Ursolic Acid Induces Beneficial Changes in Skeletal Muscle mRNA Expression and Increases Exercise Participation and Performance in Dogs with Age-Related Muscle Atrophy

Abstract

Muscle atrophy and weakness are prevalent and debilitating conditions in dogs that cannot be reliably prevented or treated by current approaches. In non-canine species, the natural dietary compound ursolic acid inhibits molecular mechanisms of muscle atrophy, leading to improvements in muscle health. To begin to translate ursolic acid to canine health, we developed a novel ursolic acid dietary supplement for dogs and confirmed its safety and tolerability in dogs. We then conducted a randomized, placebo-controlled, proof-of-concept efficacy study in older beagles with age-related muscle atrophy, also known as sarcopenia. Animals received placebo or ursolic acid dietary supplements once a day for 60 days. To assess the study's primary outcome, we biopsied the quadriceps muscle and quantified atrophy-associated mRNA expression. Additionally, to determine whether the molecular effects of ursolic acid might have functional correlates consistent with improvements in muscle health, we assessed secondary outcomes of exercise participation and T-maze performance. Importantly, in canine skeletal muscle, ursolic acid inhibited numerous mRNA expression changes that are known to promote muscle atrophy and weakness. Furthermore, ursolic acid significantly improved exercise participation and T-maze performance. These findings identify ursolic acid as a natural dietary compound that inhibits molecular mechanisms of muscle atrophy and improves functional performance in dogs.

Keywords: activity; cachexia; canine; dietary supplement; dog; exercise; muscle atrophy; sarcopenia; skeletal muscle; ursolic acid.

Figure 1

Ursolic acid inhibits atrophy-associated mRNA expression in canine skeletal muscle. Older beagle dogs with mild to moderate age-related skeletal muscle atrophy were randomized to receive either one placebo soft chew or one ursolic acid soft chew (24 mg ursolic acid/day) once a day for 60 days. Quadriceps muscle biopsies were obtained from each animal immediately prior to dietary supplementation (“Pre”) and immediately after 60 days of dietary supplementation (“Post”). RNA was then isolated from the muscle biopsies and used for quantification of mRNAs encoding the muscle atrophy mediators MuRF1/TRIM63 (A), ZFAND5 (B), 4E-BP1/EIF4EBP1 (C), FOXO4 (D), NCOR1 (E), ACVR2B (F), TRAF6 (G), FNIP1 (H), GCN5/KAT2A (I), UBR4 (J), BNIP3 (K), and MEKK4/MAP3K4 (L). Data are truncated violin plots from 8 placebo-supplemented dogs and 9 ursolic acid-supplemented dogs, with red bars denoting median values, blue bars denoting interquartile ranges, and p-values determined by two-way ANOVA with multiple comparison testing. MuRF1: Muscle-Specific RING Finger Protein 1; TRIM63: Tripartite Motif Containing 63; ZFAND5: Zinc Finger AN1-Type Containing 5; 4E-BP1: 4E-Binding Protein 1; EIF4EBP1: Eukaryotic Translation Initiation Factor 4E Binding Protein 1; FOXO4: Forkhead Box O4; NCOR1: Nuclear Receptor Corepressor 1; ACVR2B: Activin A Receptor Type 2B; TRAF6: TNF Receptor-Associated Factor 6; FNIP1: Folliculin Interacting Protein 1; GCN5: General Control of Amino Acid Synthesis Protein 5; KAT2A: Lysine Acetyltransferase 2A; UBR4: Ubiquitin Protein Ligase E3 Component N-Recognin 4; BNIP3: BCL2 Interacting Protein 3; MEKK4: MAP/ERK Kinase Kinase 4; MAP3K4: Mitogen-Activated Protein Kinase Kinase Kinase 4.

Figure 2

In canine skeletal muscle, ursolic acid decreases mRNAs encoding mediators and biomarkers of atrophy and weakness and increases mRNAs encoding mediators and biomarkers of muscle health and exercise. Older beagle dogs with mild to moderate age-related skeletal muscle atrophy were randomized to receive either one placebo soft chew or one ursolic acid soft chew (24 mg ursolic acid/day) once a day for 60 days. Quadriceps muscle biopsies were obtained from each animal immediately prior to dietary supplementation (“Initial”) and immediately after 60 days of dietary supplementation (“Final”). RNA was then isolated from the muscle biopsies, and log2 fold changes in the indicated mRNA levels were determined in each animal by comparing final mRNA levels to initial baseline mRNA levels in the same animal. Data are from 8 placebo-supplemented dogs and 9 ursolic acid-supplemented dogs. (A,B) Mean log2 fold changes in mRNAs encoding mediators and biomarkers of muscle atrophy and weakness, shown as a heat map (A) and scatter plot (B). (C,D) Mean log2 fold changes in mRNAs encoding mediators and biomarkers of muscle health and exercise, shown as a heat map (C) and scatter plot (D). In (B,D), each data point represents the mean log2 fold change in one mRNA, horizontal lines denote means of all assessed mRNA levels, and p-values were determined by one-way ANOVA with Dunnett’s multiple comparison tests.

Figure 3

Ursolic acid improves exercise participation and T-maze performance in older dogs. Older beagle dogs with mild to moderate age-related skeletal muscle atrophy were randomized to receive either one placebo soft chew or one ursolic acid soft chew (24 mg ursolic acid/day) once a day for 60 days. (A,B) Exercise participation was assessed in each animal before and after 60 days of dietary supplementation with placebo (A) or ursolic acid (B). In this assay, exercise participation is graded on a scale from 0 (no participation) to 3 (enthusiastic participation). (C,D) T-maze performance was assessed in each animal before and after 60 days of dietary supplementation with placebo (C) or ursolic acid (D). In this assay, a lower latency score indicates higher T-maze performance. (A–D) Each data point represents the value from one animal, orange lines denote paired values from the same animal, bars indicate mean values, and p-values were determined by two-tailed Wilcoxon matched-pairs signed rank tests.