Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice
James B. Papizan, … , Ning Liu, Eric N. Olson
James B. Papizan, … , Ning Liu, Eric N. Olson
Published September 5, 2017
Citation Information: J Clin Invest. 2017;127(10):3730-3740. https://doi.org/10.1172/JCI93445.
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Research Article Muscle biology

Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice

Abstract

Maintenance of muscle structure and function depends on the precise organization of contractile proteins into sarcomeres and coupling of the contractile apparatus to the sarcoplasmic reticulum (SR), which serves as the reservoir for calcium required for contraction. Several members of the Kelch superfamily of proteins, which modulate protein stability as substrate-specific adaptors for ubiquitination, have been implicated in sarcomere formation. The Kelch protein Klhl31 is expressed in a muscle-specific manner under control of the transcription factor MEF2. To explore its functions in vivo, we created a mouse model of Klhl31 loss of function using the CRISPR-Cas9 system. Mice lacking Klhl31 exhibited stunted postnatal skeletal muscle growth, centronuclear myopathy, central cores, Z-disc streaming, and SR dilation. We used proteomics to identify several candidate Klhl31 substrates, including Filamin-C (FlnC). In the Klhl31-knockout mice, FlnC protein levels were highly upregulated with no change in transcription, and we further demonstrated that Klhl31 targets FlnC for ubiquitination and degradation. These findings highlight a role for Klhl31 in the maintenance of skeletal muscle structure and provide insight into the mechanisms underlying congenital myopathies.

Authors

James B. Papizan, Glynnis A. Garry, Svetlana Brezprozvannaya, John R. McAnally, Rhonda Bassel-Duby, Ning Liu, Eric N. Olson

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Figure 3

Loss of Klhl31 causes multiple muscle abnormalities.

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Loss of Klhl31 causes multiple muscle abnormalities. (A) Laminin immunos...
(A) Laminin immunostaining of P10 WT and Klhl31-KO quadriceps. Scale bar: 50 μm. (B) Quantification of the mean CSA of WT and KO (n = 4 for both genotypes). **P < 0.01. Data are represented as mean ± SEM. (C) H&E staining of quadriceps muscle from 12-week-old WT (left) and KO (right) mice. Scale bar: 50 μm. (D) Desmin immunostaining of quadriceps from 12-week-old WT (left) and KO (right) mice. Scale bar: 50 μm. (E) NADH-TR staining of quadriceps from 4-week-old animals reveals a disorganized intermyofibrillary matrix, central cores (white arrowheads), and rubbed out fibers (red arrowheads) in KO. Scale bar: 50 μm. (F and G) Transmission electron microscopic images of (F) 4-week-old animals. In transverse orientation (top panel), KO mice show myofilament loss (white arrowhead) and pleomorphic dense structures (arrows). Red arrowhead denotes a myofibril with a normal pattern of myofilaments. In longitudinal sections (lower panel), numerous mitochondria are evident, Z-line streaming (white arrowhead) is abundant, and numerous Z-discs are degenerated (red arrowheads). Scale bars: 0.5 μm (upper panel); 2 μm (lower panel). (G) Transverse sectioning through quadriceps of 7-month-old KO animals (top panel) reveals a severely dilated SR (white arrowheads). A profound loss of myofilaments is evident in KO mice. Longitudinal sectioning demonstrates a loss of the sarcomere architecture and Z-disc continuity (white arrowhead in bottom panel). Scale bars: 0.2 μm (upper panel); 0.5 μm (low panel) (n = 3 for both genotypes). (H) Grip-force strength test of 6-week-old WT and KO mice. Each symbol represents the average grip force from 6 grip-force trials from an individual mouse. (n = 6 for both genotypes). **P < 0.01, ****P < 0.0001. Data are presented as mean ± SEM. Statistical analyses were performed using an unpaired 2-tailed Student’s t test.