Cellular energetics and mitochondrial uncoupling in canine aging

Abstract

The first domesticated companion animal, the dog, is currently represented by over 190 unique breeds. Across these numerous breeds, dogs have exceptional variation in lifespan (inversely correlated with body size), presenting an opportunity to discover longevity-determining traits. We performed a genome-wide association study on 4169 canines representing 110 breeds and identified novel candidate regulators of longevity. Interestingly, known functions within the identified genes included control of coat phenotypes such as hair length, as well as mitochondrial properties, suggesting that thermoregulation and mitochondrial bioenergetics play a role in lifespan variation. Using primary dermal fibroblasts, we investigated mitochondrial properties of short-lived (large) and long-lived (small) dog breeds. We found that cells from long-lived breeds have more uncoupled mitochondria, less electron escape, greater respiration, and capacity for respiration. Moreover, our data suggest that long-lived breeds have higher rates of catabolism and β-oxidation, likely to meet elevated respiration and electron demand of their uncoupled mitochondria. Conversely, cells of short-lived (large) breeds may accumulate amino acids and fatty acid derivatives, which are likely used for biosynthesis and growth. We hypothesize that the uncoupled metabolic profile of long-lived breeds likely stems from their smaller size, reduced volume-to-surface area ratio, and therefore a greater need for thermogenesis. The uncoupled energetics of long-lived breeds lowers reactive oxygen species levels, promotes cellular stress tolerance, and may even prevent stiffening of the actin cytoskeleton. We propose that these cellular characteristics delay tissue dysfunction, disease, and death in long-lived dog breeds, contributing to canine aging diversity.

Keywords: Aging; Dogs; GWAS; Mitochondria; Primary cells; Uncoupling.

Fig. 1

Thermoregulatory and mitochondrial genes associated with canine breed longevity. a Scatter plot of breeds registered with the American Kennel Club plotted average lifespan by average weight. Cells from breeds used in this study are highlighted in blue (long-lived) or orange (short-lived). b Manhattan (blue and red lines correspond to p value cutoffs of 10⁻⁵ and 10⁻⁸) and (c) quantile-quantile plots from the GWAS of breed-average life expectancy (n = 4169 individuals, 110 breeds, see Table 1). d FGF5 and RSPO2 govern variation in coat length and phenotype; this plot shows the relative distribution of coat length versus increasing breed weight (see methods). Note that the smaller the breed, the more likely it is to have longer hair and vice versa

Fig. 2

Primary canine fibroblasts retain breed-specific bioenergetic-metabolite profiles in vitro. Multiple donors were utilized from: Chihuahuas, Chihuahua-mixed, Labrador Retrievers, and Boxers for targeted mass spectrometry of organic acids, amino acids, and acylcarnitines. Raw metabolite values were normalized to protein concentrations and averaged across three replicates for every donor cell line. a Unbiased hierarchical clustering of donor metabolite profiles, note the clustering of donors by breed. b Principal component analysis of acylcarnitine metabolites. Circles denote 90% confidence interval (orange—short-lived breeds, blue—long-lived, dashed green—Labradors). c Magnification of major metabolite branch driving distinctions between breeds (see Supplemental File 2 for raw data, Fibroblast donor information)

Fig. 3

Fibroblasts from long-lived breeds have greater respiration capacity, lower mitochondrial potential, and less electron escape from electron transport chains than short-lived breeds. The Seahorse assay results are from 3 to 5 (dependent on breed) independent experiments. a Plot depicting the Seahorse assay results for metabolic potential with fold change of respiration (y-axis) and glycolysis (x-axis), standard error shown. Each point corresponds to an individual breed, and the size of the point approximately correlates to the average size of the breed. Note the clustering of breeds by longevity on the respiration y-axis. b Box plot representations of the fold change in respiration depicted in a. Note the trend of decreasing respiration fold change with decreasing breed longevity. c Box plots of the relative MitoTracker Green intensity (mitochondrial mass) in primary canine fibroblasts. d Box plots of relative TMRE (mitochondrial membrane potential—ΔΨ intensity). e Representative fluorescent microscopy images of fibroblasts stained with the MitoTracker Green and TMRE as quantified in c and d. Results are pooled from three independent experiments with standard deviations shown. Note the equivalent mitochondrial abundance between breeds but increasing potential with decreasing breed longevity. f Box plots depicting the average and relative levels of ROS in fibroblasts from long- and short-lived breeds, measured by H2DCFDA staining. g Example of H2DCFDA staining in fibroblasts from the Chihuahua and Great Dane. h Electron transport chain efficiency calculated from the ratio of oxygen consumption under maximum ETC flux (measured by the Seahorse) to ROS H2DCFDA intensity. Note short-lived breeds have and produce more ROS relative to oxygen consumed

Fig. 4

Fibroblasts from long-lived breeds have greater stress tolerance and consume more oxygen in low-nutrient conditions. Box plots showing relative survival of fibroblasts after (a) nutrient deprivation and (b) rotenone exposure. c Example of flow cytometry plots (annexin-V by PI staining) used to quantify survival of fibroblasts as depicted in a and b. d Baseline respiration rates measured by the Seahorse under low-nutrient conditions. e Respiration capacity measured by the Seahorse assay under low-nutrient conditions, also shown is the capacity under nutrient-replete conditions. Cells from long-lived donors have elevated baseline and respiration capacity under low-nutrient conditions

Fig. 5

Bioenergetic metabolites differ significantly between the Boxers and Chihuahuas. Boxplots from left to right: Chihuahuas, Chihuahua-mixed, Labradors, and Boxers. Raw metabolite values were normalized to protein concentrations and averaged across three replicates for every donor cell line. Asterisk indicates significant difference, in the respective metabolite, between the Boxers and Chihuahuas. a Glycolysis and citric acid cycle metabolite pathway schematic. b Fold enrichment of significantly different acylcarnitines between the Boxers and Chihuahuas (Labradors had intermediate concentrations for all acylcarnitines, see Supplemental File 2 for raw data and fibroblast donor information)

Fig. 6

Model. We hypothesize that smaller dogs, due to extra demand for thermogenesis, evolved or responded to have more uncoupled mitochondria, which in turn results in lowered electrical potential (due to proton leak) and elevated β-oxidation and catabolism (in order to fuel uncoupled mitochondria). We suggest that the secondary changes associated with these adaptations happen to be beneficial for longevity; these include decreased production of reactive oxygen species, metabolic profiles which are less favorable for tumorigenesis, increased cytoskeletal flexibility, and resistance to stress (affording superior tissue homeostasis and longevity). Conversely, larger breeds might preferentially use energy for biosynthesis to maintain their greater mass. Overall, our data suggest that lifespan variation across canine breeds is partially regulated by innate cellular properties