LC and show that a single bout of non‐damaging SHC does not stimulate SC activity.

Eccentric exercise in aging and diseased skeletal muscle: good or bad?

Evidence is accumulating regarding the benefits of exercise in people who are more susceptible to injury, such as the elderly, or those with a neuromuscular disease, for example Duchenne muscular dystrophy (DMD). There appears to be a consensus that exercise can be safely performed in aging and diseased muscles, but the role of eccentric exercise is not as clear. Eccentric (lengthening) contractions have risks and benefits. Eccentric contractions are commonly performed on a daily basis, and high-force voluntary eccentric contractions are often employed in strength training paradigms with excellent results; however, high-force eccentric contractions are also linked to muscle damage. This minireview examines the benefits and safety issues of using eccentric exercise in at-risk populations. A common recommendation for all individuals is difficult to achieve, and guidelines are still being established. Some form of exercise is generally recommended with aging and even with diseased muscles, but the prescription (frequency, intensity, and duration) and type (resistance vs. aerobic) of exercise requires personal attention, as there is great diversity in the functional level and comorbidities in the elderly and those with neuromuscular disease.

Mechanisms of enhanced force production in lengthening (eccentric) muscle contractions

In contrast to isometric and shortening contractions, many observations made on actively lengthening muscles cannot be readily explained with the sliding filament and cross-bridge theory. Specifically, residual force enhancement, the persistent increase in force following active muscle lengthening, beyond what one would expect based on muscle length, has not been explained satisfactorily. Here, we summarize the experimental evidence on residual force enhancement, critically evaluate proposed mechanisms for the residual force enhancement, and propose a mechanism for residual force enhancement that explains all currently agreed upon experimental observations. The proposed mechanism is based on the engagement of the structural protein titin upon muscle activation and an increase in titin's resistance to active compared with passive stretching. This change in resistance from the passive to the active state is suggested to be based on 1) calcium binding by titin upon activation, 2) binding of titin to actin upon activation, and 3) as a consequence of titin-actin binding--a shift toward stiffer titin segments that are used in active compared with passive muscle elongation. Although there is some experimental evidence for the proposed mechanism, it must be stressed that much of the details proposed here remain unclear and should provide ample research opportunities for scientists in the future. Nevertheless, the proposed mechanism for residual force enhancement explains all basic findings in this area of research.

Acute blood flow restricted exercise to treat Duchenne muscular dystrophy: would it be efficacious?

The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD), which is an X-linked disorder affecting 1 in 3500 newborn males across the world. DMD is caused by the lack of dystrophin which is a protein that provides stability to the sarcolemma and is likely involved in the transmission of force between the extracellular matrix and the intracellcular contractile apparatus (Lovering and Brooks, 2013). Due to the absence of this protein, the sarcolemma is easily damaged in response to muscle contraction (Markert et al., 2012). Although there is no cure for DMD, exercise has been proposed as a possible treatment; however, the risk of muscle damage is a large concern for this population (Markert et al., 2012). Therefore, traditional strength training which includes high load eccentric contractions may need to be avoided in patients with DMD due to the susceptibility of muscle damage with lengthening contractions. In addition, although submaximal exercise may exert some benefits for this population, the low load exercise is unlikely to be an optimal stimulus for maintaining or increasing muscle function. Interestingly, there are numerous studies (>40) in healthy subjects which suggest that submaximal exercise in combination with blood flow restriction (BFR) can elicit muscle adaptations similar to that observed with higher load resistance training (Loenneke et al., 2012c) without increasing indices of muscle damage (Loenneke et al., 2011). Briefly, BFR is a stimulus commonly applied with specialized pressure cuffs placed at the top of a limb which are inflated to a set pressure throughout exercise. The pressure applied should be high enough to occlude venous return from the muscle but low enough to maintain arterial inflow into the muscle. Available evidence indicates that the pressure applied should be based on the size of the limb (i.e., bigger the limb higher the pressure) (Loenneke et al., 2012b). The proposed mechanisms (Loenneke et al., 2012a) behind the effects of low load exercise in combination with BFR on skeletal muscle include acute muscle cell swelling, increased fiber type recruitment from metabolic accumulation, decreased myostatin (48 h after last training session), decreased atrogenes (8 h post exercise), and the proliferation of satellite cells (8 days into training intervention, 3 and 10 days after cessation of training) (Nielsen et al., 2012). A recent review discussed 5 mechanisms of DMD pathology which may improve or worsen as a result of exercise training which included: (1) mechanical weakening of the sarcolemma; (2) inappropriate calcium influx; (3) aberrant cell signaling (angiogenesis); (4) increased oxidative stress; and (5) recurrent muscle ischemia (Markert et al., 2012). The purpose of this manuscript is to discuss each one of these mechanisms as it relates to what is known about low load resistance exercise in combination with BFR.

