Abstract:  

Nemaline myopathy (NM) is a congenital skeletal muscle disorder characterized by hypotonia, muscle weakness, and atrophy. NM is clinically and genetically heterogeneous and its occurrence is frequently implicated with defects in actin thin filaments of the sarcomere. There have been described thirteen genes whose mutations cause NM. Within the NM genes, KLHL40, KLHL41, and KBTBD13 belong to the Kelch-like gene family whose gene products affect protein turnover, bringing a different mechanistic perspective to the disease. The Kelch-like proteins are involved in the ubiquitination of target proteins as a substrate-recognizing component of a cullin3 RING ubiquitin ligase. Kelch proteins participate in a variety of biological processes including cytoskeleton organization, cell morphology, and extracellular communication. Here we report a novel role of KLHL41 as a regulator of sarco/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1) and ryanodine receptor 1 (RYR1). When we knocked down the expression of KLHL41 in C2C12 myotubes, it led to an increase in levels of SERCA1 and RYR1 by several folds. Overexpression of a recombinant KLHL41 reduced the expression of SERCA1 and RYR1 considerably. This reduction seems to involve the proteasome. Enhanced levels of SERCA1 and RYR1 in KLHL41-depleted myotubes caused a decrease in resting cytosolic Ca2+ concentration and an increase in Ca2+ release from the sarcoplasmic reticulum (SR) upon UTP treatment, suggesting that the SR is overloaded with Ca2+. Interestingly, knockdown and overexpression of KLHL41 led to a decrease and an increase of the levels of autophagic marker LC3B-II, respectively. We generated stable zebrafish klhl41 KO lines using CRISPR/Cas9 and analyzed their phenotypes. In zebrafish, KLHL41 happens as a pair of orthologue genes called klhl41a and klhl41b, which share 80% similarity with the human gene. klhl41a knockout (KO) alone caused no evident phenotype, while klhl41b or klhl41a;klhl41b double KO (dKO) caused strong phenotypes. The klhl41b and the dKO mutants presented with disorganized and thinner myofibrils, actin aggregation, reduced muscle integrity, and a smaller muscle area. In addition, levels of SERCA1 increased in klhl41 KO embryos. Furthermore, the cisternae of the sarcoplasmic reticulum (SR) were enlarged in the mutants. We conclude that KLHL41 is crucial for maintaining proper levels of two key Ca2+ handlers and is also a key regulator of autophagy in skeletal muscle. In zebrafish, we conclude that klhl41 is crucial for the organization of the sarcomeric thin filament as well as the homeostasis of the SR. 

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