Dysferlinopathy is an age-dependent muscular dystrophy caused by loss of the membrane-associated protein dysferlin. Disease severity increases with age and selectively affects specific muscle groups, yet the molecular basis for this vulnerability remains unclear. Since lipid remodeling is a hallmark of dysferlinopathy and aging, we investigated how age, sex, and muscle fiber type interact to shape the muscle lipidome in dysferlin-deficient mice. We performed omics-scale lipid profiling across 738 lipid species and 30 lipid classes in quadriceps and gastrocnemius (fast-twitch muscles exhibiting pronounced pathology), soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch, relatively spared) muscles from male and female dysferlin-deficient (BLA/J) and wildtype C57BL/6J (WT) mice aged 3, 10, and 26 months. Normal aging was associated with broad lipid remodeling, however, in the absence of dysferlin markedly amplified this remodeling, leading to elevations in specific triglycerides, diglycerides, cholesterol esters, gangliosides, ceramides, and sphingomyelin compared to WT muscle. Interestingly, we observed minimal sex differences between dysferlin-deficient muscles. Fast-twitch muscles, particularly quadriceps and gastrocnemius, exhibited the most extensive lipid alterations, whereas the slow-twitch soleus muscle showed relative lipid stability even at advanced age. Thus, fast-glycolytic muscles are more susceptible to age- and dysferlin-dependent lipid dysregulation than slow oxidative muscle. The preferential vulnerability of fast-twitch muscles to muscle wasting in dysferlinopathy suggests that fiber-type-dependent lipid handling contributes to selective muscle degeneration. This work defines a comprehensive lipidomic signature of disease progression and provides a framework for understanding how aging, sex, and muscle phenotype interact in muscular dystrophy.

This two part study on the afferent connectivity of lumbar spinal motor neurons in normal ageing mice investigates; Study 1: time course analysis of age-related changes in the synaptic coverage of lumbar spinal cords of male C57BL/BJ mice at 4,15,18 and 24 months of age and Study 2: the effect of long term 8-month resistance wheel exercise (RWE) on lumbar spinal cords of male C57BL/6J mice exercised from 15 to 23 months of age. Uniquely, each study used spinal cords obtained from the same mice that had previously been analysed for changes in skeletal muscles and sciatic nerves in a parallel series of time course and exercise studies. Input to presumed alpha motor neurons was investigated by quantifying VGLUT1 immunoreactive synaptic contacts known to be derived from proprioceptive muscle afferents. Here we found no significant changes in the percentage of synaptic VGLUT1 coverage of motor neurons from 4 to 24 months. Importantly, this differs from our previous results (Krishnan et al., Biogerontology 19:385–399, 2018) where there was about 50

Duchenne Muscular Dystrophy (DMD) is an X-linked disease characterised by chronic inflammation, progressive muscle damage, and muscle loss. Typically, initial symptoms affect lower limb muscles, including the gastrocnemius (GA), tibialis anterior (TA), and extensor digitorum longus (EDL). During the acute phase of DMD, particularly in boys aged 2–8 years, muscle damage resulting in necrosis (myonecrosis) involves a complex immune-inflammatory response. Prostaglandin D2 (PGD2) is recognised for enhancing pro-inflammatory chemokine and interleukin signalling and recruiting infiltrating immune cells such as pro-inflammatory macrophages, exacerbating myonecrosis. To reduce levels of PGD2, a novel hematopoietic prostaglandin D2 synthase (HPGDS) inhibitor, PK007, was characterised (i) for potency and pharmacokinetic profiles and then tested in the mdx mouse model of DMD during the acute early onset of disease progression. Juvenile mdx and wild type (WT) C57Bl/10Scsn mice were orally treated with PK007 and control vehicle solution for 10 days, from postnatal day 18 to 28. This builds upon a previous study with PK007 with (ii) additional analyses of disease progression assessed for muscle grip strength, metabolic and locomotor activity, myonecrosis in a wide range of muscles (3 from hindlimb, diaphragm, heart, and tongue), macrophage infiltration and pro-inflammatory cytokines (TNF-α, IL-1β and iNOS). PK007 exhibited high potency (17.23 ± 12 nM), a long half-life (3.0 ± 0.3 h), and good oral bioavailability (81

