Authors
  • Kocha, Katrinka M.
  • Genge, Christine Elizabeth
  • Moyes, Christopher D.

Summary

Do the transcriptional mechanisms that control an individual's mitochondrial content, PGC1α (peroxisome proliferator-activated receptor γ coactivator-1α) and NRF1 (nuclear respiratory factor-1), also cause differences between species? We explored the determinants of cytochrome c oxidase (COX) activities in muscles from 12 rodents differing 1,000-fold in mass. Hindlimb muscles differed in scaling patterns from isometric (soleus, gastrocnemius) to allometric (tibialis anterior, scaling coefficient = −0.16). Consideration of myonuclear domain reduced the differences within species, but interspecies differences remained. For tibialis anterior, there was no significant scaling relationship in mRNA/g for COX4-1PGC1α, or NRF1, yet COX4-1 mRNA/g was a good predictor of COX activity (r2 = 0.55), PGC1α and NRF1 mRNA correlated with each other (r2 = 0.42), and both could predict COX4-1 mRNA (r2 = 0.48 and 0.52) and COX activity (r2 = 0.55 and 0.49). This paradox was resolved by multivariate analysis, which explained 90% of interspecies variation, about equally partitioned between mass effects and PGC1α (or NRF1) mRNA levels, independent of mass. To explore the determinants of PGC1α mRNA, we analyzed 52 mammalian PGC1α proximal promoters and found no size dependence in regulatory element distribution. Likewise, the activity of PGC1αpromoter reporter genes from 30 mammals showed no significant relationship with body mass. Collectively, these studies suggest that not all muscles scale equivalently, but for those that show allometric scaling, transcriptional regulation of the master regulators, PGC1α and NRF1, does not account for scaling patterns, though it does contribute to interspecies differences in COX activities independent of mass.

Methodology

Tissue Samples

In addition to published sequences, we used tissues from various sources to generate DNA sequence, promoter constructs, enzyme measurements, and RNA analyses (Table 1). All collections from live animals were made in accordance with an approved Queen's University Animal Care Committee protocol.

Enzyme and RNA analyses

Animals used for tissue enzyme and RNA analyses were derived from both lab and field collections. Charles River Canada supplied lab mice (Mus musculus CD-1 males), gerbils (Meriones unguiculatus), and hamsters (Mesocricetus auratus). Rats (Rattus norvegicus Sprague-Dawley) were sentinel males from the animal care centre at Queen's University. These samples were euthanized using pentobarbital 150 mg/kg. Female guinea pigs (Cavia porcellus) were control animals used in the Reynolds and Brien lab at Queen's University and were anaesthetized and decapitated. Dwarf hamsters (Phodopus campbelli) were retired from the Wynne-Edwards colony at Queen's University and were killed by cervical dislocation. Groundhogs (Marmota monax) muscles were collected on our behalf by Cornell University staff from their colony.

Wild rodents were trapped using Sherman box traps (North American deer mice, Peromyscus maniculatus; eastern chipmunks, Tamias striatus) or Havahart traps (eastern grey squirrels, Sciurus carolinensis; American red squirrels, Tamiasciurus hudsonicus). Animals were euthanized using 150 mg/kg pentobarbital after being removed from the trap. Beaver (Castor canadensis) samples were contributed by a local trapper as part of a pest removal, killed by rifle.

Location