Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Evaluation of a rapid von Willebrand factor activity latex immuno assay for monitoring of patients with von Willebrand disease (VWD) receiving DDAVP or VWF replacement therapy.

Haemostatic management of surgery in patients with von Willebrand disease (VWD) includes DDAVP or von Willebrand factor (VWF)-containing concentrates. Although the recommendations are for monitoring by VWF activity assays, it is quite common for clinicians to use factor VIII due usually to longer turnaround times required for VWF ristocetin cofactor assay (VWF:RCo) measurements. The aim of this study was to evaluate use of the rapid HaemosIL VWF activity (VWF:Act) latex immuno assay (LIA) on an automated coagulometer (ACL TOP(™) 700; Instrumentation Laboratory, Bedford, MA, USA) compared to platelet-based VWF:RCo assays in this setting. One hundred and sixty-seven plasma samples from 42 patients [Type 1 (n = 22), Type 2A (n = 2), Type 2B (n = 3), Type 2M (n = 10), Type 3 (n = 3)] and acquired von Willebrand syndrome (n = 2) with VWD treated with DDAVP or VWF-containing concentrates were included in the study. Method comparison and method bias were evaluated by Bland-Altman analysis (BA) and Passing and Bablok regression modelling respectively. BA of baseline samples (n = 39) showed a mean difference of -3.0 (±1.96 SD -25.2 to +19.4). Post (treatment) samples (n = 120) were separated into two groups. Group 1 contained samples with VWF:RCo levels 10 to ≤175 IU dL(-1) (n = 97) and group 2, samples with VWF:RCo levels >175 IU dL(-1) (n = 23). BA of group 1 postsamples showed a mean difference of +3.4 (±1.96 SD -44.6 to +51.5), and the BA of Group 2 samples was -23.9 (±1.96 SD -136.1 to +88.3). In conclusion, use of HaemosIL VWF:Act LIA test on an automated coagulometer is a reproducible and rapid assay that can be used as an alternative test for monitoring VWF replacement therapy, facilitating dose adjustments on a real-time basis.

Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.

1. Differences in the effect of age on structure-function relationships of limb muscles of mdx (dystrophin null) and control mice have not been resolved. We tested the hypotheses that, compared with limb muscles from age-matched control mice, limb muscles of 6- to 17-month-old mdx mice are larger but weaker, with lower normalised force and power, whereas those from 24- to 28-month-old mdx mice are smaller and weaker. 2. The maximum isometric tetanic force (P(o)) and power output of limb muscles from 6-, 17-, 24- and 28-month-old mdx and control mice were measured in vitro at 25 degrees C and normalised with respect to cross-sectional area and muscle mass, respectively. 3. Body mass at 6 and 28 months was not significantly different in mdx and control mice, but that of control mice increased 16 % by 17 months and then declined 32 % by 28 months. The body masses of mdx mice declined linearly with age with a decrease of 25 % by 28 months. From 6 to 28 months of age, the range in the decline in the masses of EDL and soleus muscles of mdx and control mice was from 16 to 28 %. The muscle masses of mdx mice ranged from 9 % to 42 % greater than those of control mice at each of the four ages and, even at 28 months, the masses of EDL and soleus muscles of mdx mice were 17 % and 22 % greater than control values. 4. For mdx mice of all ages, muscle hypertrophy was highly effective in the maintenance of control values for absolute force for both EDL and soleus muscles and for absolute power of soleus muscles. Throughout their lifespan, muscles of mdx mice displayed significant weakness with values for specific P(o) and normalised power approximately 20 % lower than values for control mice at each age. For muscles of both strains, normalised force and power decreased approximately 28 % with age, and consequently weakness was more severe in muscles of old mdx than in those of old control mice.

Severity of contraction-induced injury is affected by velocity only during stretches of large strain.

Our purpose was to investigate the effect of velocity of stretch on contraction-induced injury to whole skeletal muscles. Single stretches provide an effective method for studying factors that initiate contraction-induced injury. We tested the null hypothesis that the severity of injury is not dependent on the velocity of the stretch. From the plateau of maximum isometric contractions, extensor digitorum longus muscles of mice were administered single stretches in situ of 30--50% strain relative to muscle fiber length (L(f)) at rates of 1--16 L(f)/s. The magnitude of injury was represented by the isometric force deficit 1--10 min after the stretch. Although the null hypothesis was not supported because the force deficit was affected by velocity (r(2) = 0.09), the effect was relatively weak and was not significant except at the largest strain. Velocity had no effect on peak or average force or work input, factors established to have significant relationships with the force deficit. Velocity may play a minor role in contraction-induced injury, but its importance is negligible relative to that of strain.

