Eccentric Contraction-induced Damage Research Articles

Phytoecdysteroids Accelerate Recovery of Skeletal Muscle Function Following in vivo Eccentric Contraction-Induced Injury in Adult and Old Mice.

Background: Eccentric muscle contractions are commonly used in exercise regimens, as well as in rehabilitation as a treatment against muscle atrophy and weakness. If repeated multiple times, eccentric contractions may result in skeletal muscle injury and loss of function. Skeletal muscle possesses the remarkable ability to repair and regenerate after an injury or damage; however, this ability is impaired with aging. Phytoecdysteroids are natural plant steroids that possess medicinal, pharmacological, and biological properties, with no adverse side effects in mammals. Previous research has demonstrated that administration of phytoecdysteroids, such as 20-hydroxyecdysone (20E), leads to an increase in protein synthesis signaling and skeletal muscle strength.Methods: To investigate whether 20E enhances skeletal muscle recovery from eccentric contraction-induced damage, adult (7–8 mo) and old (26–27 mo) mice were subjected to injurious eccentric contractions (EC), followed by 20E or placebo (PLA) supplementation for 7 days. Contractile function via torque-frequency relationships (TF) was measured three times in each mouse: pre- and post-EC, as well as after the 7-day recovery period. Mice were anesthetized with isoflurane and then electrically-stimulated isometric contractions were performed to obtain in vivo muscle function of the anterior crural muscle group before injury (pre), followed by 150 EC, and then again post-injury (post). Following recovery from anesthesia, mice received either 20E (50 mg•kg−1 BW) or PLA by oral gavage. Mice were gavaged daily for 6 days and on day 7, the TF relationship was reassessed (7-day).Results: EC resulted in significant reductions of muscle function post-injury, regardless of age or treatment condition (p < 0.001). 20E supplementation completely recovered muscle function after 7 days in both adult and old mice (pre vs. 7-day; p > 0.05), while PLA muscle function remained reduced (pre vs. 7-day; p < 0.01). In addition, histological markers of muscle damage appear lower in damaged muscle from 20E-treated mice after the 7-day recovery period, compared to PLA.Conclusions: Taken together, these findings demonstrate that 20E fully recovers skeletal muscle function in both adult and old mice just 7 days after eccentric contraction-induced damage. However, the underlying mechanics by which 20E contributes to the accelerated recovery from muscle damage warrant further investigation.

High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.

Duchenne muscular dystrophy (DMD) is a genetic disorder that causes progressive muscle weakness, ultimately leading to early mortality in affected teenagers and young adults. Previous work from our lab has shown that a small transmembrane protein called sarcospan (SSPN) can enhance the recruitment of adhesion complex proteins to the cell surface. When human SSPN is expressed at three-fold levels in mdx mice, this increase in adhesion complex abundance improves muscle membrane stability, preventing many of the histopathological changes associated with DMD. However, expressing higher levels of human SSPN (ten-fold transgenic expression) causes a severe degenerative muscle phenotype in wild-type mice. Since SSPN-mediated stabilization of the sarcolemma represents a promising therapeutic strategy in DMD, it is important to determine whether SSPN can be introduced at high levels without toxicity. Here, we show that mouse SSPN (mSSPN) can be overexpressed at 30-fold levels in wild-type mice with no deleterious effects. In mdx mice, mSSPN overexpression improves dystrophic pathology and sarcolemmal stability. We show that these mice exhibit increased resistance to eccentric contraction-induced damage and reduced fatigue following exercise. mSSPN overexpression improved pulmonary function and reduced dystrophic histopathology in the diaphragm. Together, these results demonstrate that SSPN overexpression is well tolerated in mdx mice and improves sarcolemma defects that underlie skeletal muscle and pulmonary dysfunction in DMD.

How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse.

Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.

Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice.

Approaches targeting nitric oxide (NO) signaling show promise as therapies for Duchenne and Becker muscular dystrophies. However, the mechanisms by which NO benefits dystrophin-deficient muscle remain unclear, but may involve nNOSβ, a newly discovered enzymatic source of NO in skeletal muscle. Here we investigate the impact of dystrophin deficiency on nNOSβ and use mdx mice engineered to lack nNOSμ and nNOSβ to discern how the loss of nNOS impacts dystrophic skeletal muscle pathology. In mdx muscle, nNOSβ was mislocalized and its association with the Golgi complex was reduced. nNOS depletion from mdx mice prevented compensatory skeletal muscle cell hypertrophy, decreased myofiber central nucleation and increased focal macrophage cell infiltration, indicating exacerbated dystrophic muscle damage. Reductions in muscle integrity in nNOS-null mdx mice were accompanied by decreases in specific force and increased susceptibility to eccentric contraction-induced muscle damage compared with mdx controls. Unexpectedly, muscle fatigue was unaffected by nNOS depletion, revealing a novel latent compensatory mechanism for the loss of nNOS in mdx mice. Together with previous studies, these data suggest that localization of both nNOSμ and nNOSβ is disrupted by dystrophin deficiency. They also indicate that nNOS has a more complex role as a modifier of dystrophic pathology and broader therapeutic potential than previously recognized. Importantly, these findings also suggest nNOSβ as a new drug target and provide a new conceptual framework for understanding nNOS signaling and the benefits of NO therapies in dystrophinopathies.

