Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy
Michaela Yuen, et al.
Michaela Yuen, et al.
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Research Article

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Abstract

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

Authors

Michaela Yuen, Sarah A. Sandaradura, James J. Dowling, Alla S. Kostyukova, Natalia Moroz, Kate G. Quinlan, Vilma-Lotta Lehtokari, Gianina Ravenscroft, Emily J. Todd, Ozge Ceyhan-Birsoy, David S. Gokhin, Jérome Maluenda, Monkol Lek, Flora Nolent, Christopher T. Pappas, Stefanie M. Novak, Adele D’Amico, Edoardo Malfatti, Brett P. Thomas, Stacey B. Gabriel, Namrata Gupta, Mark J. Daly, Biljana Ilkovski, Peter J. Houweling, Ann E. Davidson, Lindsay C. Swanson, Catherine A. Brownstein, Vandana A. Gupta, Livija Medne, Patrick Shannon, Nicole Martin, David P. Bick, Anders Flisberg, Eva Holmberg, Peter Van den Bergh, Pablo Lapunzina, Leigh B. Waddell, Darcée D. Sloboda, Enrico Bertini, David Chitayat, William R. Telfer, Annie Laquerrière, Carol C. Gregorio, Coen A.C. Ottenheijm, Carsten G. Bönnemann, Katarina Pelin, Alan H. Beggs, Yukiko K. Hayashi, Norma B. Romero, Nigel G. Laing, Ichizo Nishino, Carina Wallgren-Pettersson, Judith Melki, Velia M. Fowler, Daniel G. MacArthur, Kathryn N. North, Nigel F. Clarke

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Figure 6

LMOD3 localizes to actin thin filaments in the muscle sarcomere.

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LMOD3 localizes to actin thin filaments in the muscle sarcomere. (A) TMO...
(A) TMOD1 pointed end localization and (B and C) 2 examples of LMOD3 localization in stretched human control quadriceps muscles. Actin filaments were colabeled with Alexa Fluor 488–conjugated phalloidin (blue), α-actinin (Z-discs, red), and LMOD3 (green). (D) At higher magnification, LMOD3 pointed end staining can occasionally be resolved into 2 distinct bands. (E) Measurements of LMOD3 and phalloidin signal breadth taken at the half peak height (LLMOD and LPhal, respectively), using methods illustrated in F. LLMOD and LPhal are indistinguishable in fast fibers (NS, paired t test), but LLMOD is shorter than LPhal in slow fibers (***P < 0.0001, paired t test), suggesting that LMOD3 binding ends before the pointed end cap in slow fibers. (G) Measurements of the distance between the LMOD3 peak located near the pointed end and the Z-disc (fast and slow fibres) and the distance between the TMOD1 peak and the Z-disc (p-p, peak-to-peak), as illustrated in H. Thin filament lengths were measured in the same sarcomeres from phalloidin staining, as illustrated in F. The peak of the LMOD3 signal was significantly closer to the Z-disc in slow myofibers (***P = 0.0009, 2-tailed unpaired t test) and fast myofibers (*P = 0.0472, 2-tailed unpaired t test) as compared with TMOD1. The peak of LMOD3 staining localized closer to the Z-disc in slow muscle myofibers compared with fast myofibers (**P = 0.0043, 2-tailed unpaired t test). (I) Diagram showing the hypothesized positions of LMOD3 pointed end binding relative to other thin filament proteins. Scale bar: 7.5 μm. Graphs show mean ± SD.