Lengthening contractions are not required to induce protection from contraction-induced muscle injury.

We tested the hypothesis that lengthening contractions and subsequent muscle fiber degeneration and/or regeneration are required to induce exercise-associated protection from lengthening contraction-induced muscle injury. Extensor digitorum longus muscles in anesthetized mice were exposed in situ to repeated lengthening contractions, isometric contractions, or passive stretches. Three days after lengthening contractions, maximum isometric force production was decreased by 55%, and muscle cross sections contained a significant percentage (18%) of injured fibers. Neither isometric contractions nor passive stretches induced a deficit in maximum isometric force or a significant number of injured fibers at 3 days. Two weeks after an initial bout of lengthening contractions, a second identical bout produced a force deficit (19%) and a percentage of injured fibers (5%) that was smaller than those for the initial bout. Isometric contractions and passive stretches also provided protection from lengthening contraction-induced injury 2 wk later (force deficits = 35 and 36%, percentage of injured fibers = 12 and 10%, respectively), although the protection was less than that provided by lengthening contractions. These data indicate that lengthening contractions and fiber degeneration and/or regeneration are not required to induce protection from lengthening contraction-induced injury.

Conditioning of skeletal muscles in adult and old mice for protection from contraction-induced injury.

The purpose of this study was to design a conditioning program that protected muscles in both adult and old mice from a protocol of contractions that previously caused a significant number of damaged fibers and a deficit in force. Hind-limb dorsiflexor muscles of adult (7 months) and old (22 months) female B6D2F1 mice were exposed once a week to a protocol of repeated forced stretches while maximally activated in vivo. By week 4, muscles of adult, but not old, mice showed no force deficit. Conditioning was continued for 6 weeks, when both age groups showed no force deficit for two consecutive weeks. Three days after the sixth contraction protocol, when morphological damage and force deficits are most severe, the numbers of damaged fibers in muscles of adult and old mice were not different from those in uninjured control muscles and the force deficits were reduced dramatically compared with unconditioned muscles. We conclude that muscles of both adult and old mice conditioned successfully, but muscles of old mice conditioned more slowly than those of adult mice.

Tibialis anterior muscles in mdx mice are highly susceptible to contraction-induced injury.

Skeletal muscles of patients with Duchenne muscular dystrophy (DMD) and mdx mice lack dystrophin and are more susceptible to contraction-induced injury than control muscles. Our purpose was to develop an assay based on the high susceptibility to injury of limb muscles in mdx mice for use in evaluating therapeutic interventions. The assay involved two stretches of maximally activated tibialis anterior (TA) muscles in situ. Stretches of 40% strain relative to muscle fiber length were initiated from the plateau of isometric contractions. The magnitude of damage was assessed one minute later by the deficit in isometric force. At all ages (2-19 months), force deficits were four- to seven-fold higher for muscles in mdx compared with control mice. For control muscles, force deficits were unrelated to age, whereas force deficits increased dramatically for muscles in mdx mice after 8 months of age. The increase in susceptibility to injury of muscles from older mdx mice did not parallel similar adverse effects on muscle mass or force production. The in situ stretch protocol of TA muscles provides a valuable assay for investigations of the mechanisms of injury in dystrophic muscle and to test therapeutic interventions for reversing DMD.

Measurement of recovery of function following whole muscle transfer, myoblast transfer, and gene therapy.

For a skeletal muscle tissue engineer, the most important issue following an experimental intervention is the evaluation of the recovery of the functional capabilities of the tissue, relative to those of the control tissue. Whether investigators perform whole muscle transfers with spontaneous (1) or surgical (2) vascular and nerve repair, myoblast transfers (3), or manipulations of muscle-specific genes (4,5), the question remains the same: Has the intervention impaired, maintained, or enhanced the functional capabilities of the skeletal muscles involved? In each case, determining structure-function relationships is of vital importance because structure-function relationships are frequently disrupted following an intervention, so that muscle mass and total muscle fiber cross-sectional area (CSA) are not different from control values, but function is impaired, or both are impaired, but with different magnitudes of impairment.

