Location of cMyBP-C in cardiac muscle
Previous attempts to label heart muscle with antibody to MyBP-C have been thwarted by the difficulty in getting the antibodies into the cells in sufficient concentration to be clearly seen in electron micrographs. The thick basal lamina inhibits the entry of the antibody. We circumvented this problem by using freshly prepared adult rat cardiomyocytes whose basal lamina is at least partly digested during preparation. In addition, a polyclonal antibody against cardiac MyBP-C17
with a very strong affinity was used and the labelling was enhanced with a secondary antibody to the first. shows electron micrographs of thin sections of adult rat cardiomyocytes labelled in this way; a shows a low-magnification overview of the labelling pattern in each half A-band. The enlarged sarcomeres in b show that the labelling occurs over nine stripes. The positions of these stripes correspond to those of the nine stripes (stripes 3 to 11) seen in rabbit soleus muscle12
(discussed later in relation to ).
Fig. 2 Determining the precise location of cMyBP-C in cardiac muscle. (a and b) Electron micrographs of thin sections of rat cardiomyocytes immunolabelled with antibody to cMyBP-C. (a) An overview of the labelling pattern in each half A-band. (b) The labelling (more ...)
Fig. 4 Comparison of mean profile plots in muscle; the left panel shows an example A-band electron micrograph used to calculate the average profile plots in the right panel. The profile plots are precisely lined up at the centre of the M-line and the edge of (more ...)
Procedure for averaging A-band profile plots
The axial distribution of MyBP-C and its relation to the myosin head array can be analyzed by averaging profile plots of the A-band. illustrates the procedure used for averaging profile plots of the A-band. a shows an electron micrograph of a negative-stained cryosection of rat papillary muscle. The image shows clear M-line stripes and cross bridge bands. Several half A-band regions like the one boxed were selected; the profile plots were calculated and aligned by cross-correlation and the mean profile plot was calculated (b). After averaging, half A-band profile plots typically have well-defined symmetrical M-band profiles comprising three or five strong peaks and a marked end of the A-band (both indicated with a green line). The profile plot comprises distinct regular peaks in the cross bridge region. Starting from the edge of the bare zone, we can draw 11 equally spaced lines that coincide with the major peaks. The peaks correspond to the major stripes seen in the micrographs. They are labelled from 1 to 11 as in previous studies.12
We identify the range from stripes 3 to 11 as the cardiac C-zone. Hence, we refer to the remainder of the A-band, between stripes 1 and 3, as the P-zone (proximal) and the region distal from stripe 11 to the edge of the A-band as the D-zone (see legend). Continuing the equally spaced lines distally in the D-zone takes us to line 17 just before the edge of the A-band. There are no obvious 43-nm features in this region. Electron microscopy studies of striated muscle are greatly aided by the fact that X-ray diffraction of live muscle gives a measure of the cross bridge periodicity present (43 nm).6
Using this value for internal calibration overcomes the problems of sample shrinkage due to fixation, dehydration and embedding and electron microscope radiation.19
Hence, the periodicity of the stripes is assumed to be 43 nm. In practice, we used the third order of the 43-nm reflection in the Fourier transform (see later), at 14.3 nm, for internal calibration, as it is much clearer.
Relaxed muscle samples were used in this study and it is important to note that MyBP-C stripes, which are quite labile, are only clearly seen in this state (authors' unpublished observations). Well-preserved samples show two sub-bands of ~ 14.3-nm periodicity within each 43-nm period. This is especially clear in the frog profile plot in c. We have identified these sub-bands as myosin head “crowns” by electron tomography of this sample (Luther et al., manuscript in preparation).
The average profile plot is ideal for measuring the length of the thick filament, a value not well known for cardiac muscle. In this study, we measure the length from the profile plot starting from the centre of the M-band to the A-band edge (half peak height). Our measurements show that the average A-band length in cardiac muscle [rat and wild type (wt) mouse] is 1.585 ± 0.011 μm.
Analysis of A-bands: skeletal, cardiac and cMyBP-C-ko
The main results of this study are shown in , with redrawn as d. Electron micrographs of the different samples are shown in the left panel. They are arranged so that the A-band sizes approximately match in each image. Each image in this panel was used along with others of the same sample to produce the average plot profiles shown in the right panel.
