Thea Shavlakadze

Striking Denervation of Neuromuscular Junctions without Lumbar Motoneuron Loss in Geriatric Mouse Muscle

Reasons for the progressive age-related loss of skeletal muscle mass and function, namely sarcopenia, are complex. Few studies describe sarcopenia in mice, although this species is the mammalian model of choice for genetic intervention and development of pharmaceutical interventions for muscle degeneration. One factor, important to sarcopenia-associated neuromuscular change, is myofibre denervation. Here we describe the morphology of the neuromuscular compartment in young (3 month) compared to geriatric (29 month) old female C57Bl/6J mice. There was no significant difference in the size or number of motoneuron cell bodies at the lumbar level (L1–L5) of the spinal cord at 3 and 29 months. However, in geriatric mice, there was a striking increase (by ∼2.5 fold) in the percentage of fully denervated neuromuscular junctions (NMJs) and associated deterioration of Schwann cells in fast extensor digitorum longus (EDL), but not in slow soleus muscles. There were also distinct changes in myofibre composition of lower limb muscles (tibialis anterior (TA) and soleus) with a shift at 29 months to a faster phenotype in fast TA muscle and to a slower phenotype in slow soleus muscle. Overall, we demonstrate complex changes at the NMJ and muscle levels in geriatric mice that occur despite the maintenance of motoneuron cell bodies in the spinal cord. The challenge is to identify which components of the neuromuscular system are primarily responsible for the marked changes within the NMJ and muscle, in order to selectively target future interventions to reduce sarcopenia.

IMPLICATIONS OF CROSS-TALK BETWEEN TUMOUR NECROSIS FACTOR AND INSULIN-LIKE GROWTH FACTOR-1 SIGNALLING IN SKELETAL MUSCLE

1 Inflammation, particularly the pro‐inflammatory cytokine tumour necrosis factor (TNF), increases necrosis of skeletal muscle. Depletion of inflammatory cells, such as neutrophils, cromolyn blockade of mast cell degranulation or pharmacological blockade of TNF reduces necrosis of dystrophic myofibres in the mdx mouse model of the lethal childhood disease Duchenne muscular dystrophy (DMD). 2 Insulin‐like growth factor‐1 (IGF‐1) is a very important cytokine for maintenance of skeletal muscle mass and the transgenic overexpression of IGF‐1 within muscle cells reduces necrosis of dystrophic myofibres in mdx mice. Thus, IGF‐1 usually has the opposite effect to TNF. 3 Activation of TNF signalling via the c‐Jun N‐terminal kinase (JNK) can inhibit IGF‐1 signalling by phosphorylation and conformational changes in insulin receptor substrate (IRS)‐1 downstream of the IGF‐1 receptor. Such silencing of IGF‐1 signalling in situations where inflammatory cytokines are elevated has many implications for skeletal muscle in vivo. 4 The basis for these interactions between TNF and IGF‐1 is discussed with specific reference to clinical consequences for myofibre necrosis in DMD and also for the wasting (atrophy) of skeletal muscles that occurs in very old people and in cachexia associated with inflammatory disorders.

A growth stimulus is needed for IGF-1 to induce skeletal muscle hypertrophy in vivo

Here, we characterise new strains of normal and dystrophic (mdx) mice that overexpress Class 2 IGF-1 Ea in skeletal myofibres. We show that transgenic mice have increased muscle levels of IGF-1 (~13-26 fold) and show striking muscle hypertrophy (~24-56% increase in mass). Adult normal muscles were resistant to elevated IGF-1; they reached adult steady state and maintained the same mass from 3 to 12 months. By contrast, dystrophic muscles from mdx/IGF-1(C2:Ea) mice continued to increase in mass during adulthood. IGF-1 signalling was evident only in muscles that were growing as a result of normal postnatal development (23-day-old mice) or regenerating in response to endogenous necrosis (adult mdx mice). Increased phosphorylation of Akt at Ser473 was not evident in fasted normal adult transgenic muscles, but was 1.9-fold higher in fasted normal young transgenic muscles compared with age-matched wild-type controls and fourfold higher in fasted adult mdx/IGF-1(C2:Ea) compared with mdx muscles. Muscles of adult mdx/IGF-1(C2:Ea) mice showed higher p70S6K(Thr421/Ser424) phosphorylation and both young transgenic and adult mdx/IGF-1(C2:Ea) mice had higher phosphorylation of rpS6(Ser235/236). The level of mRNA encoding myogenin was increased in normal young (but not adult) transgenic muscles, indicating enhanced myogenic differentiation. These data demonstrate that elevated IGF-1 has a hypertrophic effect on skeletal muscle only in growth situations.