Rapid recovery following contraction-induced injury to in situ skeletal muscles in mdx mice.

The muscles of mdx mice lack the subsarcolemmal protein dystrophin, and as a consequence may be more susceptible to damage induced by contractions. The purpose of this study was to characterize the response of muscles in mdx mice to contraction-induced injury in situ. The hypothesis tested was that following a protocol of repeated stretches of maximally activated muscles, the magnitude of the injury is greater for muscles in mdx mice than for muscles in C57BL/10 control mice, and consequently, the muscles in mdx mice recover more slowly. Each stretch was of 20% strain relative to muscle fibre length (Lf) at 0.5 Lf s-1 and was initiated from the force plateau of an isometric contraction. The protocol consisted of a total of ten contractions, with one contraction occurring every ten seconds. The time-course of injury and recovery was determined through measurements of in situ force production at 10, 30, 45 and 60 minutes, and either 12, 24, 48 or 72 hours after the contraction protocol. The initial injury, as assessed by the decrease in force production both immediately and 60 minutes after the contraction protocol, was significantly greater for the muscles in mdx mice compared with those in control mice. Over the next three days, a value for maximum isometric force of approximately 80% of the pre-injury value was maintained for muscles in control mice, whereas within three days muscles in mdx mice showed complete recovery of force. For muscles in mdx mice, the greater decrease in force during the contraction protocol and the more rapid recovery indicates an increased susceptibility to contraction-induced injury but an enhanced rate of recovery.

The magnitude of the initial injury induced by stretches of maximally activated muscle fibres of mice and rats increases in old age.

1. Our purpose was to compare the susceptibilities of muscles in animals of different ages to the injuries induced by stretching the contracting muscle. Single stretches provide an effective method for studying the factors that contribute to the initiation of contraction-induced injury. We hypothesized that, for maximally activated muscles in old compared with young or adult mice, the work input during a single stretch of any given strain is not different, but for a given work input the magnitude of the injury is greater. 2. The force deficit resulting from each single stretch was calculated as the decrease in the maximum isometric force expressed as a percentage of the maximum force prior to the stretch. Force deficits were compared 1 min after single stretches of in situ extensor digitorum longus (EDL) muscles of young, adult and old mice. In addition, measurements of force deficits immediately following single stretches of single permeabilized fibre segments from EDL muscles of young and old rats permitted investigation of the initial injury at the level of the contractile apparatus. 3. For maximally activated EDL muscles in young, adult and old mice, no differences were observed for the work input during stretches of any given strain. Furthermore, the relationships between the work and the resultant force deficit were not different for muscles in young and adult mice. In contrast, compared with the work-force deficit relationships for muscles in either young or adult mice, the relationship was significantly steeper for muscles in old mice. For single permeabilized fibres from muscles of old rats, the force deficits immediately after single stretches were greater than those observed for fibres from muscles of young rats. We conclude that the increased susceptibility of muscles in old animals to contraction-induced injury resides at least in part within the myofibrils.

Muscle atrophy and weakness with aging: contraction-induced injury as an underlying mechanism.

In old compared with young animals, muscle mass is decreased by 30% to 40%, and maximum force and power are decreased to an even greater extent. The age-related declines in muscle mass and muscle function are similar to those that occur with decreased physical activity. Despite the similarities, we conclude that the losses in muscle mass, force, and power are not due solely to old animals being less active, but rather accrue from intrinsic age-related changes in muscles and in muscle fibers that appear to be immutable and irreversible. The intrinsic changes are associated with denervation of fast fatigable fibers and motor units and motor unit remodeling, which may be initiated by contraction-induced injury. The mechanisms remain unresolved for the weakness, the fatigability, the high susceptibility to contraction-induced injury, and the impaired recovery from injury demonstrated by the skeletal muscles of old animals.

Injury to muscle fibres after single stretches of passive and maximally stimulated muscles in mice.

