The Phosphorylation Profile of Myosin Binding Protein-C Slow is Dynamically Regulated in Slow-Twitch Muscles in Health and Disease.

Maegen A Ackermann,Christopher W Ward,Aikaterini Kontrogianni-Konstantopoulos,Brendan King,Jaclyn P Kerr

doi:10.1038/srep12637

Abstract

Myosin Binding Protein-C slow (sMyBP-C) is expressed in skeletal muscles where it plays structural and regulatory roles. The functions of sMyBP-C are modulated through alternative splicing and phosphorylation. Herein, we examined the phosphorylation profile of sMyBP-C in mouse slow-twitch soleus muscle isolated from fatigued or non-fatigued young (2-4-months old) and old (~14-months old) wild type and mdx mice. Our findings are two-fold. First, we identified the phosphorylation events present in individual sMyBP-C variants at different states. Secondly, we quantified the relative abundance of each phosphorylation event, and of sMyBP-C phospho-species as a function of age and dystrophy, in the presence or absence of fatigue. Our results revealed both constitutive and differential phosphorylation of sMyBP-C. Moreover, we noted a 10–40% and a 25–35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx soleus muscles, respectively. On the contrary, we observed a 5–10% and a 20–25% increase in the phosphorylation levels of specific sites in young fatigued wild type and mdx soleus muscles, respectively. Overall, our studies showed that the phosphorylation pattern of sMyBP-C is differentially regulated following reversible (i.e. fatigue) and non-reversible (i.e. age and disease) (patho)physiological stressors.

Highlights

Myosin Binding Protein-C (MyBP-C) comprises a family of accessory proteins in striated muscles that contributes to the assembly and stabilization of thick filaments and modulates the formation of actomyosin cross-bridges[1,2,3,4,5,6,7,8,9]
We used wild type and phospho-ablated recombinant peptides corresponding to the NH2-terminus of m-isoform[3] (GST-sMyBP-C NH2 aa1-285) as it contains all known phosphorylation sites, including mSer-59 and mSer-204 present only in select variants (Fig. 1)
We used an established model of muscular dystrophy, aging in both the mdx and wild type background, as well as an acute in vitro fatigue protocol to reveal the dynamic range of phosphorylation events in sMyBP-C in slow-twitch muscle

Summary

Introduction

Myosin Binding Protein-C (MyBP-C) comprises a family of accessory proteins in striated muscles that contributes to the assembly and stabilization of thick filaments and modulates the formation of actomyosin cross-bridges[1,2,3,4,5,6,7,8,9]. Fourteen sMyBP-C transcripts have been identified in the human transcriptome, encoding fourteen unique variants, which differ by small segments of amino acids within the Pro/Ala rich motif, the M-motif, Ig domain C7, and the extreme COOH-terminus[10]. The different sMyBP-C variants are co-expressed in variable amounts and combinations in both slow and fast twitch skeletal muscles, and can co-exist within a single myofiber exhibiting distinct topographies and functions[1,2,8]. The overall phosphorylation levels of sMyBP-C remain relatively unchanged between young and old wild type and mdx soleus muscles, we observed qualitative and quantitative differences in individual phosphorylation events or combinations thereof as a result of age, fatigue, and/or disease. Our studies are the first to demonstrate that the phosphorylation pattern of sMyBP-C is differentially regulated in response to age, fatigue, and disease in the slow-twitch soleus muscle

Methods

Results

Conclusion