Skeletal muscle is a plastic tissue with known gender dimorphism, especially at the metabolic level. A proteomic comparison of male and female murine biceps brachii was undertaken, resolving an average of 600 protein spots of MW 15-150 kDa and pI 5-8. Twenty-six unique full-length proteins spanning 11 KOG groups demonstrated statistically significant (p<0.05) abundance differences between genders; the majority of these proteins have metabolic functions. Identified glycolytic enzymes demonstrated decreased abundance in females, while abundance differences in identified oxidative phosphorylation enzymes were specific to the proteins rather than to the functional group as a whole. Certain cytoskeletal and stress proteins showed specific expression differences, and all three phosphorylation states of creatine kinase showed significant decreased abundance in females. Expression differences were significant but many were subtle (≤ 2-fold), and known hormonally-regulated proteins were not identified. We conclude that while gender dimorphism is present in non-exercised murine skeletal muscle, the proteome comparison of male and female biceps brachii in exercise-naïve mice indicates subtle differences rather than a large or obviously hormonal dimorphism.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors 1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.) 2,3 . Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction 4 . The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise 5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle.In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging 6 . This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p < 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS) 5 . This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

Skeletal muscle is a plastic tissue that adapts to exercise in many ways. Differential proteome profiling can be used to obtain a global view of proteins that change expression following a repeat bout of eccentrically‐biased exercise, as well as its gender specificity. Exercise‐naive male and female mice (C57BL/10ScSnJ 000476 +/+) ran downhill twice one week apart (‐15°) on a treadmill at 25 m s−1 for 15 min and 800 ug of total biceps brachii extract was electrophoresed on pH 5‐8 2‐D gels. The resulting spots that changed at least +/‐ 2‐fold and were statistically significantly different (p<0.05) relative to the unexercised controls using PDQuest 8.0 were analyzed by LC/MS‐MS and identified using BioWorks 3.3.1. Significant proteome remodeling occurs subsequent to the repeat bout. The expression change patterns were followed at 0, 24, 48, 72 and 168 hrs post‐repeat bout; significant changes were observed in myofibrillar and cytoskeletal proteins, creatine kinase and many of the glycolytic enzymes. The cellular stress responses were unique to individual heat shock and oxidative stress proteins. More protein spots changed in females (299) than in males (236); the female changes peaked at 24 hr post‐exercise, whereas the male changes did not show much variation over the week following the repeat bout. Supported by NSF 0420971, the Smith College Blakeslee Fund and HHMI.

Skeletal muscle is a plastic tissue with known gender dimorphism, especially at the metabolic level. A proteomic comparison of male (n=5) and female (n=5) murine (C57BL/10ScSnJ000476 +/+) biceps brachii was undertaken, resolving an average of 600 protein spots of MW 15–150 kDa and pI 5–8. 24 unique full‐length proteins spanning 11 KOG groups demonstrated statistically significant (p<.05) abundance differences between genders; the majority of these proteins have metabolic functions. Identified glycolytic enzymes demonstrated decreased abundance in females, while identified oxidative phosphorylation enzyme abundance differences were specific to the proteins rather than to the function group as a whole. Myoglobin and GRP78 showed the largest abundance increases of 3.9 and 5.8‐fold, respectively, whereas dihydrolipoamide S‐succinyltransferase showed the largest decrease of 5.9‐fold in the females. All three phosphorylation states of creatine kinase were decreased in females. Expression differences were significant but many subtle, usually 2‐fold or less. We conclude that while gender dimorphism is present in non‐exercised murine skeletal muscle, the proteome comparison of male and female biceps brachii in exercise‐naïve mice does not indicate any apparent hormonal regulatory basis for the dimorphism. Supported by NSF 0420971, the Smith College Blakeslee Fund and HHMI.

Identifying the array of proteins that change expression following a single bout of eccentrically‐biased exercise in skeletal muscle will shed light on the molecular mechanisms underlying adaptation, as well as their gender specificity. Exercise‐naive male and female mice (C57BL/10ScSnJ 000476 +/+) ran downhill (‐15°) on a treadmill at 25 m s−1 for 15 min and 800 ug of total biceps brachii extract was electrophoresed on pH 5‐8 2‐D gels. The resulting spots that changed at least +/‐ 2‐fold and were significantly different (p<0.05) relative to unexercised controls of the appropriate gender using PDQuest 8.0 were analyzed by LC/MS‐MS and identified using BioWorks 3.3.1. This analysis showed significant proteome remodeling subsequent to downhill running. The expression change patterns were followed at 0, 24, 48, 72 and 168 hrs post‐exercise (PX) and significant changes were observed in 4 NCBI KOG groups. Both myofibrillar and cytoskeletal protein remodelings were noted, as well as decreased expression of creatine kinase. Nearly all of the glycolytic enzymes were up‐regulated. The cellular heat shock protein responses were significant and specific. Roughly the same number of protein spots changed in females and males, but the females peaked at 24 hr PX, whereas in males the peak was at 72 hr PX. Supported by NSF 0420971, the Smith College Blakeslee Fund and HHMI.