1. Our purpose was to investigate the initial mechanisms responsible for contraction-induced injury. Most studies of mechanisms of contraction-induced injury have been based on observations made either shortly after many repeated contractions at the peak of fatigue, or days after, at the peak of delayed onset injury. As a result, conclusions based on these studies are complicated by interactions of mechanical and biochemical events, as well as the passage of time. We studied the initial mechanical events associated with contraction-induced injury immediately following single stretches of whole skeletal muscles of mice in situ. 2. We tested the hypothesis that immediately following a single stretch, the severity of contraction-induced injury is a function of both strain and average force. Consequently, the work done to stretch the muscle would be the best predictor of the magnitude of injury. Extensor digitorum longus muscles were adjusted to optimum length for force (L(o)). Passive (not stimulated) and maximally activated muscles were exposed to single stretches of 10, 20, 30, 50 or 60% strain, relative to muscle fibre length (Lf), at a rate of 2 Lf s-1. 3. The magnitude of injury was represented by the force deficit 1 min after the stretch expressed as a percentage of the maximum force prior to the stretch. The occurrence of injury was confirmed directly by electron microscopic analysis of the ultrastructure of muscle fibres that were fixed immediately following single stretches. 4. For active muscles, a single stretch of only 30% strain produced a significant force deficit, whereas for passive muscles, a larger strain was required. Stretches of greater than 50% strain resulted in greater force deficits for passive than for maximally activated muscles. For either condition, the work done to stretch the muscle was the best predictor of the magnitude of injury, accounting for 76% of the variability in the force deficit for maximally activated muscles, and 85% for passive muscles.

Muscle fatigue in old animals. Unique aspects of fatigue in elderly humans.

Muscle atrophy, weakness, injury, and fatigue are inevitable and immutable concomitants of old age. Atrophy results from a gradual process of fiber denervation with loss of some fibers and atrophy of others. Fast fibers show more denervation and atrophy than slow fibers. Some fast fibers are reinnervated by axonal sprouting from slow fibers resulting in remodeling of motor units. With aging, the decreases in strength and power are greater than expected from the loss in muscle mass. Contraction-induced injury is proposed as a mechanism of the fast fiber denervation. With atrophy and weakness, human beings show a dramatic decrease in endurance and increase in fatigability with aging, but strength and endurance training slows the process.

Isometric, shortening, and lengthening contractions of muscle fiber segments from adult and old mice.

In old animals, skeletal muscle force decreases during both isometric and shortening contractions. In contrast, force during lengthening appears to be unaffected by aging. We hypothesized that with aging single permeabilized muscle fibers would demonstrate the same impairments in force as are observed for whole muscles. For single permeabilized fibers from extensor digitorum longus muscles of adult and old mice, forces were measured during isometric, shortening, and lengthening contractions performed at 15 degrees C. Maximum isometric forces normalized for fiber area were not different for fibers from adult and old mice. During submaximal isometric contractions a decreased calcium sensitivity resulted in lower forces for fibers from old compared with adult mice. In contrast to a lack of difference in forces developed by fibers from old and adult mice during shortening contractions, during lengthening contractions fibers from old mice developed forces approximately 30% higher than those of adult mice. We conclude that the impairments in force of whole muscles with aging are not the result of impairments in intrinsic force-generating capacity of cross bridges, but changes do occur in single permeabilized muscle fibers of old mice that result in higher forces during stretch.

Skeletal muscle weakness in old age: underlying mechanisms.

Maintenance of muscle mass and strength contributes to mobility which impacts on quality of life. Although muscle atrophy, declining strength, and physical frailty are generally accepted as inevitable concomitants of aging, the causes are unknown. Clarification of the mechanisms responsible for these changes would enhance our understanding of the degree to which they are preventable or treatable. The decline in muscle function between maturity and old age is similar for muscles of many different animals including human beings, and is typified by the decreases of approximately 35% in maximum force, approximately 30% in maximum power, and 20% in normalized force (kN.m-2) and power (W.kg-1) of extensor digitorum longus (EDL) muscles in old compared with adult mice. Much of the age-associated muscle atrophy and declining strength may be explained by motor unit remodeling which appears to occur by selective denervation of muscle fibers with reinnervation by axonal sprouting from an adjacent innervated unit. Muscles in old mice appear more susceptible to injury than muscles in young or adult mice and have a decreased capacity for recovery. The process of age-related denervation may be aggravated by an increased susceptibility of muscles in old animals to contraction-induced injury coupled with impaired capacity for regeneration.

