Myosin Binding Protein-C Slow Phosphorylation is Altered in Duchenne Dystrophy and Arthrogryposis Myopathy in Fast-Twitch Skeletal Muscles

Abstract

Myosin Binding Protein-C slow (sMyBP-C), encoded by MYBPC1, comprises a family of regulatory proteins of skeletal muscles that are phosphorylated by PKA and PKC. MYBPC1 missense mutations are linked to the development of Distal Arthrogryposis-1 (DA-1). Although structure-function details for this myopathy are evolving, function is undoubtedly driven by sequence variations and post-translational modifications in sMyBP-C. Herein, we examined the phosphorylation profile of sMyBP-C in mouse and human fast-twitch skeletal muscles. We used Flexor Digitorum Brevis (FDB) isolated from young (~2-months old) and old (~14-months old) wild type and mdx mice, and human Abductor Hallucis (AH) and gastrocnemious muscles carrying the DA-1 mutations. Our results indicate both constitutive and differential phosphorylation of sMyBP-C in aged and diseased muscles. We report a 7-35% reduction in the phosphorylation levels of select sites in old wild type and young or old mdx FDB mouse muscles, compared to young wild type tissue. Similarly, we observe a 30-70% decrease in the phosphorylation levels of all PKA and PKC phospho-sites in the DA-1 AH, but not gastrocnemius, muscle. Overall, our studies show that the phosphorylation pattern of sMyBP-C is differentially regulated in response to age and disease, suggesting that phosphorylation plays important roles in these processes.

Figure 1. The NH₂-terminus of sMyBP-C is subjected to phosphorylation.

Schematic representation of the NH₂-terminus of the known human and mouse sMyBP-C variants with the phosphorylation sites highlighted. The Pro/Ala rich and M- motifs are denoted as dark and light grey rectangles, respectively, and the first Ig domain (C1) is shown as a white oval. Colored rectangles indicate short stretches of amino acids that are products of alternatively spliced regions.

Figure 2. Evaluation of the phosphorylation profile of sMyBP-C in young and old wild type and mdx FDB muscles.

(a) Standard SDS-PAGE western blot analysis of protein lysates prepared from young (~2 months) and old (~14 months) wild type and mdx FDB muscles. Lysates were probed with a α-pan-sMyBP-C (top panel) recognizing all sMyBP-C variants, and α-Hsp90 (bottom panel) to ensure equal loading. (a’) Percent expression of total sMyBP-C in young and old wild type and mdx FDB muscles, after normalization to the levels of Hsp90, and relative to the percent expression of sMyBP-C in young wild type tissue, which was set as baseline. Significance was calculated via student’s t-test (p < 0.01). The star (*) symbol denotes significance compared to young wild type. (b) Separation of the same protein lysates used in panel (a) via phosphate affinity SDS-PAGE followed by immunoprobing with the α-pan-sMyBP-C antibody. Colored dots represent immunoreactive bands of low (red and yellow dots), intermediate (bright yellow, dark green, turquoise, dark blue, purple, and brown dots), and high (pink dot) mobility. Four animals (n = 4) were used per group.

Figure 3. Examination of the phosphorylation levels of mSer59, mSer62, mThr84, and mSer204 of sMyBP-C in fast-twitch FDB muscles of young and old wild type and mdx mice.

Western blot analyses were performed using standard SDS-PAGE and lysates prepared from young (~2 months) and old (~14 months) wild type and mdx FDB muscles. Samples were probed with α-sMyBP-C mSer59P (a), α-sMyBP-C mSer62P (b), α-sMyBP-C mThr84P (c), and α-sMyBP-C mSer204P (d) recognizing the four known phospho-sites of sMyBP-C. The levels of each phosphorylated residue, mSer59 (a’), mSer62 (b’), mThr84 (c’), and mSer204 (d’) in the different FDB muscles were calculated as percent expression relative to those in young wild type tissue, as described in Fig. 2. Four animals (n = 4) were used per group. Significance was calculated via student’s t-test (p < 0.01). The symbols, *, #, and @ denote significance compared to young wild type, young mdx, and old wild type, respectively.

Figure 4. Identification of distinct phosphorylation events present in individual sMyBP-C forms in fast-twitch FDB muscles of young and old wild type and mdx mice.

Western blot analysis using phosphate affinity SDS-PAGE blots of protein lysates obtained from young (~2 months) and old (~14 months) wild type and mdx FDB muscles were probed with α-sMyBP-C mSer59P (a), α-sMyBP-C mSer62P (b), α-sMyBP-C mThr84P (c), and α-sMyBP-C mSer204P (d). Colored dots denote low, intermediate, and high mobility sMyBP-C species identified with each antibody, and correspond to those shown in Fig. 2. Four animals (n = 4) were used per group.

Figure 5. Relative abundance and phosphorylation profile of distinct sMyBP-C forms in fast-twitch FDB muscles of young and old wild type and mdx mice.

