Diurnal Differences in Human Muscle Isometric Force In Vivo Are Associated with Differential Phosphorylation of Sarcomeric M-Band Proteins.

We investigated whether diurnal differences in muscle force output are associated with the post-translational state of muscle proteins. Ten physically active men (mean ± SD; age 26.7 ± 3.7 y) performed experimental sessions in the morning (08:00 h) and evening (17:00 h), which were counterbalanced in order of administration and separated by at least 72 h. Knee extensor maximal voluntary isometric contraction (MVIC) force and peak rate of force development (RFD) were measured, and samples of vastus lateralis were collected immediately after exercise. MVIC force was greater in the evening (mean difference of 67 N, 10.2%; p < 0.05). Two-dimensional (2D) gel analysis encompassed 122 proteoforms and discovered 6 significant (p < 0.05; false discovery rate [FDR] = 10%) diurnal differences. Phosphopeptide analysis identified 1693 phosphopeptides and detected 140 phosphopeptides from 104 proteins that were more (p < 0.05, FDR = 22%) phosphorylated in the morning. Myomesin 2, muscle creatine kinase, and the C-terminus of titin exhibited the most robust (FDR < 10%) diurnal differences. Exercise in the morning, compared to the evening, coincided with a greater phosphorylation of M-band-associated proteins in human muscle. These protein modifications may alter the M-band structure and disrupt force transmission, thus potentially explaining the lower force output in the morning.

Age- and Activity-Related Differences in the Abundance of Myosin Essential and Regulatory Light Chains in Human Muscle.

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM). We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping.