Cryo-electron tomography of cardiac myofibrils reveals a contraction-induced lattice twist in the Z-discs

The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has been elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actinactinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin showed a contraction-induced swing motion along with a global twist in the actin lattice. Our observation suggests that the elasticity and the integrity of the Z-disc during the muscle contraction cycle are maintained by the structural flexibility within the actin-actinin complex.


Introduction 28
The Z-disc defines the boundary between the two adjacent sarcomeres by crosslinking the 29 two anti-parallel thin myofilaments. The Z-discs transmit tension generated by muscle 30 contraction through the crosslinks mainly composed of a-actinin 1, 2 . The a-actinin belongs 31 to the spectrin family and forms a rod-shaped antiparallel homodimer of length ~35 nm. 32 The a-actinin monomer has the N-terminal actin-binding domain, which is composed of 33 two calponin homology (CH) domains; the central rod domain, which is composed of four 34 spectrin-like repeats; and the C-terminal tandem EF-hand domains, which are insensitive to 35 calcium in the muscle-type isoforms 3-5 . 36

37
The structure of vertebrate Z-disc has been intensively studied using conventional ultra-thin 38 section electron microscopy for decades [6][7][8][9][10][11] . In the transverse sections, the Z-discs show 39 two distinct structural states: the "small square" form and the "basket-weave" form. In the 40 classical studies of fixed muscle tissues, it has been proposed that the small square and the 41 basket-weave forms represent the relaxed and the active contracted states, respectively 7, 11, 42 4 structure of the Z-discs. In this study, therefore, we performed cryo-electron tomography of 48 isolated myofibrils in the presence of either EGTA+ATP or calcium+ATP (Ca+ATP). Under 49 the ion-controlled conditions, we visualized the 3D structural changes within the Z-discs. 50 51

Results 52
Thin-section microscopy of cardiac myofibrils 53 Before conducting the cryo-electron microscopy, we observed the isolated myofibrils using 54 conventional ultra-thin section electron microscopy because the structure of the Z-disc in 55 the Ca+ATP-activated state has not been reported. Although the Z-discs in the EGTA+ATP 56 (relaxed) state showed a square lattice as expected ( Fig. 1A and C), the Z-discs in the 57 Ca+ATP state exhibited a diamond-shaped lattice with inter-axial angles of 80° and 100° 58 ( Fig. 1B and D). When we carefully examined the previous studies, however, the reported 59 "small square" lattice is not always square and diamond-shaped "offset-square" lattices 60 have often been reported in mammalian Z-discs 8,9,15 . Thus, it is possible that the Z-discs 61 under the Ca+ATP condition are in a fully-activated state, which takes a diamond-shaped 62 lattice rather than a square lattice. We acquired tomograms of ultra-thin sections of the 63 myofibrils and used the averaged subtomograms for the initial references in the subsequent 64 cryo-electron tomography. 65 66

Cryo-electron tomography of native cardiac myofibrils 67
Cryo-electron tomography of myofibrils has been challenging due their large sizes 68 (diameter of ~2 µm). However, we happened to notice that cardiac myofibrils are often 69 branched into thin (300~500 nm) sub-fibrils when intervened by mitochondria or thick 70 collagen bundles 16 . We purified these "thin" myofibrils by gentle homogenization and with a ~13° turn about its long axis. The conformation of the a-actinin in the EGTA+ATP 112 state was similar to the proposed model of the basket-weave form 8,14,18 . Although it is 113 difficult to compare the previously reported 3D structure of the "small square" Z-disc 21 114 with our Ca+ATP map due to the limited resolution of the previous thin-section tomogram, 115 the straightened conformation of the a-actinin in the top view is consistently observed in 116 both observations. These motions of the a-actinin caused sliding of the actin-binding 117 domain along the surface of the F-actin (Fig. 5C, right). Together with the first eigenvector 118 motions (Fig. 4B), it is likely that the interface between the F-actin and the actin-binding 119 domain of the a-actinin is not strictly defined, which is similar to the F-actin-tropomyosin 120 association 22-24 . 121 122

