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Unravelling protein turnover in Duchenne muscular dystrophy: one protein at a time

Skeletal muscle plays an essential role in metabolic health and physical function, maintaining general health status, independence and quality of life. Hence, degenerative health conditions that profoundly reduce the quantity and quality of skeletal muscle have received significant attention. Duchenne muscular dystrophy (DMD), an X-linked recessive disorder caused by the absence of the dystrophin protein due to mutations in the DMD gene, is the most common and relentless neuromuscular disorder, affecting ∼1 in 5000–6000 live male births (Duan et al. 2021). While treatments exist to alleviate symptoms of DMD, there is currently no cure. Therefore, even with optimal care, which can be financially crippling for patients and their families, patients rarely survive beyond ∼40 years – mainly due to cardiac and respiratory complications. Patients with DMD have less muscle and of poorer quality. A reduction in the contractile components of skeletal muscle occurs concomitantly with lipid and fibrotic tissue accumulation. The lack of the dystrophin protein and thus the disassociation of its larger eponymous structure, the dystrophin-associated protein complex, causes impairments in signalling events, reduces cellular structural integrity, and underpins poor muscle regenerative capacity (Duan et al. 2021). Together, the extensive reduction in the amount and quality of skeletal muscle and the dysregulated signalling have dire consequences for skeletal muscle strength, function, ambulation, quality of life and lifespan. While proxy biomarkers (e.g. creatine kinase) of muscle status exist for diagnostic purposes, monitoring disease progression and therapeutic efficacy by tracking changes in skeletal muscle mass (SMM) in patients with DMD would be tremendously advantageous. The clinical tools currently employed to assess SMM – magnetic resonance imaging, computed tomography and dual-energy X-ray absorptiometry (DXA) – have considerable drawbacks (Clark et al. 2018). These drawbacks include exposure to ionizing radiation, use of ostensibly representative ‘sentinel’muscle cross-sectional areas, expense, access to specialist equipment and personnel, and time-consuming post-scan image processing. DXA, while convenient, does not measure muscle per se but assesses fatand bone-free lean soft tissue and is often used as a proxy for SMM. The recent development of a stable isotope-labelled method – deuterium labelled creatine (D3-Cr) – provides a safe, non-invasive and highly accurate tool to assess SMM (Clark et al. 2018). The D3-Cr method requires no specific diet, is not dependent on renal function, is not influenced by hydration status, and requires only the ingestion of labelled creatine and the collection of a fasted-state urine sample. By capitalizing on creatine biology with an innovative and non-invasive method, in a recent issue of The Journal of Physiology Evans and colleagues report profound differences in whole-body functional SMM between boys with DMD (∼17%, and as low as 3%, of total body mass) and healthy controls (∼41% of total body mass) (Evans et al. 2021). Muscle mass in these patients with DMD was also negatively related to some physical functional assessments (e.g. time to rise from supine). Therefore, D3-Cr appears to be a superior assessment tool to traditional methods, particularly due to its non-invasive nature and its association with functional outcomes (Clark et al. 2014). Muscle biopsies and stable isotope tracers, employed in physiological investigations for many years, have substantially advanced our understanding of the regulatory processes involved in muscle hypertrophy and atrophy. However, much of what we currently know about skeletal muscle remodelling is derived from acute stable isotopic tracer-labelled amino acids in a controlled laboratory setting. SMM is primarily dictated by the balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB). In healthy non-diseased individuals, changes in SMM are primarily the result of changes in MPS. However, given the severe nature of DMD, few exploratory investigations have been performed on patients with DMD, and only one investigation has conducted MPS assessments in patients with DMD. In the previous study, the authors report a reduced rate of mixed MPS as a primary cause of growth failure in DMD (Rennie et al. 1982). Since this initial investigation (Rennie et al. 1982), there have been substantial advances in tracer techniques and analytical methodology, and given the burgeoning interest in muscle protein turnover in ageing and diseased populations, scientists must capitalize on these developments. Deuterated or ‘heavy’ water (i.e. H2O) provides an unbiased strategy to quantitatively assess the turnover rate of hundreds of proteins across the proteome over days to weeks to months, which, by capturing typical daily perturbations, is much more reflective of meaningful changes when compared to traditional isotope methods. Using H2O and a ‘virtual biopsy’ approach (Hellerstein & Evans, 2017), which involves assessing the synthetic rates of proteins in the plasma and urine that mirror rates in the muscle, Evans and colleagues showed that the synthetic rate of contractile proteins (i.e. titin and myosin light chain 1/3) in patients with DMD was substantially lower than in the controls (Evans et al. 2021). In contrast, sarcoplasmic protein synthesis rates were not different. Interestingly, the synthetic rate of some extracellular matrix proteins (e.g. fibronectin) was greater in patients with DMD, possibly as a compensatory response. A controversy in patients with DMD is whether increased proteolysis, reduced synthesis or (likely) some combination of both drives the drastic reduction in SMM. In the present investigation, much like situations of age-related anabolic resistance and immobilization, patients with DMD displayed reduced synthesis of vital