A Laing distal myopathy–associated proline substitution in the β-myosin rod perturbs myosin cross-bridging activity
Massimo Buvoli, … , Julien Ochala, Leslie A. Leinwand
Massimo Buvoli, … , Julien Ochala, Leslie A. Leinwand
Published May 1, 2024
Citation Information: J Clin Invest. 2024;134(9):e172599. https://doi.org/10.1172/JCI172599.
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Research Article Muscle biology

A Laing distal myopathy–associated proline substitution in the β-myosin rod perturbs myosin cross-bridging activity

Abstract

Proline substitutions within the coiled-coil rod region of the β-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

Authors

Massimo Buvoli, Genevieve C.K. Wilson, Ada Buvoli, Jack F. Gugel, Abbi Hau, Carsten G. Bönnemann, Carmen Paradas, David M. Ryba, Kathleen C. Woulfe, Lori A. Walker, Tommaso Buvoli, Julien Ochala, Leslie A. Leinwand

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Figure 1

Transgenic mice expressing the myosin mutant R1500P have reduced muscle strength.

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Transgenic mice expressing the myosin mutant R1500P have reduced muscle ...
(A) Pie chart shows the relative percentage of the different myosin isoforms expressed in TA muscles of NTG mice, transgenic mice expressing WT MYH7 (βWT), and mice expressing mutant MYH7 (R1500P), quantified by myosin unique peptide proteomics. Six- to 7-month-old male mice were studied (n = 3 mice/group). (B) Average ratio of TA muscle/body weight (left) and TA muscle weight (right); Six- to 10-month-old male mice were studied (n = 20 mice/group). (C) Representative SDH activity of TA muscle cross-sections isolated from 8-month-old male βWT (left) and R1500P (right) animals. Scale bar: 200 μm. (D) Quantification of SDH-positive fibers (left), and fiber CSA, reported in μm2 divided by 100 (right). Representative images of sections (fields) used in the CSA quantification are shown in E and Supplemental Figure 2. (E) Myofibrils from TA muscles from 6-month-old male βWT (left) and R1500P (right) mice were prepared and immunostained with the Myc antibody. Samples were imaged on a confocal spinning-disk microscope (Nikon Ti-E; ×100 silicon immersion objective). Scale bar: 5 μm. (F) Four-limb hanging (left) and grip strength (right) tests. The hanging time for each animal was calculated after 3 trials. Eight-month-old male mice were used (n = 6 mice/group). Measurements of forelimb grip strength were carried out with a computerized grip strength meter. Seven- to 8-month-old male mice (n = 6 mice/group). All data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, by 2-tailed, unpaired Student’s t test with Welch’s correction.