(a) The relative abundance of each immunoreactive band detected with the α-pan-sMyBP-C antibody following separation of protein lysates by phosphate affinity SDS-PAGE (Fig. 2b) was calculated as percent expression of the total protein content within each sample. Significance is as described in Fig. 2a’. (b) The phosphorylation events detected in each sMyBP-C form across the different FDB muscle groups were identified by comparative evaluation of the phosphate affinity SDS-PAGE immunoblots shown in Fig. 4. Notably, colored dots correspond to sMyBP-C immunoreactive bands with specific electrophoretic mobility, as determined by phosphate affinity SDS-PAGE, and thus may represent different (i.e. bands denoted with purple and pink dots) or the same (bands denoted with red, yellow, bright yellow, dark green, turquoise, dark blue, and white dots) phospho-species among FDB samples.

Figure 6. Examination of the phosphorylation profile of sMyBP-C in Abductor Hallucis (AH) and gastrocnemius muscles from human patients carrying the DA-1 mutations.

(a) Alignment of sMyBP-C NH₂-termini between mouse isoform 3 and human v1. Dark grey, white, and light grey boxes mark the Pro/Ala rich motif, domain C1, and the M-motif, respectively. The peptide sequences that were used for the generation of the four phospho-antibodies are marked with a line; notably, the sequence similarity and identity for each antigenic peptide between mouse and human are 100% and 90% for the Ser59 peptide, 82% and 45% for the Ser62 peptide, 100% and 85% for the Thr84 peptide, and 100% and 100% for the Ser204 peptide, respectively. (b) Standard SDS-PAGE western blot analysis of protein lysates prepared from human biopsies of AH and gastrocnemius muscles carrying the Y856H and W236R DA-1 mutations, respectively, or control muscles; for details on the DA-1 and control muscles please see the Materials and Methods section. Lysates were probed with α-pan-sMyBP-C (top panel) and α-Hsp90 (bottom panel). (b’) Percent expression of total sMyBP-C in DA-1 AH and gastrocnemius muscles after normalization to the levels of Hsp90, and relative to the percent expression of sMyBP-C in matching controls. Significance was calculated via student’s t-test (p < 0.01). The star (*) symbol denotes significance compared to controls. (c) Separation via phosphate affinity SDS-PAGE of the same protein lysates used in panel (b) followed by probing with the α-pan-sMyBP-C antibody. Colored dots represent immunoreactive bands of low (red, yellow, and blue dots), intermediate (light yellow, olive green, light green, turquoise, dark blue, and purple dots), and high (light purple dot) mobility.

Figure 7. Examination of the phosphorylation levels of hSer61, hThr64, hSer85, and hSer206 of sMyBP-C in AH and gastrocnemius muscles from human patients carrying the DA-1 mutations.

Western blot analyses were performed using standard SDS-PAGE of lysates prepared from human biopsies of AH and gastrocnemius muscles carrying the Y856H and W236R DA-1 mutations, respectively, and matched control tissues. Samples were probed with α-sMyBP-C mSer59P (a), α-sMyBP-C mSer62P (b), α-sMyBP-C mThr84P (c), and α-sMyBP-C mSer204P (d) recognizing the four known phospho-sites of sMyBP-C in humans, too. The levels of each phosphorylated residue, hSer61 (a’), hThr64 (b’), hSer85 (c’), and hSer206 (d’) in the DA-1 samples were calculated as percent expression relative to those in matching control tissues, as described in Fig. 6. Significance was calculated via student’s t-test (p < 0.01). The star (*) symbol denotes significance compared to controls.

Figure 8. Identification of distinct phosphorylation events present in individual sMyBP-C forms in AH and gastrocnemius muscles from human patients carrying the DA-1 mutations.

Western blot analysis using phosphate affinity SDS-PAGE blots of protein lysates obtained from human biopsies of AH and gastrocnemius muscles carrying the Y856H and W236R DA-1 mutations, respectively, and control muscles were probed with α-sMyBP-C mSer59P (a), α-sMyBP-C mSer62P (b), α-sMyBP-C mThr84P (c), and α-sMyBP-C mSer204P (d). Colored dots denote low, intermediate, and high mobility sMyBP-C species identified with each antibody, and correspond to those shown in Fig. 6.

Figure 9. Relative abundance and phosphorylation profile of sMyBP-C forms present in AH and gastrocnemius muscles from human patients carrying the DA-1 mutations.

(a) The relative abundance of each immunoreactive band detected with the α-pan-sMyBP-C antibody following separation of protein lysates by phosphate affinity SDS-PAGE (Fig. 6b) was calculated as percent expression of the total protein content within each sample. Significance is as described in Fig. 6a’. (b) The phosphorylation events detected in each sMyBP-C form within the different samples were determined by comparative evaluation of the phosphate affinity SDS-PAGE immunoblots shown in Fig. 8. The colored dots correspond to sMyBP-C immunoreactive bands with specific electrophoretic mobility as determined by phosphate affinity SDS-PAGE, and thus may represent different (i.e. bands denoted with red, yellow, tan, and dark blue dots) or the same (bands denoted with light yellow, olive green, light green, turquoise, purple, and light purple dots) phospho-species across the human samples.