Effects of selective breeding for voluntary exercise, chronic exercise, and their interaction on muscle attachment site morphology in house mice
Authors: Castro Alberto A. , Karakostis Fotios Alexandros , Copes Lynn E. , McClendon Holland E. , Trivedi Aayushi P. , Schwartz Nicole E. , Garland Theodore 
Published: September 2021

Skeletal muscles attach to bone at their origins and insertions, and the interface where tendon meets bone is termed the attachment site or enthesis. Mechanical stresses at the muscle/tendon‐bone interface are proportional to the surface area of the bony attachment sites, such that a larger attachment site will distribute loads over a wider area. Muscles that are frequently active and/or are of larger size should cause attachment sites to hypertrophy (training effect); however, experimental studies of animals subjected to exercise have provided mixed results. To enhance our ability to detect training effects (a type of phenotypic plasticity), we studied a mouse model in which 4 replicate lines of High Runner (HR) mice have been selectively bred for 57 generations. Selection is based on the average number of wheel revolutions on days 5 & 6 of a 6‐day period of wheel access as young adults (6–8 weeks old). Four additional lines are bred without regard to running and serve as non‐selected controls (C). On average, mice from HR lines voluntarily run ~3 times more than C mice on a daily basis. For this study, we housed 50 females (half HR, half C) with wheels (Active group) and 50 (half HR, half C) without wheels (Sedentary group) for 12 weeks starting at weaning (~3 weeks old). We tested for evolved differences in muscle attachment site surface area between HR and C mice, plastic changes resulting from chronic exercise, and their interaction. We used a precise, highly repeatable method for quantifying the three‐dimensional (3D) surface area of four muscle attachment sites: the humerus deltoid tuberosity (the insertion point for the spinodeltoideus, superficial pectoralis, and acromiodeltoideus), the femoral third trochanter (the insertion point for the quadratus femoris), the femoral lesser trochanter (the insertion point for the iliacus muscle), and the femoral greater trochanter (insertion point for the middle gluteal muscles). In univariate analyses, with body mass as a covariate, mice in the Active group had significantly larger humerus deltoid tuberosities than Sedentary mice, with no significant difference between HR and C mice and no interaction between exercise treatment and linetype. These differences between Active and Sedentary mice were also apparent in the multivariate analyses. Surface areas of the femoral third trochanter, femoral lesser trochanter, and femoral greater trochanter were unaffected by either chronic wheel access or selective breeding. Our results, which used robust measurement protocols and relatively large sample sizes, demonstrate that muscle attachment site morphology can be (but is not always) affected by chronic exercise experienced during ontogeny. However, contrary to previous results for other aspects of long bone morphology, we did not find evidence for evolutionary coadaptation of muscle attachments with voluntary exercise behavior in the HR mice.