Cortical and Spinal Excitability during and after Lengthening Contractions of the Human Plantar Flexor Muscles Performed with Maximal Voluntary Effort

This study was designed to investigate the sites of potential specific modulations in the neural control of lengthening and subsequent isometric maximal voluntary contractions (MVCs) versus purely isometric MVCs of the plantar flexor muscles, when there is enhanced torque during and following stretch. Ankle joint torque during maximum voluntary plantar flexion was measured by a dynamometer when subjects (n = 10) lay prone on a bench with the right ankle tightly strapped to a foot-plate. Neural control was analysed by comparing soleus motor responses to electrical nerve stimulation (M-wave, V-wave), electrical stimulation of the cervicomedullary junction (CMEP) and transcranial magnetic stimulation of the motor cortex (MEP). Enhanced torque of 17±8% and 9±8% was found during and 2.5–3 s after lengthening MVCs, respectively. Cortical and spinal responsiveness was similar to that in isometric conditions during the lengthening MVCs, as shown by unchanged MEPs, CMEPs and V-waves, suggesting that the major voluntary motor pathways are not subject to substantial inhibition. Following the lengthening MVCs, enhanced torque was accompanied by larger MEPs (p≤0.05) and a trend to greater V-waves (p≤0.1). In combination with stable CMEPs, increased MEPs suggest an increase in cortical excitability, and enlarged V-waves indicate greater motoneuronal output or increased stretch reflex excitability. The new results illustrate that neuromotor pathways are altered after lengthening MVCs suggesting that the underlying mechanisms of the enhanced torque are not purely mechanical in nature.

Decreased nerve growth factor upregulation is a mechanism for reduced mechanical hyperalgesia after the second bout of exercise in rats

Delayed onset muscle soreness (DOMS) is reduced when the same exercise is repeated after a certain interval. However, the mechanism for this adaptation, called a repeated bout effect, is still not well understood. Recently, we showed that upregulated nerve growth factor (NGF) triggered by B2 bradykinin receptor (B2R) activation in exercised muscle was responsible for DOMS. In this study, we investigated whether NGF upregulation was reduced after repeated bouts of exercise in rats, and if so, whether this change occurred upstream of B2R. A bout of 500 lengthening contractions (LC) was applied on day 0 and again 5 days later. DOMS was evaluated by the mechanical withdrawal threshold of the exercised extensor digitorum longus (EDL) muscle. Mechanical hyperalgesia and NGF mRNA upregulation in EDL were observed after the first LC, but not after the second LC. We then injected HOE140, a B2R antagonist with effects lasting only several hours, once before the first LC. This blocked the development of mechanical hyperalgesia and NGF mRNA upregulation not only after the first LC but also after the second LC. This suggests that adaptation occurred upstream of B2R, as the influence of the first LC was limited to that area by HOE140.

Characteristics of myogenic response and ankle torque recovery after lengthening contraction-induced rat gastrocnemius injury

BackgroundAlthough muscle dysfunction caused by unfamiliar lengthening contraction is one of most important issues in sports medicine, there is little known about the molecular events on regeneration process. The purpose of this study was to investigate the temporal and spatial expression patterns of myogenin, myoD, pax7, and myostatin after acute lengthening contraction (LC)-induced injury in the rat hindlimb.MethodsWe employed our originally developed device with LC in rat gastrocnemius muscle (n = 24). Male Wistar rats were anesthetized with isoflurane (aspiration rate, 450 ml/min, concentration, 2.0%). The triceps surae muscle of the right hindlimb was then electrically stimulated with forced isokinetic dorsi-flexion (180°/sec and from 0 to 45°). Tissue contents of myoD, myogenin, pax7, myostatin were measured by western blotting and localizations of myoD and pax7 was measured by immunohistochemistry. After measuring isometric tetanic torque, a single bout of LC was performed in vivo.ResultsThe torque was significantly decreased on days 2 and 5 as compared to the pre-treatment value, and recovered by day 7. The content of myoD and pax7 showed significant increases on day 2. Myogenin showed an increase from day 2 to 5. Myostatin on days 5 and 7 were significantly increased. Immunohistochemical analysis showed that myoD-positive/pax7-positive cells increased on day 2, suggesting that activated satellite cells play a role in the destruction and the early recovery phases.ConclusionWe, thus, conclude that myogenic events associate with torque recovery after LC-induced injury.