Oxidative stress as a therapeutic target during muscle wasting: considering the complex interactions

Purpose of reviewThe aim of this overview is to highlight the multiple ways in which oxidative stress could be exacerbating muscle wasting. Understanding these interactions in vivo will assist in identifying opportunities for more targeted therapies to reduce skeletal muscle wasting. Recent findingsThere are many excellent reviews describing how oxidative stress can damage cellular macromolecules, as well as cause deleterious effects through the modulation of signalling pathways. In this overview, we highlight the potential for complex and possibly paradoxical interactions in vivo. Signalling pathways are discussed, using examples involving nuclear factor-kappa B, apoptosis signal-regulating kinase 1 and Akt. Oxidative stress may also be involved in complex interactions with other factors capable of stimulating the loss of muscle mass, possibly through amplifying feedback cycles. This is discussed using examples related to calcium and tumour necrosis factor. SummaryThere is convincing evidence that oxidative stress can increase protein catabolism. The challenge is to demonstrate that oxidative stress is a significant player in the complex interplay that leads to the in-vivo muscle wasting that is caused by a range of conditions and diseases.

Age influences the early events of skeletal muscle regeneration: Studies of whole muscle grafts transplanted between young (8 weeks) and old (13-21 months) mice.

Injured skeletal muscle generally regenerates less efficiently with age, but little is understood about the effects of ageing on the very early inflammatory and neovascular events in the muscle repair process. This study used a total of 174 whole muscle grafts transplanted within and between young and old mice to analyse the effects of ageing on the early inflammatory response in two strains of mice (BALB/c and SJL/J). There was a very slight delay in the early inflammatory response, and in the appearance of myotubes at day 4 in BALB/c muscle grafted into an old host environment (implicating systemic events). In SJL/J mice, the initial speed of the inflammatory response was slightly delayed with old muscle grafts regardless of host age (implicating muscle-derived factors), while an old host environment transiently affected myogenesis (myotube formation). The slight delays in inflammatory and neovascular responses in old mice did not dramatically impact on the overall formation of new muscle. The neovascular response to injured young and old muscle tissue was further analysed using the corneal micropocket assay. This showed a very clear 1-2 day delay in angiogenesis induced by old versus young BALB/c muscle tissue implanted into the young rat cornea, indicating that new blood vessel formation is at least partly determined by muscle-derived factors. Taken together these results indicate that, while there are slight age-associated delays in inflammation and neovascularisation in response to injured muscle, there is no detrimental effect on myogenesis in the mouse model used in this study.

Insulin-like growth factor I slows the rate of denervation induced skeletal muscle atrophy

Loss of the nerve supply to skeletal muscle results in a relentless loss of muscle mass (atrophy) over time. The ability of insulin-like growth factor-1 to reduce atrophy resulting from denervation was examined after transection of the sciatic nerve in transgenic MLC/mIGF-1 mice that over-express mIGF-1 specifically in differentiated myofibres. The cross sectional area (CSA) of all types of myofibres and specifically type IIB myofibres was measured in tibialis anterior muscles from transgenic and wild-type mice at 28 days after denervation. There was a marked myofibre atrophy ( approximately 60%) in the muscles of wild-type mice over this time with increased numbers of myofibres with small CSA. In the muscles of MLC/mIGF-1 mice, over-expression of mIGF-1 reduced the rate of denervation induced myofibre atrophy by approximately 30% and preserved myofibres with larger CSA, compared to wild-type muscles. It is proposed that the protective effect of mIGF-1 on denervated myofibres might be due to reduced protein breakdown.