The plot profiles in the right panel are arranged to precisely line up the centre of the M-bands and the edges of the A-band as shown by the two continuous green lines drawn through the whole panel. Intervals of 43 nm, numbered 1 to 17, are drawn for each profile plot and they run continuously over the whole panel.
a shows an electron micrograph of rabbit psoas muscle (a fast muscle) labelled with a polyclonal antibody against fast MyBP-C. This result was first shown by Bennett et al.
here we are re-evaluating the pattern in relation to the new data in this figure. The electron micrograph shows enhanced labelling over seven stripes from stripe 5 to 11 and hence identifies the C-zone in this muscle. Bennett et al.
showed that slow muscle has a wider C-zone spanning nine stripes from 3 to 11.
b shows the analysis for anti-cMyBP-C-labelled cardiac muscle from isolated rat cardiomyocytes. cMyBP-C is located at nine positions, from stripe 3 to 11. The positions of the outer seven labelled peaks match the positions of the peaks in the rabbit psoas (fast skeletal) muscle in (a). In b, the labelling at stripe 4 is located a little (~ 6 nm towards the Z-line) off the 43-nm banding pattern for all the other stripes. We have frequently observed weaker density and slightly variable location at stripe 4 in unlabelled cardiac and skeletal muscles.
c shows the plot profile for fast skeletal muscle (frog sartorius). The plot is particularly clear, as this sample had the best preparation technique in this study (fast freezing and freeze substitution). The antibody labelling in (a) identifies the C-zone between stripes 5 and 11. Of special note here is that the native stripes in this muscle match precisely with the anti-MyBP-C peaks in a. This is an important result, as it is consistent with the conclusion that most of the MyBP-C molecule is located at the native 43-nm stripes. Between each pair of the 43-nm stripes in the C-zone are two minor peaks. We show elsewhere that these two minor peaks are due to the myosin cross bridge crowns, which we label crowns 2 and 3, with crown 1 being located at the 43-nm stripe (Luther et al., unpublished data). The A-band length in this muscle is 1.578 ± 0.007 μm.
The analysis for rat cardiac muscle cryosections is shown in d. The native peaks in this muscle up to stripe 11 match precisely the peaks in skeletal muscle (c). With the match of the M-line centre and A-band edge, this shows that cardiac and skeletal muscles have similar structure and organisation at this level. As in skeletal muscle, the 43-nm stripes coincide with the antibody-labelled positions for cMyBP-C (b), showing further similarity between skeletal and cardiac muscle.
An electron micrograph of a cryosection of mouse wt papillary muscle is shown in e. This figure has lower contrast overall and lower density in the D-zone, probably due to the staining method used (ammonium molybdate). However, when averaged over a large area, the C-zone shows peaks at locations that match the rat cardiac sample.
In f, we show the analysis for cryosections of mouse cardiac MyBP-C-ko muscle. Of the main stripes in the profile plot, only the first and second 43-nm stripes have strong peaks. In the C-zone, the major stripe values are greatly diminished and this occurs for all of the peaks 3 to 11. Hence, the densities at the positions identified as cMyBP-C location by immunolabelling (b), viz., stripes 3 to 11, are greatly reduced in MyBP-C-deficient mice.
The sub-banding attributed to myosin crowns is also seen in the rodent cardiac profiles. In c–f, C1, C2 and C3 identify examples of three peaks within a 43-nm period. It is important to note that C1 is much stronger than C2 and C3 in frog skeletal muscles and in the cardiac muscles. However, in the knockout, C1 is much weaker than C2 and C3, suggesting that the heads at this crown may be considerably disordered.
The mean length of the A-band in both cryosections and plastic sections (data not shown) in mouse wt cardiac and mouse cMyBP-C-ko muscle was found to be 1.584 ± 0.011 and 1.574 ± 0.008 μm, respectively. Hence, the A-band length is not affected by the ablation of cMyBP-C.
