Susan V. Brooks

Contractile properties of skeletal muscles from young, adult and aged mice.

1. Comparisons were made in vitro at 25 degrees C among soleus and extensor digitorum longus (EDL) muscles from young (2‐3 months), adult (9‐10 months), and aged (26‐27 months) male mice. We tested the hypotheses that, compared with soleus and EDL muscles of young and adult mice, those from aged mice develop decreased maximum tetanic force (P0, mN) and specific P0 (N/cm2), and that no significant differences occur for contraction time, half‐relaxation time, or force‐velocity relationship. 2. For the aged mice, the P0 of the soleus muscles and EDL muscles were 78 and 73% respectively of the values for adult mice. The specific P0 of EDL muscles of aged mice was 78% of the value of 23 N/cm2 obtained for young and adult mice. For soleus muscles, the specific P0 of 21 N/cm2 did not change with age. 3. Compared to values for young and adult mice, the contraction and half‐relaxation times of soleus muscles from aged mice were increased, but the overall force‐velocity relationships of soleus and EDL muscles did not change. The pooled values for the maximum velocity of unloaded shortening extrapolated from the force‐velocity relationship of soleus and EDL muscles were 4.6 and 10.1 fibre lengths/s, respectively. 4. The decrease in the specific P0 of the EDL muscle with ageing must result from either a decrease in the number of cross‐bridges in the driving stroke or a decrease in the force developed by each cross‐bridge.

Modular flexibility of dystrophin: Implications for gene therapy of Duchenne muscular dystrophy

Attempts to develop gene therapy for Duchenne muscular dystrophy (DMD) have been complicated by the enormous size of the dystrophin gene. We have performed a detailed functional analysis of dystrophin structural domains and show that multiple regions of the protein can be deleted in various combinations to generate highly functional mini- and micro-dystrophins. Studies in transgenic mdx mice, a model for DMD, reveal that a wide variety of functional characteristics of dystrophy are prevented by some of these truncated dystrophins. Muscles expressing the smallest dystrophins are fully protected against damage caused by muscle activity and are not morphologically different from normal muscle. Moreover, injection of adeno-associated viruses carrying micro-dystrophins into dystrophic muscles of immunocompetent mdx mice results in a striking reversal of histopathological features of this disease. These results demonstrate that the dystrophic pathology can be both prevented and reversed by gene therapy using micro-dystrophins.

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.

AGE‐RELATED CHANGES IN THE STRUCTURE AND FUNCTION OF SKELETAL MUSCLES

1 For animals of all ages, during activation of skeletal muscles and the subsequent contraction, the balance between the force developed by the muscle and the external load determines whether the muscle shortens, remains at fixed length (isometric) or is lengthened. With maximum activation, the force developed is least during shortening, intermediate when muscle length is fixed and greatest during lengthening contractions. During lengthening contractions, when force is high, muscles may be injured by the contractions. 2 ‘Frailty’ and ‘failure to thrive’ are most frequently observed in elderly, physically inactive people. A ‘frail’ person is defined as one of small stature, with muscles that are atrophied, weak and easily fatigued. The condition of ‘failure to thrive’ is typified by a lack of response to well‐designed programmes of nutrition and physical activity. 3 With ageing, skeletal muscle atrophy in humans appears to be inevitable. A gradual loss of muscle fibres begins at approximately 50 years of age and continues such that by 80 years of age, approximately 50% of the fibres are lost from the limb muscles that have been studied. For both humans and rats, the observation that the timing and magnitude of the loss of motor units is similar to that for muscle fibres suggests that the mechanism responsible for the loss of fibres and the loss of whole motor units is the same. The degree of atrophy of the fibres that remain is largely dependent on the habitual level of physical activity of the individual. 4 ‘Master athletes’ maintain a high level of fitness throughout their lifespan. Even among master athletes, performance of marathon runners and weight lifters declines after approximately 40 years of age, with peak levels of performance decreased by approximately 50% by 80 years of age. The success of the master athletes and of previously sedentary elderly who undertake well‐designed, carefully administered training programmes provide dramatic evidence that age‐associated atrophy, weakness and fatigability can be slowed, but not halted.

