A ‘virtual’ revolution: non‐invasive methods to probe skeletal muscle metabolism in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by a mutation in the DMD gene that results in the absence of the protein dystrophin in muscle cells. Dystrophin is a component of the dystrophin glycoprotein complex that is crucial to the maintenance of muscle membrane integrity. Mutations of the DMD gene disrupt the dystrophin glycoprotein complex and cause membrane instability, resulting in enhanced susceptibility to muscle damage, intramuscular fibrosis, mitochondrial dysfunction, impaired muscle stem cell function and lipid accumulation. These changes result in a progressive loss of muscle mass, strength and functional capacity. Currently, there is no effective cure for DMD. The progressive weakening of the neuromuscular system ultimately leads to the inability to walk and premature loss of life. Changes in muscle mass (MM) are dictated primarily by the balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB). When rates of MPS exceed rates of MPB, the result is a positive net protein balance and muscle protein accretion. Alternatively, when rates of MPB exceed rates of MPS, the result is a negative net protein balance and muscle protein loss. Muscle loss can occur owing to a reduction in MPS rates, increased MPB rates, or some combination thereof. Current understanding of the dynamic nature of protein metabolism in health and disease has come from research using stable-isotope tracers (e.g. 13C and 2H). Traditional approaches to measure MPS and MPB have relied on substrate-specific stable-isotope tracers (e.g. l-[ring-2H5]-phenylalanine). These experiments require sterile infusions along with repeated blood and skeletal muscle biopsy sample collection in a controlled laboratory environment over short time periods (e.g. several hours). These acute substrate-specific tracer studies coupled with invasive tissue sampling have generated a wealth of information on how muscle protein metabolism responds to factors such as nutrition, exercise and ageing. However, the invasive nature of these studies prevents their application in young children and vulnerable populations. Less-invasive approaches to study the dynamic nature of muscle protein metabolism are emerging based on the application of the orally administered tracer deuterium oxide (D2O). With this technique, 2H from body water is incorporated into newly synthesized amino acids (e.g. 2H-alanine), enabling the assessment of integrative MPS rates in ‘free-living’ conditions over days to weeks. Most studies to date incorporating D2O to investigate muscle protein metabolism have relied on needle biopsies to collect muscle samples, although Shankaran et al. (2016) recently developed and validated a method to assess MPS rates using a ‘virtual muscle biopsy’ approach whereby 2H-labelled muscle proteins that enter the circulation are sampled via blood collection to determine integrative fractional synthesis rates (FSRs) of individual proteins across the muscle proteome. In addition to measures of MPS and MPB, accurate measures of muscle mass are crucially important in research and clinical practice. Although techniques such as dual-energy X-ray absorptiometry, magnetic resonance imaging, computed tomography and bio-electrical impedance analysis are implemented routinely to assess body composition, some of these techniques do not measure muscle mass, expose the body to ionizing radiation and are expensive. The application of methyl-[D3]-creatine (D3Cr) is a stable-isotope based approach to quantify functional skeletal muscle mass (FMM) that is non-invasive, inexpensive and accurate (Clark et al. 2014). A small amount of D3Cr is ingested orally, and Cr and creatinine (Crn) concentrations and D3Crn enrichments (corrected for D3Cr spillage) are assessed in fasted urine samples taken 48–72 h later to determine the size of the whole-body Cr pool. The FMM can then be calculated based on the assumption of 4.3 g Cr/kg of muscle (Clark et al. 2014). In this issue of The Journal of Physiology, Evans et al. (2021) applied D2O with mass spectrometry-based proteomic techniques to determine the FSR of individual muscle proteins across the proteome, and the D3Cr dilution method to assess whole-body FMM non-invasively in boys with DMD and healthy age-matched boys using blood and urine samples. The 14-day study consisted of 10 subjects with DMD (6–17 years of age) and nine age-matched control subjcts who consumed an initial dose of D3Cr and D2O on day 1. The D2O was then provided daily for 2H labelling of all newly synthesized proteins in conjunction with saliva sampling at 2 h and 7, 10 and 14 days to measure 2H enrichment in body water. Urine samples were collected on day 2 for determination of D3Crn enrichment and assessment of FMM. On day 14, blood and urine were collected to measure the FSR of individual muscle proteins that ‘spill’ into biological fluids using the virtual biopsy approach. The FSR of two key contractile proteins, titin and myosin light chain 1/3, were found to be 73% and 89% lower in DMD subjects (P < 0.001) vs. age-matched control subjects. These findings align with results reported by Rennie et