Impaired Autophagosome Formation and Autophagy in Skeletal Muscle of Aged American Quarter Horses
Authors: Li Chengcheng , White Sarah , Warren Lori , Wohlgemuth Stephanie 
Published: November 2023

We have previously reported that mitochondrial function decreased in both gluteus medius (GM) and triceps brachii (TB) muscle from aged American Quarter Horses; and that activity of the mitochondrial enzyme citrate synthase (CS) in the TB was lower in aged compared to young horses, suggesting a muscle‐specific decrease in mitochondrial content with age (Li et al. 2016 doi: 10.1152/japplphysiol.01077.2015). One underlying cause for the age‐associated decrement of mitochondrial content and function could be an impairment of mitochondrial quality control. Mitochondrial biogenesis and selective degradation of damaged mitochondria (through autophagy) are two of the most prominent quality control mechanisms that have been described. A balance between those two processes is necessary to maintain a healthy population of mitochondria. The objective of the present study was to investigate potential underlying mechanisms that could have caused the age‐associated decline in mitochondrial content and function in equine skeletal muscle by analyzing the expression of factors associated with mitochondrial biogenesis and autophagy pathways. Muscle biopsies of the GM and TB were collected from young (1.8±0.1 yr; n=24) and aged (20±5 yr; n=12) American Quarter Horses, and gene and protein expressions analyzed by qRT‐PCR and immunoblotting. Statistical differences between ages and muscle groups were analyzed using Two‐Way ANOVA and Holm‐Sidak post hoc analysis (Sigmaplot vs. 12.0; Systat Software Inc., San Jose, CA). Consistent with its activity, we found that protein expression of CS was decreased in aged TB (P<0.05), but not in aged GM. Moreover, mtDNA copy number was decreased with age in TB (P<0.05) but not in GM, which further confirmed the age‐related decline in mitochondrial content indicated by CS protein expression and enzymatic activity. The mRNA level of PGC‐1α, a master regulator of mitochondrial biogenesis, was lower in aged compared to young TB (P<0.05). Consistently, the mRNA level of Tfam, which is involved in mitochondrial biogenesis and regulated by PGC‐1α, was decreased exclusively in aged TB (P<0.05). Gene expression of the autophagy protein Microtubule‐Associated Protein Light Chain 3 (LC3) was higher in aged GM (P<0.001), but in either muscle age had no effect on gene expression of the autophagy‐associated Lysosomal Membrane Associated Protein‐2 (LAMP‐2), the transcription factor Nuclear Respiratory Factor‐1 (NRF‐1), and subunits of mitochondrial electron transport complexes ND1, COX1, and COX2. On a protein level, we observed an accumulation of the autophagosomal cargo protein SQSTM/p62 in both muscles (P<0.05 for both), and, concomitantly, a decrease in the autophagy related protein Atg5 (P<0.05) and the autophagosome‐bound form of LC3 (LC3‐II; P<0.05) in TB, suggesting impaired autophagosome formation in aged skeletal muscle. Taken together, our data suggest that mitochondrial biogenesis and autophagic activity were impacted by age in equine skeletal muscle, and that the TB is more susceptible to damage than the GM.