Abstract
Muscle aging is accompanied by blunted muscle regeneration in response to injury and disuse. Oxidative stress likely underlies this diminished response, but muscle redox sensors that act in regeneration have not yet been characterized. Calmodulin contains multiple redox sensitive methionines whose oxidation alters the regulation of numerous cellular targets. We have used the CRISPR-Cas9 system to introduce a single amino acid substitution M109Q that mimics oxidation of methionine to methionine sulfoxide in one or both alleles of the CALM1 gene, one of three genes encoding the muscle regulatory protein calmodulin, in C2C12 mouse myoblasts. When signaled to undergo myogenesis, mutated myoblasts failed to differentiate into myotubes. Although early myogenic regulatory factors were present, cells with the CALM1 M109Q mutation in one or both alleles were unable to withdraw from the cell cycle and failed to express late myogenic factors. We have shown that a single oxidative modification to a redox-sensitive muscle regulatory protein can halt myogenesis, suggesting a molecular target for mitigating the impact of oxidative stress in age-related muscle degeneration.
Highlights
Aging causes a decline in muscle power and speed due to muscle atrophy and weakness; by the age of 80, humans lose 40% of muscle mass and 50% of muscle power [1]
CALM1 gene editing in mouse myoblasts. (A) Methionine can be oxidized to methionine sulfoxide by cellular oxidants; glutamine substitution mimics methionine sulfoxide. (B) Structural models of CaM bound to the ryanodine receptor (RyR) peptide with four bound Ca2+ atoms; CaM Met 109 and Met 124 sidechains are shown (PDB: 1BCX). (C) CRISPR-Cas 9 editing of C2C12 myoblasts
Given the importance of Ca2+ signaling in myogenesis and the central role of CaM in interpreting the Ca2+ signal, we hypothesized that the muscle regulatory protein CaM acts as a cellular redox sensor that blocks myogenesis in response to oxidative stress
Summary
Aging causes a decline in muscle power and speed due to muscle atrophy and weakness; by the age of 80, humans lose 40% of muscle mass and 50% of muscle power [1]. Given CaM’s central role in Ca2+ signaling and CaM’s structural and functional sensitivity to oxidation, we hypothesize that CaM serves as a muscle redox sensor that blocks myogenesis when the redox balance tips toward oxidative stress. We have used the CRISPR-Cas system to knock-in a single amino acid mutation that mimics the oxidative modification of a single Met to Met sulfoxide (M109Q) in one or both alleles of the CALM1 gene, one of three genes encoding the muscle regulatory protein CaM in mammalian cells. We have shown that a single oxidative modification to a redox-sensitive muscle regulatory protein can halt myogenesis, suggesting a molecular target for mitigating the impact of oxidative stress in age-related muscle degeneration
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