Overexpression of SERCA1a in the mdx Diaphragm Reduces Susceptibility to Contraction-Induced Damage

Although the precise pathophysiological mechanism of muscle damage in dystrophin-deficient muscle remains disputed, calcium appears to be a critical mediator of the dystrophic process. Duchenne muscular dystrophy patients and mouse models of dystrophin deficiency exhibit extensive abnormalities of calcium homeostasis, which we hypothesized would be mitigated by increased expression of the sarcoplasmic reticulum calcium pump. Neonatal adeno-associated virus gene transfer of sarcoplasmic reticulum ATPase 1a to the mdx diaphragm decreased centrally located nuclei and resulted in reduced susceptibility to eccentric contraction-induced damage at 6 months of age. As the diaphragm is the mouse muscle most representative of human disease, these results provide impetus for further investigation of therapeutic strategies aimed at enhanced cytosolic calcium removal.

Skeletal muscle repair/regeneration after eccentric contraction-induced damage: effects of S1P

Skeletal muscle repair/regeneration after eccentric contraction-induced damage: effects of S1P

Between channels and tears: aim at ROS to save the membrane of dystrophic fibres

Lengthening during contraction allows muscles to develop force well above the levels reached in isometric conditions. Such enhanced performance has, however, a negative counterpart: damage which is revealed by pain (DOMS), structural and ultra-structural alterations and reduced ability to develop force (Asmussen, 1952). The phenomenon has been demonstrated in vitro and in vivo, in amphibian as well as in mammalian muscles and two main factors have been identified as determinants of eccentric contraction-induced damage: myofibrillar disorganization (Z line streaming and sarcomere length heterogeneity) (Allen, 2001) and calcium homeostasis alteration, possibly related to T tubule disruption (Warren et al. 1993). About 15 years ago Petrof and coworkers showed that mdx dystrophic muscles are highly sensitive to eccentric contraction damage as they show greater loss of force and cellular damage, indicated by sarcolemmal disruption and procion orange penetration, than wild-type muscles (Petrof et al. 1993). Although only sparse evidence is available, the phenomenon seems to be specific to mdx mice and not present in other murine models of muscular dystrophy (Head et al. 2004). Since Petrof's paper was published, many studies aimed to identify the mechanism responsible for the extra damage of dystrophin-lacking fibres exposed to eccentric contractions. It was assumed that the sarcolemma, made fragile by the lack of dystrophin, was easily broken by the intense mechanical strain generated by eccentric contraction. This led to the ‘delta lesions’ early observed in DMD fibres (Pestronk et al. 1982), which were sufficient to give access to large extracellular molecules. Calcium influx and subsequent increase of intracellular free calcium were considered the trigger for degeneration and death of the muscle fibres. The scenario was considerably shifted by more recent studies which suggested that calcium entry could follow a more physiological pathway. The involvement of channels of the TRPC family, which support both stretch activated and store operated calcium influx, was demonstrated (Vandebrouck et al. 2002) and inhibitors of the stretch-activated channels were proved sufficient to control the calcium influx (Yeung et al. 2005). Thus, calcium influx might be a cause and not a direct effect of the membrane tears, and the activation of a relatively slow intracellular mechanism is needed to make the connection (Yeung et al. 2005). The paper published by Whitehead et al. (2008) in this issue of The Journal of Physiology unveils such a connection. Some years ago it was shown that mdx muscle fibres are more susceptible to ROS damage (Rando et al. 1998) and show signs of increased lipid and protein oxidation in spite of the enhanced expression of antioxidant enzymes (Hauser et al. 1995). Whitehead and coworkers demonstrate that an active antioxidant treatment based on N-acetylcysteine (NAC), which is membrane permeant and known to be active in muscle fibres, significantly reduces the impact of eccentric contraction on mdx muscle fibres when given either in vivo or in vitro. Thus, ROS may represent the missing intracellular link between calcium entrance and membrane tears. Two important questions follow: where are ROS produced and what are their targets?Whitehead et al. (2008) suggest that the two main sites of ROS production are mitochondria, whose activity is stimulated in response to increase of intracellular calcium, and NADPH oxidase. The targets of ROS are, obviously, many, but one is of special interest for the fate of dystrophic fibres. Sarcolemma can be destabilized by the lack of β-dystroglycan (BDG), which is essential for the structure of costamere, the skeleton of the muscle membrane. Src kinase activated by ROS (Chen et al. 2005) phosphorylates BDG leading to its internalization. This process is counteracted by dystrophin and utrophin and enhanced by caveolin 3. Besides, lipid peroxidation and membrane protein oxidation might be involved. The proposed interpretation receives strong support from the results of the NAC treatment, which prevents the eccentric contraction damage in mdx, but not in wild-type, muscles, restores the presence of BDG and decreases the amount of caveolin 3. All together, this represents a substantial contribution to maintaining the structural and functional integrity of the membrane. Whether the mechanism outlined above is the only one responsible for the triggering of degenerative processes in muscles lacking dystrophin is still to be defined, but it may represent, in any case, a significant component of the worsening of the pathological phenotype. As suggested by the experiments reported by Whitehead and coworkers the control of oxidative stress may also become an important avenue to study therapies to improve the dystrophic muscle conditions.