Sarcopenia is the accelerated loss of skeletal muscle mass and function commonly, but not exclusively, associated with advancing age. It is observed across many species including humans in whom it can lead to decline in physical function and mobility as well as to increased risk of adverse outcomes including falls, fractures and premature mortality. Although prevalence estimates vary because sarcopenia has been defined in different ways, even using a conservative approach, the prevalence is between 5% and 10% in the general population. A life course framework has been proposed for understanding not only the occurrence of sarcopenia in later life but also influences operating at earlier life stages with potentially important implications for preventive strategies. Harnessing progress in understanding the hallmarks of ageing has been key to understanding sarcopenia pathophysiology. Considerable convergence in approaches to diagnosis of sarcopenia has occurred over the last 10 years, with a growing emphasis on the central importance of muscle strength. Resistance exercise is currently the mainstay of treatment; however, it is not suitable for all. Hence, adjunctive and alternative treatments to improve quality of life are needed. An internationally agreed approach to definition and diagnosis will enable a step change in the field and is likely to be available in the near future through the Global Leadership Initiative in Sarcopenia.

Skeletal muscle tissue function is governed by the mechanical properties and organization of its components, including myofibers, extracellular matrix, and adipose tissue, which can be modified by the onset and progression of many disorders. This study used a novel combination of quantitative micro-elastography and clearing-enhanced three-dimensional (3D) microscopy to assess 3D micro-scale elasticity and micro-architecture of muscles from two muscular dystrophies: dysferlinopathy and Duchenne muscular dystrophy, using male BLA/J and mdx mice, respectively, and their wild-type (WT) controls. We examined three muscles with varying proportions of slow- and fast-twitch myofibers: the soleus (predominantly slow), extensor digitorum longus (EDL; fast), and quadriceps (mixed), from BLA/J and WTBLA/J mice aged 3, 10, and 24 months, and mdx and WTmdx mice aged 10 months. Both dysferlin deficiency and age reduced the elasticity and variability of elasticity of the soleus and quadriceps, but not EDL. Overall, the BLA/J soleus was 20% softer than WT and less mechanically heterogeneous (-14% in standard deviation of elasticity). The BLA/J quadriceps at 24 months was 72% softer than WT and less mechanically heterogeneous (-59% in standard deviation), with substantial adipose tissue accumulation. While mdx muscles did not differ quantitatively from WT, regional heterogeneity was evident in micro-scale elasticity and micro-architecture of quadriceps (e.g., 11.2 kPa in a region with marked pathology vs 3.8 kPa in a less affected area). These results demonstrate differing biomechanical changes in hind-limb muscles of two distinct muscular dystrophies, emphasizing the potential for this novel multimodal technique to identify important differences between various myopathies.

