Alterations in osteopontin modify muscle size in females in both humans and mice

Abstract

Purpose: An osteopontin (OPN; SPP1) gene promoter polymorphism modifies disease severity in Duchenne muscular dystrophy, and we hypothesized that it might also modify muscle phenotypes in healthy volunteers.

Methods: Gene association studies were carried out for OPN (rs28357094) in the FAMuSS cohort (n = 752; mean ± SD age = 23.7 ± 5.7 yr). The phenotypes studied included muscle size (MRI), strength, and response to supervised resistance training. We also studied 147 young adults that had carried out a bout of eccentric elbow exercise (age = 24.0 ± 5.2 yr). Phenotypes analyzed included strength, soreness, and serum muscle enzymes.

Results: In the FAMuSS cohort, the G allele was associated with 17% increase in baseline upper arm muscle volume only in women (F = 26.32; P = 5.32 × 10), explaining 5% of population variance. In the eccentric damage cohort, weak associations of the G allele were seen in women with both baseline myoglobin and elevated creatine kinase. The sexually dimorphic effects of OPN on muscle were also seen in OPN-null mice. Five of seven muscle groups examined showed smaller size in OPN-null female mice, whereas two were smaller in male mice. The query of OPN gene transcription after experimental muscle damage in mice showed rapid induction within 12 h (100-fold increase from baseline), followed by sustained high-level expression through 16 d of regeneration before falling to back to baseline.

Conclusion: OPN is a sexually dimorphic modifier of muscle size in normal humans and mice and responds to muscle damage. The OPN gene is known to be estrogen responsive, and this may explain the female-specific genotype effects in adult volunteers.

Figure 1. OPN genotype is associated with untrained upper arm muscle size in young adult female volunteers

Young adult (average age 24 yrs) volunteers enrolled in a unilateral upper arm resistance training study were genotyped for the OPN polymorphism, and males and females separately assessed for associations of OPN genotype with baseline and trained muscle strength and size. Upper arm total muscle volume measured by volumetric MRI showed strong association with OPN genotype in females, but not males, where the GT/GG genotype muscle volume was 15–17% larger than the TT genotype carriers. The untrained, dominant arm, showed similar associations before and after the 12 wks training bout, whereas the trained, non-dominant arm, showed association only at baseline. Standard errors are shown.

Figure 2. A single OPN GG homozygote female shows evidence of unusual muscle damage following a challenge of upper arm eccentric activity

Young adults were given a challenge of elbow eccentric contractions, then blood sampled at 4, 7 and 10 days after the bout of eccentric activity. There were two GG homozygote females in the 147 volunteer cohort, and one of these showed extraordinary elevations of serum creatine kinase (CK), reflective of muscle damage, at 7 days post-bout (green data points). The average elevation of CK in females was 500 IU/L at 7 days, whereas the GG female showed 25,000 IU/L. Standard errors are shown.

Figure 3. Muscle weights normalized to body weight in OPN null mice and wild-type controls

Shown is the average and standard error for males and females in the indicated muscle groups (n = 6 per group). OPN null female mice showed significant reduction in normalized muscle weights for five of the seven muscle groups examined, whereas male mice showed significant reduction in two of the 10 groups. Muscle weight to body weight ratio is calculated as (milligram muscle/gram body weight) × 10. * p< 0.05; ** p<0.01

Figure 4. OPN is strongly induced by experimental muscle degeneration and regeneration

Shown is mRNA expression profile data from 54 mice at defined time points during staged degeneration and regeneration. Time 0 is prior to injection, and the days of harvest of muscle are shown through 40 days (X axis). Shown is the fold-change in OPN mRNA expression in each mouse muscle relative to baseline (time 0) (Y axis). OPN shows low baseline expression at time 0, but then is rapidly induced to about 100-times baseline levels by 1 day following experimental muscle damage. High level OPN is sustained throughout the time period of muscle regeneration, falling back to baseline levels after 16 days when regeneration is largely complete. Shown are fold-change relative to averaged baseline (time 0) for each muscle (two per time point; squares), and the average of the two muscles (circles and line).