Early Regeneration of Whole Skeletal Muscle Grafts Is Unaffected by Overexpression of IGF-1 in MLC/mIGF-1 Transgenic Mice

Early myogenic events in regenerating whole muscle grafts were compared between transgenic MLC/mIGF-1 mice with skeletal muscle-specific overexpression of the Exon-1 Ea isoform of insulin-like growth factor-1 (mIGF-1) and control FVB mice, from day 3 to day 21 after transplantation. Immunocytochemistry with antibodies against desmin showed that skeletal muscle-specific overexpression of IGF-1 did not affect the pattern of myoblast activation or proliferation or the onset and number of myotubes formed in regenerating whole muscle grafts. Hypertrophied myotubes were observed in MLC/mIGF grafts at day 7 after transplantation, although such hypertrophy was transient, and the transgenic and control grafts had a similar appearance at later time points (days 10, 14, and 21). Immunostaining with antibodies to platelet endothelial cell adhesion molecule-1, which identifies endothelial cells, demonstrated no difference in the formation of new vascular network in grafts of transgenic and control mice. Skeletal muscle-specific overexpression of mIGF-1 does not appear to stimulate the early events associated with myogenesis during regeneration of whole muscle grafts. (J Histochem Cytochem 52:873–883, 2004)

Growing muscle has different sarcolemmal properties from adult muscle: A proposal with scientific and clinical implications

We hypothesise that the sarcolemma of an actively growing myofibre has different properties to the sarcolemma of a mature adult myofibre. Such fundamentally different properties have clinical consequences for the onset, and potential therapeutic targets, of various skeletal muscle diseases that first manifest either during childhood (e.g. Duchenne muscular dystrophy, DMD) or after cessation of the main growth phase (e.g. dysferlinopathies). These characteristics are also relevant to the selection of both tissue culture and in vivo models employed to study such myopathies and the molecular regulation of adult myofibres. During growth, multinucleated myofibres increase enormously in size and volume with dramatic increases in length (up to ~600 mm). This is in striking contrast with most mononucleated cells such as fibroblasts, that remain at a relatively small size (~10-20 µm diameter). The consequences of a dynamic, expanding sarcolemma during growth, compared with that of an adult myofibre of a fixed length, are discussed with respect to various aspects of muscle biology.

Quantification of Ceroid and Lipofuscin in Skeletal Muscle

Ceroid and lipofuscin are autofluorescent granules thought to be generated as a consequence of chronic oxidative stress. Because ceroid and lipofuscin are persistent in tissue, their measurement can provide a lifetime history of exposure to chronic oxidative stress. Although ceroid and lipofuscin can be measured by quantification of autofluorescent granules, current methods rely on subjective assessment. Furthermore, there has not been any evaluation of variables affecting quantitative measurements. The article describes a simple statistical approach that can be readily applied to quantitate ceroid and lipofuscin. Furthermore, it is shown that several factors, including magnification tissue thickness and tissue level, can affect precision and sensitivity. After optimizing for these factors, the authors show that ceroid and lipofuscin can be measured reproducibly in the skeletal muscle of dystrophic mice (ceroid) and aged mice (lipofuscin).

Lifelong exercise and locally produced insulin-like growth factor-1 (IGF-1) have a modest influence on reducing age-related muscle wasting in mice

The age‐related loss of skeletal muscle mass and function is termed sarcopenia and has been attributed to a decline in concentrations of insulin‐like growth factor‐1 (IGF‐1). We hypothesized that constitutively expressed IGF‐1 within skeletal muscles with or without exercise would prevent sarcopenia. Male transgenic mice that overexpress IGF‐1 Ea in skeletal muscles were compared with wild‐type littermates. Four‐month‐old mice were assigned to be sedentary, or had access to free‐running wheels, until 18 or 28 months of age. In wild‐type mice, the mass of the quadriceps muscles was reduced at 28 months and exercise prevented such loss, without affecting the diameter of myofibers. Conversely, increased IGF‐1 alone was ineffective, whereas the combination of exercise and IGF‐1 was additive in maintaining the diameter of myofibers in the quadriceps muscles. For other muscles, the combination of IGF‐1 and exercise was variable and either increased or decreased the mass at 18 months of age, but was ineffective thereafter. Despite an increase in the diameter of myofibers, grip strength was not improved. In conclusion, our data show that exercise and IGF‐1 have a modest effect on reducing aged‐related wasting of skeletal muscle, but that there is no improvement in muscle function when assessed by grip strength.