Zebrafish cardiac muscle thick filaments: isolation technique and three-dimensional structure

Abstract

To understand how mutations in thick filament proteins such as cardiac myosin binding protein-C or titin, cause familial hypertrophic cardiomyopathies, it is important to determine the structure of the cardiac thick filament. Techniques for the genetic manipulation of the zebrafish are well established and it has become a major model for the study of the cardiovascular system. Our goal is to develop zebrafish as an alternative system to the mammalian heart model for the study of the structure of the cardiac thick filaments and the proteins that form it. We have successfully isolated thick filaments from zebrafish cardiac muscle, using a procedure similar to those for mammalian heart, and analyzed their structure by negative-staining and electron microscopy. The isolated filaments appear well ordered with the characteristic 42.9 nm quasi-helical repeat of the myosin heads expected from x-ray diffraction. We have performed single particle image analysis on the collected electron microscopy images for the C-zone region of these filaments and obtained a three-dimensional reconstruction at 3.5 nm resolution. This reconstruction reveals structure similar to the mammalian thick filament, and demonstrates that zebrafish may provide a useful model for the study of the changes in the cardiac thick filament associated with disease processes.

Figure 1

(A) Light micrograph of isolated zebrafish heart showing the atrium, ventricle, and bulbous arteriosus (average weight of 2.75 mg per heart, 0.3 mm long and 0.1 mm width for each heart), and (B) cardiac thick filaments of zebrafish in a low magnification field. (C) A group of thick filaments at a higher magnification showing the frequent binding of actin thin filaments (black arrows). (D) Gallery of selected filament cross-bridge regions at high magnification, showing the highly periodic cross-bridge arrangement (bare zone at bottom).

Figure 2

(A–D) A gallery of Fourier transforms (FFTs) from individual isolated thick filaments and (E) an averaged transform obtained from averaging the transforms of 15 different isolated thick filaments. Note that, in the averaged transform, the periodic information extends to at least the 12th layer line (∼3.5 nm).

Figure 3

Four views of the three-dimensional map showing a length of a full 42.9 nm repeat rotated by 20° around the filament axis. The major projecting densities on Levels 1 and 3 are clearly observed as triangular-shaped motifs (pink and blue) consistent with the Wendt et al. (58) and Alamo et al. (50) off-state configuration of the paired myosin heads. The base of the triangular motif is also observed on Level 2, but the myosin density at this level (yellow) is not as well fit by the Wendt/Alamo motif. The bare zone is toward the bottom in all views. For the full 0–120° rotation, see Fig. S5A in the Supporting Material).

Figure 4

(A) Image of the reconstruction showing the fitting of the Alamo et al. (50) myosin-head atomic model into the three-dimensional map within the length of the full 42.9 nm axial repeat. The boxed area at the bottom of the panel shows the atomic model with the free head (f) and blocked head (b) labeled. The color scheme for free and blocked myosin heads in panels A–C are the same as was used in Wendt et al. (58) and Alamo et al. (50). For the free myosin head: (blue) myosin heavy chain; (magenta) essential light chain; (beige) regulatory light chain. For the blocked myosin head: (green) myosin heavy chain; (orange) essential light chain; (yellow) regulatory light chain. The S2 region of the atomic model was not fitted in panels A–C. (B and C) Illustration of the fitted configuration of the atomic model of the paired myosin heads at the cross-bridge levels seen in panel A. No intermolecular interaction is seen between the pairs of heads on Level 1 with those on Level 2, or the pairs of heads on Levels 2 with those on Level 3. However, there is interaction between the motor domain of the free head of Level 1 with the RLC of the blocked head on Level 3. (D) A view of the reconstruction showing the three densities to the left of the myosin-head densities on Level 1 (red arrows) that could be attributed to cMyBP-C. Bare zone is toward the bottom in panels A–D. (E) Sum of the slices within Levels 2, 3, and 1, all viewed perpendicular to the filament long axis and toward the bare zone showing that the angle going from Level 2 to 3 is 50°, from 3 to 1 is 8°, and from 1 to 2 is 62°, all in the anticlockwise direction such that the total angular displacement is 120°. (Yellow arrow) Density attributed to titin; and (red arrow) extra density observed on Level 1 and attributed to cMyBP-C.