al. (1982), who were the first to explore MPS rates in boys with DMD using substrate-specific amino acid tracer methods coupled with skeletal muscle biopsies. Mixed muscle FSR (as a percentage per hour) was 67% lower in boys with DMD in comparison to adult men, a surprising observation at the time considering that the disease progression was believed to be attributable to elevated MPB rates. Interestingly, Evans et al. (2021) reported no difference in the FSR of sarcoplasmic proteins (creatine kinase M-type and carbonic anhydrase 3) in boys with DMD vs. healthy age-matched control subjects. What is unclear from the results of Evans et al. (2021) is the extent to which the decline in FSR of contractile proteins is attributable to the disease itself. A reduction in MPS rates occurs in response to physical inactivity (e.g. owing to bed rest). Therefore, the physical inactivity associated with the disease and/or reduced total testosterone secondary to corticosteroid treatment could have contributed to the reduced contractile protein FSR seen in the DMD patients. Given that changes in MPS rates precede longer-term changes in MM, it might be possible non-invasively to evaluate the efficacy of anabolic therapies and/or monitor disease progression in DMD and other neuromuscular diseases in the clinic using urine or blood samples. Although MPS rates of contractile proteins are clearly suppressed in DMD, whether there are changes in MPB rates in DMD is unclear. Elevated rates of MPB in conjunction with reduced rates of MPS would result in rapid and pronounced muscle loss. Compared with information on the response of MPS to various interventions, less information is available on the response of MPB, partly because its assessment using traditional stable-isotope methodologies is difficult and invasive. Recently, methyl-[D3]-3-methylhistidine (D3-3MH) has been applied along with D2O and D3Cr as part of a combined stable-isotope approach to assess MM, MPS and MPB in vivo (Cegielski et al. 2021). 3-Methylhistidine can be sampled in blood or urine and is a post-translational modification of contractile protein histidine residues, which are not used for MPS because there is no aminoacyl-tRNA for 3-MH. The use of D3-3MH might provide a useful non-invasive assessment of whole-body MPB in boys with DMD. Alternatively, an intriguing question is whether it is possible to use D2O to measure MPB rates non-invasively on a protein-by-protein basis using biological fluids. Ultimately, a complete picture of muscle protein turnover requires assessment of both MPS and MPB. Non-invasive stable-isotope methodologies are emerging that are ideally suited to advance research and understanding of muscle metabolism in vulnerable populations, such as younger patients with neuromuscular disease. In addition to applying D2O to measure MPS rates virtually across the proteome, Evans et al. (2021) were the first to apply the D3Cr dilution method to measure FMM in boys with DMD. This non-invasive approach revealed that MM and MM/body weight (bw) ratios were dramatically lower in DMD subjects vs. healthy control subjects, with the lowest value of FMM reported to be merely 3% of total body mass in one DMD subject. In DMD subjects, the percentage MM/bw and percentage fat-free mass (FFM)/bw were substantially lower in comparison to control subjects (MM, 17 ± 10 vs. 41 ± 9; FFM, 54 ± 19 vs. 74 ± 9). Although the D3Cr dilution method provides a direct estimate of FMM and has several advantages over other methods, the work of Evans et al. (2021) highlighted some important remaining questions. The calculation of Cr pool size to MM is based on healthy muscle containing an average of 4.3 g Cr/kg of muscle; the Cr content of muscle might be different in individuals with DMD. In the future, it would be of immense interest to apply the D3Cr dilution method longitudinally to monitor disease progression and/or evaluate the efficacy of therapies targeting muscle anabolism. In conclusion, Evans et al. (2021) present fascinating data on two key parameters of skeletal muscle metabolism, MPS and FMM, using a minimally invasive stable-isotope approach in boys with DMD. The incorporation of these new techniques to attain both a ‘virtual’ protein-by-protein measurement of MPS and an estimate of FMM is perfectly suited to the study of compromised dystrophic muscle in younger patients. With these techniques it might be possible to predict the longer-term anabolic effects of a therapeutic intervention on skeletal muscle in individuals based on measurement of the shorter-term changes in the FSR of select muscle proteins in a blood or urine sample. Changes in FMM could then be assessed longitudinally to monitor disease progression or the efficacy of treatments designed to improve muscle mass in patients with DMD. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. The authors declare no conflict of interest and do not have any financial disclosures. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship and all those who qualify for authorship are listed. We appreciate the feedback of Dr Tyler A. Churchward-Venne during the preparation of this manuscript.

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