Muscle-specific deletion of SOCS3 increases the early inflammatory response but does not affect regeneration after myotoxic injury

Swiderski et al. Skeletal Muscle (2016) 6:36 DOI 10.1186/s13395-016-0108-4 RESEARCH Open Access Muscle-specific deletion of SOCS3 increases the early inflammatory response but does not affect regeneration after myotoxic injury Kristy Swiderski, Savant S. Thakur, Timur Naim, Jennifer Trieu, Annabel Chee, David I. Stapleton, René Koopman and Gordon S. Lynch* Abstract Background: Muscles of old animals are injured more easily and regenerate poorly, attributed in part to increased levels of circulating pro-inflammatory cytokines. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade is a key mediator of inflammatory cytokine action, and signaling via this pathway is increased in muscles with aging. As a negative regulator of JAK/STAT signaling, a key mediator of myogenic proliferation and differentiation, altered expression of suppressor of cytokine signaling (SOCS3) is likely to have important consequences for muscle regeneration. To model this scenario, we investigated the effect of SOCS3 deletion within mature muscle fibers on injury and repair. We tested the hypothesis that reduced SOCS3 function would alter the inflammatory response and impair muscle regeneration after myotoxic injury. Methods: Mice with a specific deletion of SOCS3 within mature skeletal muscle fibers were used to assess the effect of SOCS3 deletion on muscle injury and repair. Twelve-week-old or 24-month-old SOCS3 muscle-specific knockout (SOCS3 MKO) mice and littermate controls were either left uninjured or injured with a single injection of notexin (10 μg/ml) into the right tibialis anterior (TA) muscle. At 1, 2, 3, 5, 7, or 14 days post-injury, the right TA muscle was excised and subjected to histological, western immunoblotting, and gene expression analyses. Force production and fatigue were assessed in uninjured muscles and at 7 days post-notexin injury. Results: In uninjured muscles, SOCS3 deletion decreased force production during fatigue but had no effect on the gross or histological appearance of the TA muscles. After notexin injury, deletion of SOCS3 increased STAT3 phosphorylation at day 1 and increased the mRNA expression of the inflammatory cytokine TNF-α, and the inflammatory cell markers F4/80 and CD68 at day 2. Gene expression analysis of the regeneration markers Pax7, MyoD, and Myogenin indicated SOCS3 deletion had no effect on the progression of muscle repair after notexin injury. Inflammation and regeneration were also unchanged in the muscles of 24-month-old SOCS3 MKO mice compared with control. Conclusions: Loss of SOCS3 expression in mature muscle fibers increased the inflammatory response to myotoxic injury but did not impair muscle regeneration in either adult or old mice. Therefore, reduced SOCS3 expression in muscle fibers is unlikely to underlie impaired muscle regeneration. Further investigation into the role of SOCS3 in other cell types involved in muscle repair is warranted. Keywords: SOCS3, Muscle, Regeneration, Inflammation, MCK * Correspondence: Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Melbourne 3010, Australia © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Swiderski et al. Skeletal Muscle (2016) 6:36 Background Muscles of old animals are more susceptible to injury and regenerate poorly. Cycles of repeated damage and incomplete repair contributes to muscle atrophy and weakness with age [1–5]. A well-regulated inflammatory response is critical for the initiation of muscle repair through muscle stem cell activation and necessary for myogenic differentiation [6–11]. In contrast, chronic low-grade systemic inflammation is thought to interfere with effective regeneration in older individuals. Although increased levels of circulating pro-inflammatory cytokines, such as interleukin −6 (IL-6), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) are associated with muscle wasting and are increased in aged muscle [12], the signaling mechanisms controlling degeneration, regeneration, and inflammation are not well understood. Inflammatory cytokines exert many effects via activation of the Janus kinase/Signal transducers and activators of transcription (JAK/STAT) signaling pathway [13]. The suppressors of cytokine signaling (SOCS) protein family consisting of eight members including cytokine-induced STAT inhibitor (CIS) and SOCS1-7 are key negative regulators of JAK/STAT signaling. SOCS3 is one of the best characterized SOCS proteins and an important regulator of JAK1/STAT3 signaling and inflammation in many cell types via direct interactions with both the gp130 receptor and JAK1 [14–17]. Properly regulated JAK/STAT signaling is required for progression through myogenic differentiation and critical for muscle regeneration [18]. In the absence of inflammation, SOCS3 is expressed at very low levels but is thought to play a role in various muscle resident cells including hematopoietic cells, muscle stem cells, and mature muscle fibers [17, 19–23]. In vitro adenoviral overexpression of SOCS3 in human myotubes indicated a role for SOCS3 in directing the expression of genes regulating myogenic differentiation, myotube maturation, and cell survival [24], demonstrating a potential role for SOCS3 in regulating myogenesis. Transgenic muscle-specific overexpression of SOCS3, driven by the myosin light chain (MLC) promoter, impairs muscle morphology and ambulation, associated with disrupted calcineurin signaling and defects in sarcoplasmic reticulum and mitochondrial function [25]. In another study, transgenic overexpression of SOCS3 driven by the muscle creatine kinase (MCK) promoter impaired glucose and insulin tolerance as a result of suppressed leptininduced activation of the AMP-regulated protein kinase (AMPK) [23]. In contrast, mice with MCK-Cre-mediated SOCS3 deletion had normal muscle development and functional performance and, consistent with a role of SOCS3 in inhibition of insulin signaling, had improved insulin sensitivity and glucose homeostasis [26]. As JAK/STAT signaling is implicated in the regulation of inflammation, anabolic signaling, and myogenic Page 2 of 15 differentiation, altered regulation of this signaling is likely to have important consequences for muscle health and effective regeneration. Increased STAT3 phosphorylation and SOCS3 messenger RNA (mRNA) expression has been reported in skeletal muscles f (...truncated)