Abstract Background Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by gene mutations resulting in deficiency of the membrane-associated protein dysferlin. They manifest post-growth and are characterised by muscle wasting (primarily in the limb and limb-gridle muscles), inflammation, and replacement of myofibres with adipose tissue. The precise pathomechanism for dysferlinopathy is currently unclear; as such there are no treatments currently available. Glucocorticoids (GCs) are widely used to reduce inflammation and treat muscular dystrophies, but when administered to patients with dysferlinopathy, they have unexpected adverse effects, with accelerated loss of muscle strength. Methods To investigate the mechanistic basis for the adverse effects of GCs in dysferlinopathy, the potent GC dexamethasone (Dex) was administered for 4–5 weeks (0.5–0.75 µg/mL in drinking water) to dysferlin-deficient BLA/J and normal wild-type (WT) male mice, sampled at 5 (Study 1) or 10 months (Study 2) of age. A wide range of analyses were conducted. Metabolism- and immune-related gene expression was assessed in psoas muscles at both ages and in quadriceps at 10 months of age. For the 10-month-old mice, quadriceps and psoas muscle histology was assessed. Additionally, we investigated the impact of Dex on the predominantly slow and fast-twitch soleus and extensor digitorum longus (EDL) muscles (respectively) in terms of contractile function, myofibre-type composition, and levels of proteins related to contractile function and metabolism, plus glycogen. Results At both ages, many complement-related genes were highly expressed in BLA/J muscles, and WT mice were generally more responsive to Dex than BLA/J. The effects of Dex on BLA/J mice included (i) increased expression of inflammasome-related genes in muscles (at 5 months) and (ii) exacerbated histopathology of quadriceps and psoas muscles at 10 months. A novel observation was pronounced staining for glycogen in many myofibres of the damaged quadriceps muscles, with large pale vacuolated myofibres, suggesting possible myofibre death by oncosis. Conclusion These pilot studies provide a new focus for further investigation into the adverse effects of GCs on dysferlinopathic muscles.

Redox modifications to the plasma protein albumin have the potential to be used as biomarkers of disease progression and treatment efficacy in pathologies associated with inflammation and oxidative stress. One such pathology is Duchenne muscular dystrophy (DMD), a fatal childhood disease characterised by severe muscle wasting. We have previously shown in the mdx mouse model of DMD that plasma albumin thiol oxidation is increased; therefore, the first aim of this paper was to establish that albumin thiol oxidation in plasma reflects levels within mdx muscle tissue. We therefore developed a method to measure tissue albumin thiol oxidation. We show that albumin thiol oxidation was increased in both mdx muscle and plasma, with levels correlated with measures of dystropathology. In dystrophic muscle, albumin content was associated with areas of myonecrosis. The second aim was to test the ability of plasma thiol oxidation to track acute changes in dystropathology: we therefore subjected mdx mice to a single treadmill exercise session (known to increase myonecrosis) and took serial blood samples. This acute exercise caused a transient increase in total plasma albumin oxidation and measures of dystropathology. Together, these data support the use of plasma albumin thiol oxidation as a biomarker to track active myonecrosis in DMD.

Sarcopenia is the accelerated loss of skeletal muscle mass and function commonly, but not exclusively, associated with advancing age. It is observed across many species including humans in whom it can lead to decline in physical function and mobility as well as to increased risk of adverse outcomes including falls, fractures and premature mortality. Although prevalence estimates vary because sarcopenia has been defined in different ways, even using a conservative approach, the prevalence is between 5% and 10% in the general population. A life course framework has been proposed for understanding not only the occurrence of sarcopenia in later life but also influences operating at earlier life stages with potentially important implications for preventive strategies. Harnessing progress in understanding the hallmarks of ageing has been key to understanding sarcopenia pathophysiology. Considerable convergence in approaches to diagnosis of sarcopenia has occurred over the last 10 years, with a growing emphasis on the central importance of muscle strength. Resistance exercise is currently the mainstay of treatment; however, it is not suitable for all. Hence, adjunctive and alternative treatments to improve quality of life are needed. An internationally agreed approach to definition and diagnosis will enable a step change in the field and is likely to be available in the near future through the Global Leadership Initiative in Sarcopenia.

Ageing is a complex biological process associated with increased morbidity and mortality. Nine classic, interdependent hallmarks of ageing have been proposed involving genetic and biochemical pathways that collectively influence ageing trajectories and susceptibility to pathology in humans. Ageing skeletal muscle undergoes profound morphological and physiological changes associated with loss of strength, mass, and function, a condition known as sarcopenia. The aetiology of sarcopenia is complex and whilst research in this area is growing rapidly, there is a relative paucity of human studies, particularly in older women. Here, we evaluate how the nine classic hallmarks of ageing: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication contribute to skeletal muscle ageing and the pathophysiology of sarcopenia. We also highlight five novel hallmarks of particular significance to skeletal muscle ageing: inflammation, neural dysfunction, extracellular matrix dysfunction, reduced vascular perfusion, and ionic dyshomeostasis, and discuss how the classic and novel hallmarks are interconnected. Their clinical relevance and translational potential are also considered.