Stretch‐induced force deficits in murine extensor digitorum longus muscles after cardiotoxin injection

A leftward shift in a muscle's length-tension relationship is thought to impair myofilament overlap. We hypothesized that left-shifted muscles would incur greater eccentric contraction-induced damage compared to controls. We evaluated contractile properties and force deficits in regenerating murine extensor digitorum longus (EDL) muscles 7, 14, and 21 days after cardiotoxin (CTX) injection. Specific tension recovered to control values by 21 days. CTX-injected muscles demonstrated left-shifted length-tension curves and incurred greater contraction-induced force deficits than controls (P < 0.001) on day 7. We speculate that increased contraction-induced damage in 7-day CTX-injected muscles results from changes in myofilament overlap that occurs during early regeneration.

Adeno-Associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury

Duchenne muscular dystrophy (DMD) is the most common inherited lethal muscle degenerative disease. Currently there is no cure. Highly abbreviated microdystrophin cDNAs were developed recently for adeno-associated virus (AAV)-mediated DMD gene therapy. Among these, a C-terminal-truncated DeltaR4-R23/DeltaC microgene (DeltaR4/DeltaC) has been considered as a very promising therapeutic candidate gene. In this study, we packaged a CMV.DeltaR4/DeltaC cassette in AAV-5 and evaluated the transduction and muscle contractile profiles in the extensor digitorum longus muscles of young (7-week-old) and adult (9-month-old) mdx mice. At approximately 3 months post-gene transfer, 50-60% of the total myofibers were transduced in young mdx muscle and the percentage of centrally nucleated myofibers was reduced from approximately 70% in untreated mdx muscle to approximately 22% in microdystrophin-treated muscle. Importantly, this level of transduction protected mdx muscle from eccentric contraction-induced damage. In contrast, adult mdx muscle was more resistant to AAV-5 transduction, as only approximately 30% of the myofibers were transduced at 3 months postinfection. This transduction yielded marginal protection against eccentric contraction-induced injury. The extent of central nucleation was also more difficult to reverse in adult mdx muscle (from approximately 83% in untreated to approximately 58% in treated). Finally, we determined that the DeltaR4/DeltaC microdystrophin did not significantly alter the expression pattern of the endogenous full-length dystrophin in normal muscle. Neither did it have any adverse effects on normal muscle morphology or contractility. Taken together, our results suggest that AAV-mediated DeltaR4/DeltaC microdystrophin expression represents a promising approach to rescue muscular dystrophy in young mdx skeletal muscle.

Fiber-type susceptibility to eccentric contraction-induced damage of hindlimb-unloaded rat AL muscles.

Slow oxidative (SO) fibers of the adductor longus (AL) were predominantly damaged during voluntary reloading of hindlimb unloaded (HU) rats and appeared explainable by preferential SO fiber recruitment. The present study assessed damage after eliminating the variable of voluntary recruitment by tetanically activating all fibers in situ through the motor nerve while applying eccentric (lengthening) or isometric contractions. Muscles were aldehyde fixed and resin embedded, and semithin sections were cut. Sarcomere lesions were quantified in toluidine blue-stained sections. Fibers were typed in serial sections immunostained with antifast myosin and antitotal myosin (which highlights slow fibers). Both isometric and eccentric paradigms caused fatigue. Lesions occurred only in eccentrically contracted control and HU muscles. Fatigue did not cause lesions. HU increased damage because lesioned- fiber percentages within fiber types and lesion sizes were greater than control. Fast oxidative glycolytic (FOG) fibers were predominantly damaged. In no case did damaged SO fibers predominate. Thus, when FOG, SO, and hybrid fibers are actively lengthened in chronically unloaded muscle, FOG fibers are intrinsically more susceptible to damage than SO fibers. Damaged hybrid-fiber proportions ranged between these extremes.

Structural and mechanical basis of exercise-induced muscle injury

It is well documented in both animal and human studies that unaccustomed, particularly eccentric, muscle exercise may cause damage of muscle fiber contractile and cytoskeletal components. These injuries typically include: Z-band streaming and dissolution, A-band disruption, disintegration of the intermediate filament system, and misalignment of the myofibrils. The mechanical basis for this damage is suggested to be due to the fiber strain magnitude rather than the absolute stress imposed on the fiber. We hypothesize that eccentric contraction-induced damage occurs early in the treatment period, i.e., within the first few minutes. The structural abnormalities predominate in the fast-twitch glycolytic fibers. In the final section of this paper, we hypothesize a damage scheme, based on the muscle fiber oxidative capacity as a determining factor.