Recovery time of motor evoked potentials following lengthening and shortening muscle action in the tibialis anterior

Motor evoked potentials (MEP) at rest remain facilitated following an isometric muscle contraction. Because the pre-synaptic and post-synaptic control of shortening (SHO) and lengthening (LEN) contractions differs, the possibility exists that the recovery of the MEP is also task specific. The time course of MEP recovery was assessed in the tibialis anterior following SHO and LEN (0.26rad/s) at 25% and 80% of maximal voluntary contraction. Following LEN and SHO contractions, the MEP recovered to baseline levels within 10s. Despite task-specific differences between SHO and LEN contractions, the MEP facilitation from the augmented neurotransmitter release appears to be short lasting and not influenced by contraction type.

Repeatability of Corticospinal and Spinal Measures during Lengthening and Shortening Contractions in the Human Tibialis Anterior Muscle

Elements of the human central nervous system (CNS) constantly oscillate. In addition, there are also methodological factors and changes in muscle mechanics during dynamic muscle contractions that threaten the stability and consistency of transcranial magnetic stimulation (TMS) and perpherial nerve stimulation (PNS) measures.PurposeTo determine the repeatability of TMS and PNS measures during lengthening and shortening muscle actions in the intact human tibialis anterior.MethodsOn three consecutive days, 20 males performed lengthening and shortening muscle actions at 15, 25, 50 and 80% of maximal voluntary contraction (MVC). The amplitude of the Motor Evoked Potentials (MEPs) produced by TMS was measured at rest and during muscle contraction at 90° of ankle joint position. MEPs were normalised to Mmax determined with PNS. The corticospinal silent period was recorded at 80% MVC. Hoffman reflex (H-reflex) at 10% isometric and 25% shortening and lengthening MVCs, and V-waves during MVCs were also evoked on each of the three days.ResultsWith the exception of MEPs evoked at 80% shortening MVC, all TMS-derived measures showed good reliability (ICC = 0.81–0.94) from days 2 to 3. Confidence intervals (CI, 95%) were lower between days 2 and 3 when compared to days 1 and 2. MEPs significantly increased at rest from days 1 to 2 (P = 0.016) and days 1 to 3 (P = 0.046). The H-reflex during dynamic muscle contraction was reliable across the three days (ICC = 0.76–0.84). V-waves (shortening, ICC = 0.77, lengthening ICC = 0.54) and the H-reflex at 10% isometric MVC (ICC = 0.66) was generally less reliable over the three days.ConclusionAlthough it is well known that measures of the intact human CNS exhibit moment-to-moment fluctuations, careful experimental arrangements make it possible to obtain consistent and repeatable measurements of corticospinal and spinal excitability in the actively lengthening and shortening human TA muscle.

Residual force enhancement following eccentric induced muscle damage

During lengthening of an activated skeletal muscle, the force maintained following the stretch is greater than the isometric force at the same muscle length. This is termed residual force enhancement (RFE), but it is unknown how muscle damage following repeated eccentric contractions affects RFE. Using the dorsiflexors, we hypothesised muscle damage will impair the force generating sarcomeric structures leading to a reduction in RFE. Following reference maximal voluntary isometric contractions (MVC) in 8 young men (26.5±2.8y) a stretch was performed at 30°/s over a 30° ankle excursion ending at the same muscle length as the reference MVCs (30° plantar flexion). Surface electromyography (EMG) of the tibialis anterior and soleus muscles was recorded during all tasks. The damage protocol involved 4 sets of 25 isokinetic (30°/s) lengthening contractions. The same measures were collected at baseline and immediately post lengthening contractions, and for up to 10min recovery. Following the lengthening contraction task, there was a 30.3±6.4% decrease in eccentric torque (P<0.05) and 36.2±9.7% decrease in MVC (P<0.05) compared to baseline. Voluntary activation using twitch interpolation and RMS EMG amplitude of the tibialis anterior remained near maximal without increased coactivation for MVC. Contrary to our hypothesis, RFE increased (∼100–250%) following muscle damage (P<0.05). It appears stretch provided a mechanical strategy for enhanced muscle function compared to isometric actions succeeding damage. Thus, active force of cross-bridges is decreased because of impaired excitation–contraction coupling but force generated during stretch remains intact because force contribution from stretched sarcomeric structures is less impaired.

