Mitochondrial profiling of immortalised myoblasts from a Duchenne Muscular Dystrophy patient

C.A. Timpani,^1,2 J. White,^3,4 K. Mamchaoui,⁵ G. Butler-Browne,⁵ A. Hayes^1,2 and E. Rybalka,^1,2 ¹Institute of Sport, Exercise & Active Living (ISEAL), College of Health and Biomedicine, Victoria University, Melbourne, VIC 8001, Australia, ²Australian Institute for Musculoskeletal Science (AIMSS), Victoria University, Melbourne, VIC 8001, Australia, ³Murdoch Children’s Research Institute, Melbourne, Victoria, Australia, ⁴Melbourne Veterinary School, The University of Melbourne, Parkville, VIC 3010, Australia and ⁵Institut de Myologie, Sorbonne University, INSERM UMRS974, Paris, France.

Introduction: Mitochondria are increasingly implicated in a variety of debilitating diseases including the fatal X-linked neuromuscular disease, Duchenne Muscular Dystrophy (DMD) (Timpani et al., 2015). We and others have documented mitochondrial dysfunction, morphological anomalies and oxidative stress in skeletal muscle from the mdx mouse model of DMD (Rybalka et al., 2014; Timpani et al., 2015; Timpani et al., 2016). In this study, we aimed to establish the mitochondrial profile of human DMD myoblasts and the therapeutic potential of a metabolic (Krebs cycle) stimulant to normalize any dysfunctions.

Methods: Using immortalised myoblasts derived from the fascia lata of a 10-year-old DMD patient (DMD) and the paraspinal muscles of a 12-year-old healthy child (CON), we have generated a mitochondrial profile including respiratory function (extracellular flux analysis), pool density and viability (MitoTracker probes) and reactive oxygen species (ROS) production (MitoSOX).

Results: DMD myoblasts had a higher basal mitochondrial respiration than CON myoblasts. However, oligomycin-sensitive phosphorylating respiration was depressed by ∼60% while leak respiration was increased 5-fold compared to CON, demonstrating significant mitochondrial uncoupling. Since inducible uncoupling is linked to electron slip at Complexes I and III and consequently ROS production, we next investigated mitochondrial O₂⁻content which was ∼30% higher in DMD myoblasts. This corresponded with a 2-fold higher non-mitochondrial O₂ consumption. Metabolic stimulation actually enhanced these dysfunctions. Perhaps to overcome the reduced capacity for ATP production, the mitochondrial density of DMD myoblasts was ∼20% higher than CON myoblasts albeit viability was ∼40% lower.

Conclusions: Our data highlight significant mitochondrial dysfunction of DMD myoblasts whereby metabolic substrates are oxidised to generate ROS rather than ATP. Targeting these features of the mitochondria could be therapeutically beneficial for the treatment of DMD patients.

Rybalka E, Timpani CA, Cooke MB, Williams AD & Hayes A. (2014) Defects in mitochondrial ATP synthesis in dystrophin-deficient Mdx skeletal muscles may be caused by complex I insufficiency. PLoS ONE, 9(12):e115763. doi: 0.1371/journal.pone.0115763.

Timpani CA, Hayes A & Rybalka E. (2015) Revisiting the dystrophin-ATP connection: How half a century of research still implicates mitochondrial dysfunction in Duchenne Muscular Dystrophy aetiology. Med Hypoth 5(6): 1021-33.

Timpani CA, Trewin AJ, Stojanovska V, Robinson AM, Goodman CA, Nurgali K, Betik A, Stepto N, Hayes A, McConell GM & Rybalka E. (2016) Attempting to compensate for reduced nNOS protein with nitrate supplementation cannot overcome metabolic dysfunction but rather has detrimental effects in dystrophin-deficient mdx muscle. Neurotherapeutics 14(2): 429-446.