Dysferlinopathies are a clinically heterogeneous group of muscular dystrophies caused by a genetic deficiency of the membrane-associated protein dysferlin, which usually manifest post-growth in young adults. The disease is characterized by progressive skeletal muscle wasting in the limb-girdle and limbs, inflammation, accumulation of lipid droplets in slow-twitch myofibers and, in later stages, replacement of muscles by adipose tissue. Previously we reported myofiber-type specific differences in muscle contractile function of 10-month-old dysferlin-deficient BLAJ mice that could not be fully accounted for by altered myofiber-type composition. In order to further investigate these findings, we examined the impact of dysferlin deficiency on the abundance of calcium (Ca2+) handling and glucose/glycogen metabolism-related proteins in predominantly slow-twitch, oxidative soleus and fast-twitch, glycolytic extensor digitorum longus (EDL) muscles of 10-month-old wild-type (WT) C57BL/6J and dysferlin-deficient BLAJ male mice. Additionally, we compared the Ca2+ activation properties of isolated slow- and fast-twitch myofibers from 3-month-old WT and BLAJ male mice. Differences were observed for some Ca2+ handling and glucose/glycogen metabolism-related protein levels between BLAJ soleus and EDL muscles (compared with WT) that may contribute to the previously reported differences in function in these BLAJ muscles. Dysferlin deficiency did not impact glycogen content of whole muscles nor Ca2+ activation of the myofilaments, although soleus muscle from 10-month-old BLAJ mice had more glycogen than EDL muscles. These results demonstrate a further impact of dysferlin deficiency on proteins associated with excitation-contraction coupling and glycogen metabolism in skeletal muscles, potentially contributing to altered contractile function in dysferlinopathy.

Many neuromuscular disorders can have a differential impact on a specific myofibre type, forming the central premise of this review. The many different skeletal muscles in mammals contain a spectrum of slow- to fast-twitch myofibres with varying levels of protein isoforms that determine their distinctive contractile, metabolic, and other properties. The variations in functional properties across the range of classic 'slow' to 'fast' myofibres are outlined, combined with exemplars of the predominantly slow-twitch soleus and fast-twitch extensor digitorum longus muscles, species comparisons, and techniques used to study these properties. Other intrinsic and extrinsic differences are discussed in the context of slow and fast myofibres. These include inherent susceptibility to damage, myonecrosis, and regeneration, plus extrinsic nerves, extracellular matrix, and vasculature, examined in the context of growth, ageing, metabolic syndrome, and sexual dimorphism. These many differences emphasise the importance of carefully considering the influence of myofibre-type composition on manifestation of various neuromuscular disorders across the lifespan for both sexes. Equally, understanding the different responses of slow and fast myofibres due to intrinsic and extrinsic factors can provide deep insight into the precise molecular mechanisms that initiate and exacerbate various neuromuscular disorders. This focus on the influence of different myofibre types is of fundamental importance to enhance translation for clinical management and therapies for many skeletal muscle disorders.

This commentary provides an independent consideration of data related to the drug vamorolone (VBP15) as an alternative steroid proposed for treatment of Duchenne muscular dystrophy (DMD). Glucocorticoids such as prednisone and deflazacort have powerful anti-inflammatory benefits and are the standard of care for DMD, but their long-term use can result in severe adverse side effects; thus, vamorolone was designed as a unique dissociative steroidal anti-inflammatory drug, to retain efficacy and minimise these adverse effects. Extensive clinical trials (ongoing) have investigated the use of vamorolone for DMD, with two trials also for limb-girdle muscular dystrophies including dysferlinopathy (current), plus a variety of pre-clinical trials published. Vamorolone looks very promising, with similar efficacy and some reduced adverse effects (e.g., related to height) compared with other glucocorticoids, specifically prednisone/prednisolone, although it has not yet been directly compared with deflazacort. Of particular interest to clarify is the optimal clinical dose and other aspects of vamorolone that are proposed to provide additional benefits for membranes of dystrophic muscle: to stabilise and protect the sarcolemma from damage and enhance repair. The use of vamorolone (and other glucocorticoids) needs to be evaluated in terms of overall long-term efficacy and cost, and also in comparison with many candidate non-steroidal drugs with anti-inflammatory and other benefits for DMD.