Injury to skeletal muscle fibers during contractions: conditions of occurrence and prevention.

Contraction-induced injury results in the degeneration and regeneration of muscle fibers. Of the three types of contractions--shortening (concentric), isometric, and lengthening (eccentric)--injury is most likely to occur and the severity of the injury is greatest during lengthening contractions. The magnitude of the injury to muscle fibers may be assessed by direct measures of cellular and ultrastructural damage; by indirect measures of changes in enzyme efflux, calcium influx, ratio of oxidized to reduced glutathione, and force development; and, in human beings, by reports of muscle soreness. The sequence of events includes an initial injury that is primarily mechanical and a secondary metabolic, or biochemical, injury that peaks 1 to 3 days after the injurious contractions. The recovery from contraction-induced injury is usually complete within 30 days. Repeated exposures to protocols of lengthening contractions result in "trained" muscles that are not injured by the protocol that previously caused injury.

Injury to skeletal muscle during altitude training: induction and prevention.

A contracting skeletal muscle will shorten, remain isometric, or lengthen depending on the interaction between external load and the force developed by the muscle. Most physical activities involve shortening, isometric and lengthening contractions. The fluctuations in terrain encountered at altitude, increase both the likelihood that lengthening contractions will occur and the severity of the stretches. When performing a given amount of work, muscles lengthened during contractions expend less energy and fatigue less rapidly than muscles that shorten. Conversely, with equal activation, displacement, and velocity, the work done on a muscle during lengthening contractions is greater than the work performed by a muscle during shortening contractions, but force decreases more rapidly during lengthening. Furthermore, muscles are more likely to be injured during lengthening contractions than during shortening or isometric contractions. The occurrence of contraction-induced injury can be eliminated, or minimized, by prior training specific for the performance of lengthening contractions.

Forces and powers of slow and fast skeletal muscles in mice during repeated contractions.

1. The normalized force or power developed during each of a series of repeated contractions was averaged over the entire cycle of activity and rest to provide a measure of performance referred to as sustained force or sustained power. We tested the hypotheses that compared with slow soleus muscles, fast extensor digitorum longus (EDL) muscles would attain lower maximum values of sustained force, but higher maximum values of sustained power. 2. During repeated contractions at a stimulation frequency of 150 Hz, forces and powers of soleus and EDL muscles of mice were determined in situ at 35 degrees C. The train rate of repeated contractions was incremented every 5 min to increase duty cycle until a maximum value for sustained force or power was reached. 3. In one set of repeated contractions, each contraction was preceded by a quick stretch and thereafter muscle length was held constant. The stretch minimized active shortening of muscle fibres. Sustained force was calculated from force at constant length. The maximum sustained force developed by soleus muscles of 4.58 +/- 0.31 N cm-2 (mean +/- S.E.M.) occurred at a duty cycle of 0.48. Compared with soleus muscles, EDL muscles attained a lower (P less than 0.05) maximum value of 1.38 +/- 0.15 N cm-2 at a 0.35 duty cycle. 4. During isovelocity shortening contractions, the maximum value for sustained power developed by soleus muscles of 7.4 +/- 0.5 W kg-1 occurred at a duty cycle of 0.18. Compared with soleus muscles, EDL muscles achieved a significantly greater maximum value of 9.1 +/- 0.4 W kg-1 at a 0.21 duty cycle.

Maximum and sustained power of extensor digitorum longus muscles from young, adult, and old mice.

We tested the hypothesis that extensor digitorum longus (EDL) muscles in old mice would generate lower maximum powers and sustain lower powers than EDL muscles in young or adult mice. Power was measured in situ at 35 degrees C during single and repeated isovelocity shortening contractions through 10% of fiber length at optimum velocity for power. Sustained power was measured during repeated contractions at a stimulation frequency of 150 Hz. The train rate, and consequently the duty cycle, of repeated contractions was incremented every 5 to 10 min until force could not be maintained throughout the shortening contraction. During single contractions, the maximum absolute powers of muscles in young and old mice were 30% lower than that of adult mice, and power normalized by muscle mass (watts/kg) was 20% lower for muscles in old than for those in adult mice (p less than .05). During repeated contractions, EDL muscles in old and adult mice tolerated lower train rates and duty cycles and sustained lower absolute and normalized powers than EDL muscles in young mice.