Small-Molecule Targeting MuRF1 Protects Against Denervation-Induced Diaphragmatic Dysfunction: Underlying Molecular Mechanisms

Abstract

Background: Mechanical inactivity rapidly induces diaphragm muscle fibres' contractile dysfunction and atrophy. Diaphragm weakness can impair respiratory function, quality of life and increase risks of morbidity and mortality. Muscle RING-finger protein-1 (MuRF1) expression is upregulated during denervation and muscle inactivity and is known to target key muscle proteins for degradation. We previously reported that the small-molecule targeting MuRF1 (MyoMed-205) protects against diaphragm contractile dysfunction and atrophy after 12 h of unilateral diaphragm denervation (UDD) in rats. In this study, we investigated the mechanisms by which MyoMed-205 protects the diaphragm structure and function during early UDD in rats.

Methods: Male Wistar rats were subjected to unilateral diaphragm denervation (UDD) for 12 h. Immediately after UDD, rats received either a placebo (vehicle) or small-molecule targeting MuRF1 (MyoMed-205, 50 mg/kg bw), and outcomes were compared with sham-operated controls. Diaphragm was used for histological, morphometric, transcriptomic (RNA-seq) and protein content (Western blot) analysis.

Results: UDD induced diaphragm slow- (Type I: p = 0.03) and fast-twitch (Type IIa: p = 0.04; Type IIb/x: p = 0.02) fibres atrophy after 12 h, which was prevented by MyoMed-205 (p < 0.05). Mechanistically, UDD perturbed mechanisms involved with myofibre ultrastructure and contractility, mitochondrial function, proteolysis and tissue remodelling in the diaphragm. MyoMed-205 enhanced the activation of mechanisms required for sarcomere integrity, calcium handling, antioxidant defence, chaperone-mediated unfolded protein response and muscle growth. MyoMed-205 also mitigated intramuscular fat deposition and pro-fibrotic responses triggered by UDD.

Conclusions: Small-molecule targeting MuRF1 (MyoMed-205) protects against diaphragm muscle contractile dysfunction and atrophy after 12 h of UDD. Herein, we demonstrate that this protective effect involved augmented activation of signalling pathways controlling muscle structure and function, chaperone-mediated unfolded protein and muscle growth, while mitigating intramuscular fat deposition and pro-fibrotic responses triggered by UDD at the transcriptional and/or protein level.

Keywords: MyoMed‐205; PI3K‐Akt–mTOR pathway; TRIM63; mechanical unloading; skeletal muscle; ubiquitin‐proteasome system; unilateral diaphragm denervation.