Inflammation and oxidative stress are strongly implicated in the pathology of Duchenne muscular dystrophy (DMD), and the sulphur-containing amino acid taurine ameliorates both and decreases dystropathology in the mdx mouse model for DMD. We therefore further tested taurine as a therapy using dystrophic DMDmdx rats and dmd zebrafish models for DMD that have a more severe dystropathology. However, taurine treatment had little effect on the indices of dystropathology in both these models. While we and others have previously observed a deficiency in taurine in mdx mice, in the current study we show that the rat and zebrafish models had increased taurine content compared with wild-type, and taurine treatment did not increase muscle taurine levels. We therefore hypothesised that endogenous levels of taurine are a key determinate in potential taurine treatment efficacy. Because of this, we felt it important to measure taurine levels in DMD patient plasma samples and showed that in non-ambulant patients (but not in younger patients) there was a deficiency of taurine. These data suggest that taurine homeostasis varies greatly between species and may be influenced by age and disease progression. The potential for taurine to be an effective therapy may depend on such variables.

Skeletal muscle extracellular matrix (ECM) is critical for muscle force production and the regulation of important physiological processes during growth, regeneration, and remodelling. ECM remodelling is a tightly orchestrated process, sensitive to multi-directional tensile and compressive stresses and damaging stimuli, and its assessment can convey important information on rehabilitation effectiveness, injury, and disease. Despite its profound importance, ECM biomarkers are underused in studies examining the effects of exercise, disuse, or aging on muscle function, growth, and structure. This review examines patterns of short- and long-term changes in the synthesis and concentrations of ECM markers in biofluids and tissues, which may be useful for describing the time course of ECM remodelling following physical activity and disuse. Forces imposed on the ECM during physical activity critically affect cell signalling while disuse causes non-optimal adaptations, including connective tissue proliferation. The goal of this review is to inform researchers, and rehabilitation, medical, and exercise practitioners better about the role of ECM biomarkers in research and clinical environments to accelerate the development of targeted physical activity treatments, improve ECM status assessment, and enhance function in aging, injury, and disease.

Duchenne muscular dystrophy (DMD) is a severe childhood disease characterised by progressive muscle wasting caused by widespread myofibre necrosis. Implicated in the pathology of DMD is oxidative stress, caused by excessive generation of reactive oxygen and nitrogen species (RONS). One consequence of RONS exposure is post-translational oxidative modifications to proteins, which can cause loss of protein function. This study used the dystrophic mdx mouse model for DMD to visualise the precise location of different oxidative modifications to proteins in dystrophic muscles, including both reversible (protein thiol oxidation and s-nitrosylation) and irreversible (carbonylation and dityrosine formation) oxidation at various stages of dystrophic muscle necrosis and regeneration. High levels of protein oxidation were observed in mdx myofibres undergoing degeneration and immune cell infiltration (myonecrosis). Since irreversible protein oxidation, especially dityrosine formation, was only colocalised to areas of myonecrosis, we suggest that this specific measurement could be a useful biomarker of myonecrosis. To test this we quantified dityrosines in muscle homogenates; this analysis showed significantly higher levels of dityrosines in mdx (compared with control normal) mice aged 23 days, an age when acute onset of extensive myonecrosis occurs in mdx muscles. These results indicate a major localised role of immune cells in RONS generation in dystrophic muscle, and strongly support a role for protein oxidation in myonecrosis and associated dystropathology. Consequently, the measurement of protein oxidation (specifically dityrosines) in dystrophic muscles may be a useful biomarker for indirectly quantifying myonecrosis in research studies using mdx mice and other animal models for DMD.

Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro–adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.

Abstract Background Periodic paralysis (PP) is a rare genetic disorder in which ion channel mutation causes episodic paralysis in association with hyper‐ or hypokalaemia. An unexplained but consistent feature of PP is that a phenotype transition occurs around the age of 40, in which the severity of potassium‐induced muscle weakness declines but onset of fixed, progressive weakness is reported. This phenotype transition coincides with the age at which muscle mass and optimal motor function start to decline in healthy individuals. We sought to determine if the phenotype transition in PP is linked to the normal ageing phenotype transition and to explore the mechanisms involved. Methods A mouse model of hyperkalaemic PP was compared with wild‐type littermates across a range of ages (13–104 weeks). Only male mice were used as penetrance is incomplete in females. We adapted the muscle velocity recovery cycle technique from humans to examine murine muscle excitability in vivo. We then examined changes in potassium‐induced weakness or caffeine contracture force with age using ex vivo muscle tension testing. Muscles were further characterized by either Western blot, histology or energy charge measurement. For normally distributed data, a student's t‐test (± Welch correction) or one‐ or two‐way analysis of variance (ANOVA) was performed to determine significance. For data that were not normally distributed, Welch rank test, Mann Whitney U test or Kruskal–Wallis ANOVA was performed. When an ANOVA was significant (P < 0.05), post hoc Tukey testing was used. Results Both WT (P = 0.009) and PP (P = 0.007) muscles exhibit increased resistance to potassium‐induced weakness with age. Our data suggest that healthy‐old muscle develops mechanisms to maintain force despite sarcolemmal depolarization and sodium channel inactivation. In contrast, reduced caffeine contracture force (P = 0.00005), skeletal muscle energy charge (P = 0.004) and structural core pathology (P = 0.005) were specific to Draggen muscle, indicating that they are caused, or at least accelerated by, chronic genetic ion channel dysfunction. Conclusions The phenotype transition with age is replicated in a mouse model of PP. Intrinsic muscle ageing protects against potassium‐induced weakness in HyperPP mice. However, it also appears to accelerate impairment of sarcoplasmic reticulum calcium release, mitochondrial impairment and the development of core‐like regions, suggesting acquired RyR1 dysfunction as the potential aetiology. This work provides a first description of mechanisms involved in phenotype transition with age in PP. It also demonstrates how studying phenotype transition with age in monogenic disease can yield novel insights into both disease physiology and the ageing process itself.

Older age is associated with deteriorating health, including escalating risk of diseases such as cancer, and a diminished ability to repair following injury. This rise in age-related diseases/co-morbidities is associated with changes to immune function, including in myeloid cells, and is related to immunosenescence. Immunosenescence reflects age-related changes associated with immune dysfunction and is accompanied by low-grade chronic inflammation or inflammageing. This is characterised by increased levels of circulating pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1β and IL-6. However, in healthy ageing, there is a concomitant age-related escalation in anti-inflammatory cytokines such as transforming growth factor-β1 (TGF-β1) and IL-10, which may overcompensate to regulate the pro-inflammatory state. Key inflammatory cells, macrophages, play a role in cancer development and injury repair in young hosts, and we propose that their role in ageing in these scenarios may be more profound. Imbalanced pro- and anti-inflammatory factors during ageing may also have a significant influence on macrophage function and further impact the severity of age-related diseases in which macrophages are known to play a key role. In this brief review we summarise studies describing changes to inflammatory function of macrophages (from various tissues and across sexes) during healthy ageing. We also describe age-related diseases/co-morbidities where macrophages are known to play a key role, focussed on injury repair processes and cancer, plus comment briefly on strategies to correct for these age-related changes.