The Fourier transform meridian in normal and MyBP-C-ko muscle: analysis of “forbidden meridionals”
One-dimensional Fourier transforms of the mean profile plots of half A-bands (excluding M-band and starting at stripe 1 and ending just before stripe 17) are shown in . The purpose of this figure is to illustrate the so-called forbidden meridional reflections, at the first and second orders of 43 nm− 1 that occur in the X-ray diffraction patterns of muscle. They could arise from periodic deviations of the myosin heads from the axial repeat of 14.3 nm, from the contribution of MyBP-C or a combination of the two. The Fourier transforms for relaxed skeletal, rat and mouse wt cardiac muscles are shown in a, b and c, respectively. In these transforms, the reflections corresponding to 43 and 21.5 nm occur prominently as they do along the meridian in the X-ray patterns. The Fourier transforms of the cMyBP-C-ko muscle cryosection in d is dramatically different. The 21.5-nm reflection is completely absent and the 43-nm peak is greatly diminished, indicating that MyBP-C makes a major contribution to these reflections.
Fig. 5 Comparison of the Fourier transforms of the cross bridge regions from the mean profile plots in . The transform plots correspond to the meridional intensities in X-ray patterns; however, as these transforms are derived from half A-bands, they are (more ...)
Also seen clearly in some of these transforms is the peak corresponding to 38.5 nm due to troponin on the thin filaments. This reflection is seen in both the mouse transforms (c and d) and suggests that the sarcomere length in all the areas used for analysis were rather similar so that the troponin repeat was also reinforced. On the other hand, this reflection is not seen in the frog transform (a) and only weakly in that from the rat (b). This can be explained if the sarcomere length varied in the areas used for analysis and the troponin repeat is therefore averaged out.
Crown positions in cardiac muscle
The presence in the Fourier transforms from the mouse cardiac samples of a strong reflection corresponding to 38.5 nm arising from the troponin repeat on the thin filaments suggests that the troponin mass may interfere with the interpretation of the myosin head positions especially in the MBP-C knock out. To decrease the contribution of the troponin stripes, we have averaged the different 43-nm repeats in the C-region. Since this repeat is ~ 4–5 nm longer than that of troponin, the troponin contribution will occur at different positions within each repeat and thence over several repeats will be averaged out. The positions of the nine MyBP-C stripes, 3–11, were determined and the eight repeats within that distance averaged. The centre of the sixth and seventh stripes, identified by Zoghbi et al.3
to be at 288 and 331 nm, are within a pixel of the start of the repeat defined here. The plots in show one averaged repeat from essentially the centre of the MyBP-C peaks for frog and the three cardiac samples, rat and mouse wt and the ko mouse. The data from the frog are plotted displaced from the other three for clarity. All four plots show three peaks indicative of the three crowns of myosin heads. Rat and wt mouse plots are very similar, with the major peak at the origin in the MyBP-C position. The ko has three peaks in positions similar to that of the wt but the peak at the origin is very much reduced, confirming that the loss of stripe density corresponds to the absence of MyBP-C. It is also much weaker than the other two, further suggesting more disorder in this crown.
Fig. 6 The axial densities shown in were averaged over the MyBP-C repeats. The densities at the equivalent positions in each repeat were added together and divided by the number of repeats. For the heart muscle, this was eight 43-nm repeats from stripe (more ...)
Averaging the repeats does not change the conclusions reached by looking at the raw density plots, suggesting that the troponin mass does not grossly affect the density distribution in general, but it does reinforce the conclusions reached, especially in the case of the ko where the density pattern of some of the repeats was more variable. In particular, one crown of myosin heads is hidden under the mass of the MyBP-C. Using the convention of Al-Khayat et al.
we label the three crowns of myosin heads 1, 2 and 3 from the M-line to the Z-line. Crown 1 is at the level of the MyBP-C stripe. Between the stripes there are two levels of heads. The one on the left, crown 2, is also partly subsumed in the MyBP-C mass, while crown 3 is a clear peak. MyBP-C seems to be associated axially with the space between crown 1 and 2 in the cardiac muscle. In frog, there is a similar arrangement, although the mass is more narrowly confined to crown 1.
The position of the myosin heads in the wt A-band cannot be measured with any certainty, since the presence of MyBP-C distorts their position. The position of the three individual peaks, seen in the plot from the ko, allows us to estimate the position and axial separation of the myosin heads. Using a crude measure of the positions of the peak density, we find that the separation between crowns differs. Crowns 2 and 3 are closer together (peak separation ~ 12 nm) than 1 and 3 (~ 13 nm), while 2 and 1 are the furthest separated (~ 18 nm).