Non-Targeted Metabolomics Analysis of Golden Retriever Muscular Dystrophy-Affected Muscles Reveals Alterations in Arginine and Proline Metabolism, and Elevations in Glutamic and Oleic Acid In Vivo

Abstract

Background: Like Duchenne muscular dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, and pseudohypertrophy in specific skeletal muscles. This severe GRMD phenotype includes moderate atrophy of the biceps femoris (BF) as compared to unaffected normal dogs, while the long digital extensor (LDE), which functions to flex the tibiotarsal joint and serves as a digital extensor, undergoes the most pronounced atrophy. A recent microarray analysis of GRMD identified alterations in genes associated with lipid metabolism and energy production. Methods: We, therefore, undertook a non-targeted metabolomics analysis of the milder/earlier stage disease GRMD BF muscle versus the more severe/chronic LDE using GC-MS to identify underlying metabolic defects specific for affected GRMD skeletal muscle. Results: Untargeted metabolomics analysis of moderately-affected GRMD muscle (BF) identified eight significantly altered metabolites, including significantly decreased stearamide (0.23-fold of controls, p = 2.89 × 10−3), carnosine (0.40-fold of controls, p = 1.88 × 10−2), fumaric acid (0.40-fold of controls, p = 7.40 × 10−4), lactamide (0.33-fold of controls, p = 4.84 × 10−2), myoinositol-2-phosphate (0.45-fold of controls, p = 3.66 × 10−2), and significantly increased oleic acid (1.77-fold of controls, p = 9.27 × 10−2), glutamic acid (2.48-fold of controls, p = 2.63 × 10−2), and proline (1.73-fold of controls, p = 3.01 × 10−2). Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88 × 10−4, FDR 4.7 × 10−2), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic acid, fumaric acid, citric/isocitric acid, and succinic acid), suggesting that altered energy metabolism may be underlying the observed GRMD BF muscle dysfunction. In contrast, two pathways, inosine-5′-monophosphate (VIP Score 3.91) and 3-phosphoglyceric acid (VIP Score 3.08) mainly contributed to the LDE signature, with two metabolites (phosphoglyceric acid and inosine-5′-monophosphate) being significantly decreased. When the BF and LDE were compared, the most significant metabolite was phosphoric acid, which was significantly less in the GRMD BF compared to control and GRMD LDE groups. Conclusions: The identification of elevated BF oleic acid (a long-chain fatty acid) is consistent with recent microarray studies identifying altered lipid metabolism genes, while alterations in arginine and proline metabolism are consistent with recent studies identifying elevated L-arginine in DMD patient sera as a biomarker of disease. Together, these studies demonstrate muscle-specific alterations in GRMD-affected muscle, which illustrate previously unidentified metabolic changes.

Metabolomes of mitochondrial diseases and inclusion body myositis patients: treatment targets and biomarkers

“…A recent study on muscle metabolomics of a dog model for Duchenne muscular dystrophy reported arginine and proline metabolism as the top changed pathways (Abdullah et al , ), which is also the top pathway in the blood metabolomes of our NMD patients. These findings suggest that the blood metabolomic responses to muscular dystrophy are conserved in species and that a semiquantitative metabolomics assay—or arginine/proline content of serum—could be useful as a multi‐biomarker for treatment follow‐up in muscle dystrophies.…”

Disease-specific and glucocorticoid-responsive serum biomarkers for Duchenne Muscular Dystrophy

“…Carnosine acts as an antioxidant and reduces free radicals generated from excess lipid and sugar oxidation 36 . Untargeted metabolomic analysis of skeletal muscle revealed lower concentrations of carnosine in a dystrophin deficient canine model compared to healthy controls that might explain the oxidative stress that is associated with DMD 37 . The significance of lower concentrations of circulating CNDP1 in GC-naïve DMD patients that increased following GC treatment at levels exceeding that in the controls is not well understood.…”

Plasma Metabolomic and Lipidomic Profiling of a Genetically Modified Mouse Model of Scavenger Receptor Class B Type I

“…Similarly, Willis and his colleagues reported that altered lipid metabolism genes were relative with arginine and proline metabolism. [22] But, the precise mechanism about the dysfunctional arginine metabolism still remains unknown and deserve to be further studied.…”