Myosin binding protein-C slow: a multifaceted family of proteins with a complex expression profile in fast and slow twitch skeletal muscles

Abstract

Myosin Binding Protein-C slow (sMyBP-C) comprises a complex family of proteins expressed in slow and fast type skeletal muscles. Similar to its fast and cardiac counterparts, sMyBP-C functions to modulate the formation of actomyosin cross-bridges, and to organize and stabilize sarcomeric A- and M-bands. The slow form of MyBP-C was originally classified as a single protein, however several variants encoded by the single MYBPC1 gene have been recently identified. Alternative splicing of the 5' and 3' ends of the MYBPC1 transcript has led to the differential expression of small unique segments interspersed between common domains. In addition, the NH2-terminus of sMyBP-C undergoes complex phosphorylation. Thus, alternative splicing and phosphorylation appear to regulate the functional activities of sMyBP-C. sMyBP-C proteins are not restricted to slow twitch muscles, but they are abundantly expressed in fast twitch muscles, too. Using bioinformatic tools, we herein perform a systematic comparison of the known human and mouse sMyBP-C variants. In addition, using single fiber westerns and antibodies to a common region of all known sMyBP-C variants, we present a detailed and comprehensive characterization of the expression profile of sMyBP-C proteins in the slow twitch soleus and the fast twitch flexor digitorum brevis (FDB) mouse muscles. Our studies demonstrate for the first time that distinct sMyBP-C variants are co-expressed in the same fiber, and that their expression profile differs among fibers. Given the differential expression of sMyBP-C variants in single fibers, it becomes apparent that each variant or combination thereof may play unique roles in the regulation of actomyosin cross-bridges formation and the stabilization of thick filaments.

Keywords: MYBPC1; single fiber expression; skeletal muscle.

Figure 1

Human and mouse sMyBP-C variants. Domain architecture of human and mouse sMyBP-C variants as listed in NCBI, Ensembl, and Vega illustrating their common domains and unique segments. (A) Exons 1-33 of the human MYBPC1 gene; the 5′ and 3′UTRs are denoted in black. Dark gray boxes correspond to exons encoding the Pro/Ala rich region and the M-motif, while the white and light gray boxes represent exons that encode Ig and FNIII domains, respectively. Exons highlighted by a color are alternatively spliced and encode unique regions of the sMyBP-C variants. Alternative splicing of the sMyBP-C transcript in both humans (B) and mice (C) results in the generation of several variants that share common domains, but also contain unique short sequences in the NH₂-terminus, the M-motif, the C7 domain, and the COOH-terminus.

Figure 2

Alternative splicing scheme of human MYBPC1. MYBPC1 undergoes extensive alternative splicing within the Pro/Ala rich region (A; exons 2–5), M-motif (B; exon 10), domain C7 (C; exon 23), and at the extreme COOH-terminus (D; exons 31–33). Exon coloring corresponds to Figure 1.

Figure 3

Analysis of the expression profile of sMyBP-C variants in slow twitch soleus and fast twitch FDB skeletal myofibers. Endogenous myosin and sMyBP-C proteins were analyzed in single fibers of adult mouse soleus (A) and FDB (C) muscles. The top part of the gels was stained with SYPRO ruby total protein dye to observe the different myosin isoforms (upper panels), while the bottom part of the gels (lower panels) was processed for immunoblotting using antibodies that recognize epitopes in Ig domain C5, which is present in all known slow variants. Total protein content within each lane was evaluated by Ponceau red staining, and found to be similar. We observed thirteen and five possible expression patterns of sMyBP-C variants in the 48 soleus and 41 FDB fibers examined, respectively. Representative lanes from multiple gels and corresponding blots depicting the expression patterns of myosin and sMyBP-C are shown, and separated by a dotted line; the frequency of each sMyBP-C pattern is noted under the corresponding lane. (B–D) Slow variants were grouped according to their predicted molecular weights, and then matched to the appropriate immunoreactive band. The percent (%) expression of each group of slow variants was quantified. (E) The number of variants per fiber was calculated and reported as percent (%) occurrence.

Figure 4

The NH₂-terminus of sMyBP-C is subjected to complex phosphorylation. sMyBP-C is phosphorylated within the Pro/Ala rich region (A) and the M-motif (B). Alternating exons are noted in black and blue and phosphorylation sites are shown in red. Amino acid residues h-thr-64/m-ser-62, h-ser-85/m-ser-83, and m-thr-84 are present in constitutively expressed portions of the Pro/Ala rich region. However, h-ser-61/m-ser-59 is located within a segment that is alternatively spliced and therefore unique to h-v1, h-v2, and the NH₂-terminus of a partially characterized mouse variant (m-partial-v1) matching that of h-v1 and h-v2. Moreover, human h-ser-206/m-ser-204, located in the M-motif is present in all known sMyBP-C variants, with the exception of h-v8 in which the respective exon (exon 10) is skipped.