Rapamycin slows aging in mice

Rapamycin increases lifespan in mice, but whether this represents merely inhibition of lethal neoplastic diseases, or an overall slowing in multiple aspects of aging is currently unclear. We report here that many forms of age‐dependent change, including alterations in heart, liver, adrenal glands, endometrium, and tendon, as well as age‐dependent decline in spontaneous activity, occur more slowly in rapamycin‐treated mice, suggesting strongly that rapamycin retards multiple aspects of aging in mice, in addition to any beneficial effects it may have on neoplastic disease. We also note, however, that mice treated with rapamycin starting at 9 months of age have significantly higher incidence of testicular degeneration and cataracts; harmful effects of this kind will guide further studies on timing, dosage, and tissue‐specific actions of rapamycin relevant to the development of clinically useful inhibitors of TOR action.

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 (Po) and power output of limb muscles from 6‐, 17‐, 24‐ and 28‐month‐old mdx and control mice were measured in vitro at 25 °C and normalised with respect to cross‐sectional area and muscle mass, respectively. 3 Body mass at 6 and 28 months was not signifcantly 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 Po and normalised power ≈20 % lower than values for control mice at each age. For muscles of both strains, normalised force and power decreased ≈28 % with age, and consequently weakness was more severe in muscles of old mdx than in those of old control mice.

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.

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.

Increased superoxide in vivo accelerates age-associated muscle atrophy through mitochondrial dysfunction and neuromuscular junction degeneration

Oxidative stress has been implicated in the etiology of age‐related muscle loss (sarcopenia). However, the underlying mechanisms by which oxidative stress contributes to sarcopenia have not been thoroughly investigated. To directly examine the role of chronic oxidative stress in vivo, we used a mouse model that lacks the antioxidant enzyme CuZnSOD (Sodl). Sod1−/− mice are characterized by high levels of oxidative damage and an acceleration of sarcopenia. In the present study, we demonstrate that muscle atrophy in Sod1−/− mice is accompanied by a progressive decline in mitochondrial bioenergetic function and an elevation of mitochondrial generation of reactive oxygen species. In addition, Sod1−/− muscle exhibits a more rapid induction of mitochondrial‐mediated apoptosis and loss of myonuclei. Furthermore, aged Sod1−/− mice show a striking increase in muscle mitochondrial content near the neuromuscular junctions (NMJs). Despite the increase in content, the function of mitochondria is significantly impaired, with increased denervated NMJs and fragmentation of acetylcholine receptors. As a consequence, contractile force in aged Sod1−/− muscles is greatly diminished. Collectively, we show that Sod1−/− mice display characteristics of normal aging muscle in an accelerated manner and propose that the superoxide‐induced NMJ degeneration and mitochondrial dysfunction are potential mechanisms of sarcopenia.—Jang, Y. C., Lustgarten, M. S., Liu, Y., Muller, F. L., Bhattacharya, A., Liang, H., Salmon, A. B., Brooks, S. V., Larkin, L., Hayworth, C. R., Richardson, A., and Van Remmen, H. Increased superoxide in vivo accelerates age‐associated muscle atrophy through mitochondrial dysfunction and neuro‐muscular junction degeneration. FASEB J. 24, 1376–1390 (2010). www.fasebj.org

Functional correction of adult mdx mouse muscle using gutted adenoviral vectors expressing full-length dystrophin

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene. Delivery of functionally effective levels of dystrophin to immunocompetent, adult mdx (dystrophin-deficient) mice has been challenging because of the size of the gene, immune responses against viral vectors, and inefficient infection of mature muscle. Here we show that high titer stocks of three different gutted adenoviral vectors carrying full-length, muscle-specific, dystrophin expression cassettes are able to efficiently transduce muscles of 1-yr-old mdx mice. Single i.m. injections of viral vector restored dystrophin production to 25–30% of mouse limb muscle 1 mo after injection. Furthermore, functional tests of virally transduced muscles revealed almost 40% correction of their high susceptibility to contraction-induced injury. Our results show that functional abnormalities of dystrophic muscle can be corrected by delivery of full-length dystrophin to adult, immunocompetent mdx mice, raising the prospects for gene therapy of muscular dystrophies.