Discussion 123
Comparison with the previous reports of the Z-disc structure 124 In this study, we reported the first native 3D structure of the mammalian Z-disc. The 3D 125 structure of the native invertebrate Z-disc has previously been reported 25 , but the 126 invertebrate Z-discs are separated from the actomyosin system under a harsh condition 127 (extraction using 0.6 M potassium iodide). Moreover, the reconstructed map severely 128 suffers from the missing wedge of information because the isolated invertebrate Z-discs are 129 uniformly oriented perpendicular to the beam axis. Thus, our observations were 130 physiologically more relevant for analyzing the conformational changes of the Z-disc in the 131 context of active myofibrils. 132 Although it has been accepted that the Z-disc takes a square lattice conformation 133 irrespective of whether it is in the small square or in the basket-weave form, diamond-134 shaped lattices were observed in unstimulated rat skeletal muscle (inter-axial angles of 135 80°/100°) 26 , in dog cardiac muscles (inter-axial angles of 82°/98°) 8 , and in the nemaline 136 rods of human myopathy patients (inter-axial angles of 75°/105°) 9 . As the thin and thick 137 myofilaments in the A-band constitute a hexagonal lattice 7 , it is possible that tension 138 applied by the actomyosin contraction deforms the square lattice of the Z-discs into the 139 diamond-shaped lattice. 140 It has been demonstrated that tetanized muscle tissue induced by high-frequency electrical likely to be highly dynamic, and the other molecules such as titin or nebulin/nebulette may

Symmetry mismatch between the square lattice and the F-actin helix 163
The one-start helix of F-actin has a helical parameter of 27.5 Å-rise and 166.6°-rotation, 164 giving an approximately 90°-rotation per seven actin monomers 12, 14 . Although this innate 165 90°-rotation within the helical symmetry of the F-actin matches the square lattice model 166 ( Fig. 6, asterisks), there are large angular discrepancies at other positions. We suppose that 167 these discrepancies between the helical symmetry and the lattice angles underlies the 168 existence of the two lattice forms in the Z-disc. The one-start helix of F-actin can be 169 considered as a two-start helix with 55 Å-rise and -26.8°-rotation. If we focus on one strand 170 of this two-start long-pitch helix, two a-actinin dimers bind to the F-actin every seven actin 171 monomers (Fig. 6, red/blue) 14 . As seven is an odd number, the distance between the two 172 longitudinally-neighboring a-actinin dimers was not constant and there are two patterns; 173 165 Å-rise and -80.4°-rotation, and 220 Å-rise and 107.2°-rotation (Fig. 6, stars). We 174 believe this is another innate feature of the helical symmetry of F-actin, which gives rise to 175 the diamond-shaped lattice with 80°/100° rotation angles in the Ca+ATP state. 176 The discrepancy between the helical symmetry of the F-actin and the lattice symmetry 177 cannot be fully resolved in neither of the basket-weave and the diamond-shaped forms. The 178 observed high flexibility between the F-actin and the actin-binding domain of the a-actinin 179 ( Fig. 5C, right) could compensate the angular mismatches between the helical and the 180 lattice angles. Although it is counter-intuitive that the stout structure of the Z-disc is 181 maintained by such flexible interactions, the structural adjustability of the a-actinin is thought to be essential for the muscle tissue to withstand the high mechanical stress during 183 the contraction cycles. The pellet of large myofibrils was re-suspended in HK buffer and treated either with 2 mM 204 fibrils were then fixed with 1% glutaraldehyde for 1 hour at 4℃ and stained with 1% 206 osmium tetroxide and subsequently with 1% uranium acetate. After dehydration in ethanol 207 and acetone, the samples were embedded in Quetol 812 resin (Nissin EM, Tokyo, Japan). 208 Ultrathin sections (60-nm or 200-nm thick) were cut using a ULTRACUT microtome 209 Gauting, Germany). The nominal magnification was set to 15,000× with a physical pixel 214 size of 8.6 Å/pixel. Tilt series images were recorded using EM-TOOLs program (TVIPS). 215 The angular range of the tilt series was from -60° to 60° with 2.0° increments and the target 216 defocus was set to 2 µm. Back-projection and subtomogram averaging were conducted as 217 described below in the cryo-EM section. For the subtomogram averaging of the thin-section 218 tomograms, one of the subtomograms was used for the initial reference. Movies were subjected to beam-induced motion correction using MotionCor2 33 , and tilt 244 series images were aligned, CTF corrected, and back-projected to reconstruct 3D tomograms using the IMOD software package 34 . Tomograms were 2×binned (pixel size of 246 5.34 Å) to reduce the loads of the calculation. Cross-sections of the Z-discs were displayed 247 using the slicer option of 3dmod and the lattice points of the F-actins were manually picked 248 to define the centers of the subtomograms. Volumes with 50×50×50 pixel-dimensions were 249 cut out from 8×binned tomograms and were averaged using the PEET software suite 35 . 250 Averaged subtomograms of thin-section tomography were used for the initial reference, and 251 the alignment was repeated three times for 8×binned, twice for 4×binned, once for 252        Figure S2