KBTBD13 is an actin-binding protein that modulates muscle kinetics
Josine M. de Winter, et al.
Josine M. de Winter, et al.
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Research Article Muscle biology

KBTBD13 is an actin-binding protein that modulates muscle kinetics

Abstract

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation–induced muscle relaxation, muscle fiber– and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin — a major constituent of the thin filament — and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Authors

Josine M. de Winter, Joery P. Molenaar, Michaela Yuen, Robbert van der Pijl, Shengyi Shen, Stefan Conijn, Martijn van de Locht, Menne Willigenburg, Sylvia J.P. Bogaards, Esmee S.B. van Kleef, Saskia Lassche, Malin Persson, Dilson E. Rassier, Tamar E. Sztal, Avnika A. Ruparelia, Viola Oorschot, Georg Ramm, Thomas E. Hall, Zherui Xiong, Christopher N. Johnson, Frank Li, Balazs Kiss, Noelia Lozano-Vidal, Reinier A. Boon, Manuela Marabita, Leonardo Nogara, Bert Blaauw, Richard J. Rodenburg, Benno Küsters, Jonne Doorduin, Alan H. Beggs, Henk Granzier, Ken Campbell, Weikang Ma, Thomas Irving, Edoardo Malfatti, Norma B. Romero, Robert J. Bryson-Richardson, Baziel G.M. van Engelen, Nicol C. Voermans, Coen A.C. Ottenheijm

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Figure 3

Contractility assays to study relaxation kinetics of muscle fibers and myofibrils.

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Contractility assays to study relaxation kinetics of muscle fibers and m...
(A) EM images from muscle fibers of a control and (B) a NEM6 patient (note the areas with myofibrillar damage, indicated by asterisks). (C) Light microscopy images of a slow-twitch and (D) a fast-twitch NEM6 patient fiber that were used for contractility assays. (E) Maximal tension of both slow-twitch and (F) fast-twitch fibers from NEM6 patients was significantly lower than that of controls. (G) Typical relaxation trace of a NEM6 and a control muscle fiber. (H) The kinetics of relaxation after maximal activation — reflected by the relaxation constant Krel — were markedly slower in both slow-twitch and fast-twitch fibers of NEM6 patients when compared with controls. The orange, green, and yellow circles correspond to NEM6 patients harboring the KBTBD13K390N, KBTBD13E83Q, and KBTBD13I369M mutations, respectively. The white circles correspond to patients with the KBTBD13R408C mutation. (I) EM images indicate a myofibril (highlighted in white) from muscle fibers of control and (J) a NEM6 patient. Note that these EM images are higher magnifications of the same images in parts A and B, respectively. (K) Myofibril mounted between a force probe and length controller for contractility assays. (L) The maximal tension of myofibrils of NEM6 patients was lower than that of controls. (M) Typical example of the activating kinetics and (N) the relaxation kinetics of a NEM6 and a control myofibril; note that activation and relaxation kinetics are from the same myofibril. (O) The relaxation kinetics were slower in myofibrils of NEM6 patients than in those of controls. Student’s t tests or Mann-Whitney U tests were performed between NEM6 and controls. *P < 0.05. For each biopsy, 4 to 12 muscle fibers/myofibrils were measured. For detailed information on the number of samples and statistical tests and outcomes, please see Supplemental Table 1.