In vivo studies of motor nerve re‐growth following skeletal muscle damage by lengthening contractions

Very little is known about the pattern of changes in motor neurons that occur following skeletal muscle injury and during muscle regeneration during ageing in rodents or man. Our hypothesis is that physiologically relevant episodes of damage to muscle lead to a transient loss of innervation of the damaged muscle fibres with subsequent re‐growth of the terminal axons, interaction with the regenerating muscle fibres and formation of new neuromuscular junctions. Studies were undertaken in yellow fluorescent protein (YFP)‐H mice that only express YFP in neuronal cells. These mice provide a novel approach to assess the extent of motor neuron degeneration and regeneration. One extensor digitorum longus (EDL) muscle of adult YFP‐H mice was subjected to a protocol of 450 lengthening contractions involving a stretch to 120% of fibre length. The EDL muscle from the contralateral leg was used as control. This protocol produces an equivalent level of damage to 60–70% of the muscle fibres in muscles from adult and old mice. Maximal force generation by the EDL muscle in vivo was measured to examine the extent of functional denervation at 3, 14 and 28 days following damage. Mice were then killed, the EDL muscle was removed and the extent of loss and regrowth of peripheral motor neurons was examined by visualisation of labelled axons by fluorescence microscopy in the EDL. Supported by Research into Ageing, UK.

The generation of reactive oxygen and nitrogen species (RONS) is not different during injurious lengthening contractions and non‐damaging isometric contractions

Neither isometric contractions (IC) nor stretches without activation (passive stretches, PS) cause injury to skeletal muscle. In contrast, when muscles are activated and stretched (lengthening contractions, LC), structural damage occurs that leads to the degeneration of damaged muscle fibers by unknown mechanisms. RONS may contribute to fiber degeneration by damaging proteins and lipids or by initiating signaling pathways, but RONS generation during LC has not been examined. We hypothesized that RONS generation would be greater during LC than IC. Whole lumbrical muscles dissected from mice were subjected to a series of IC or PS that did not injure the muscle or damaging LC that caused a 28 ± 9% deficit in isometric force. RONS were monitored during the protocols with CM‐H2DCF‐DA. CM‐DCF fluorescence increased 6‐fold during IC. CM‐DCF fluorescence also increased during LC, but the increase was not different from that during IC, despite greater force generation during LC (29 ± 2 mN vs 19 ± 2 mN, P &lt; 0.001) and the resultant force deficit. CM‐DCF fluorescence did not increase with PS. We conclude that RONS generation by muscle is associated primarily with activation rather than force and does not change as muscle is damaged by LC. Since RONS generation was not different during contractions whether or not the muscle was damaged, RONS may not be required for the initiation of injury following LC. This work is supported by AG020591.

Contractile properties of mice deficient in dystrophin and the NADPH subunit p47phox

Duchenne muscular dystrophy is associated with a mutation in the gene encoding dystrophin. Dystrophin‐deficient muscle is highly susceptible to contraction‐induced injury. A report by N.P. Whitehead et al (2010) regarding mdx mice, described the efficacy of pharmacological inhibition of NADPH oxidase at decreasing contraction‐induced force deficits. This indicated that during lengthening contractions, NADPH oxidase may be a source of reactive oxygen species which contributes to the opening of stretch activated channels and the deleterious influx of extracellular calcium. To evaluate this proposal further and the role of p47phox – a subunit of NADPH oxidase – in particular, we tested the hypothesis that a stretch activated channel inhibitor (streptomycin), antioxidant (N‐acetylcysteine), calcium‐free buffer, and p47phox deficiency would decrease contraction‐induced injury for muscles of mdx mice. Mdx mice were crossed with p47phox null mice and isolated extensor digitorum longus muscles were exposed to the various conditions. Specific force was unaffected in all cases. Force deficits were reduced with the addition of streptomycin, N‐aceytcysteine, or calcium‐free buffer confirming earlier reports. A deficiency in p47phox diminished the increased muscle mass typical of mdx mice. However, lacking p47phox had no effect on force deficit. The results are consistent with reactive oxygen species and the influx of extracellular calcium via stretch activated channels mediating contraction‐induced injury in dystrophin deficient muscle. However, the findings indicate that the absence of p47phox does not reduce muscle dysfunction associated with dystrophin deficiency.