Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C((t/t))). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C((t/t)) mice. Slow and fast skeletal muscles in cMyBP-C((t/t)) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C((t/t)) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C

MyBP-C isoforms are differentially expressed in cardiac and skeletal tissue.

<p>Ten µg of total proteins were resolved on 4-15% SDS-PAGE and stained with Coomassie Brilliant Blue (<b>A</b>). Expression levels of cardiac, slow skeletal and fast skeletal MyBP-C in (t/t) and WT mice were determined in heart (h), soleus (s) and EDL (e) muscles by Western blot analysis with respective antibodies (<b>B</b>). Representative Western blot shows that the presence of cardiac isoform of MyBP-C (cMyBP-C) is exclusive to ventricular muscle of WT mice (<b>C</b>), but completely absent in the (t/t) mouse hearts. sMyBP-C was significantly expressed both in the soleus muscle and EDL muscle (<b>D</b>). Conversely, fMyBP-C was mainly detected in EDL muscle (<b>E</b>), but significantly increased in the (t/t) hearts. All values were normalized to the expression of α-sarcomeric actin (MyBP-C/actin ratio) and expressed as relative values. The summarized quantitative data were derived from n=3 with mixed gender mice (*p< 0.01 versus WT). The increased level of fMyBP-C expression in the (t/t) hearts was reconfirmed by Western blot analysis (<b>F</b>) and not found in the WT hearts, summarized in panel <b>G</b> (n=6, *p< 0.0001 versus WT). α-sarcomeric actin was used as a loading control.</p

Ultrastructural and histopathological analyses show similar sarcomeric structure in (t/t) and WT soleus and EDL skeletal muscles.

<p>Phenotype of mouse soleus (slow) and EDL (fast) muscle myofibrils observed by negative staining (<b>A</b>) and thin sectioning (<b>B</b> and <b>C</b>). The structure and organization of sarcomeres in both longitudinal section (B) and transverse section (C) are not disrupted in either soleus or EDL in (t/t) mice. A-band length, M-line width and Z-disc width confirm electron microscopy images, showing no significant change between (<b>t/t</b>) and WT mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069671#tab2" target="_blank">Table 2)</a>. I-band length variations are likely due to different lengths of the muscle during fixation. Scale bars = 200 nm. Representative hematoxylin and eosin (<b>D</b>) and Masson’s trichrome (<b>E</b>) staining images show no detectable changes in myofibrils or fibrosis in the (t/t) soleus muscles, compared to the WT control.</p

Schematic diagram of striated muscle sarcomere and the three main isoforms of MyBP-C.

<p>(<b>A</b>) The bulk of MyBP-C is oriented perpendicularly to the long axis of the myosin filaments and is restricted to the C-zone in the central 1/3 of each half A-band (vertical arrows). Titin (dashed lines) is a giant protein that spans the length of the half-sarcomere. MyBP-C is restricted to the C-zone of the A-band and connects both thick and thin filaments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069671#B1" target="_blank">1</a>]. (<b>B</b>) The three main isoforms of MyBP-C: a cardiac form (cMyBP-C) and two skeletal isoforms, slow and fast (sMyBP-C and fMyBP-C, respectively). MyBP-C isoforms each have a proline–alanine (P/A)-rich region towards the N-terminus, three fibronectin type III domains (hexagons), and seven immunoglobulin domains (circles). The cardiac isoform has an additional immunoglobulin domain, C0, at the N-terminus, phosphorylation motifs in the M domain (red vertical band), and a twenty-eight residue cardiac-specific insert in the C5 immunoglobulin domain (red vertical band). The cardiac and fast skeletal isoforms share a conserved ‘linker’ between the C4 and C5 domains (dark thick band). Distinctions between the different isoforms are highlighted.</p

Analysis of twitch and tetanic tension shows no differences between (t/t) and WT mice.

<p>Skeletal muscles were attached to a micrometer and force transducer to measure twitch and tetanus tension with mechanical stretches of varying velocities (20, 40, and 60, 100 ML/s) for 100 ms duration (see Methods). Injury from stretch was determined from fractional loss of twitch force prior to tetanus versus post-tetanus stimulation. Stiffness of the muscle was determined from change in force under tetanus. An example tracing from (t/t) soleus muscle is shown here. Measurements from functional analyses of (t/t) and WT are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069671#tab3" target="_blank">Table <b>3</b></a>.</p