Cigarette smoke and muscle catabolism in C2 myotubes

doi:10.1016/j.mad.2012.11.004

Mechanisms of Ageing and Development

Volume 134, Issues 1–2, January–February 2013, Pages 24-34

https://doi.org/10.1016/j.mad.2012.11.004 Get rights and content

Abstract

Previous studies have revealed evidence of muscular damage and up-regulation of genes associated with impaired muscle maintenance in smokers. Cigarette smoking has also been associated with sarcopenia, the age-related loss of muscle mass and strength. In order to investigate the cellular mechanisms by which cigarette smoke (CS) promotes muscle catabolism, C2 myotubes from an in vitro skeletal muscle cell line, were exposed to different levels of whole vapor phase CS using a controlled CS exposure apparatus. Exposure of C2 myotubes to CS caused a reduction in diameter of myotubes and a time- and dose-dependent degradation of myosin heavy chain. Also, CS exposure resulted in increased intracellular oxidative stress and p38 MAPK phosphorylation, which led to up-regulation of the muscle specific E3 ubiquitin ligases: MAFbx/atrogin-1 and MuRF1. Pretreatment with the antioxidant N-acetylcysteine and inhibition of p38 MAPK by SB203580 prevented CS induced catabolism. In conclusion, our results demonstrate that exposure of skeletal myotubes to CS leads to increased oxidative stress and activation of the p38 MAPK pathway resulting in muscle cell atrophy and breakdown of muscle protein mediated by muscle specific E3 ubiquitin ligases. Our findings provide a possible molecular mechanism for the catabolic effects of CS in skeletal muscle.

Highlights

► Cigarette smoke (CS) exposure caused a reduction in diameter of myotubes. ► CS exposure caused degradation of myosin heavy chain (MyHC) protein. ► CS increased intracellular oxidative stress and activated the p38 MAPK pathway. ► CS up-regulated the muscle specific E3 ligases: MAFbx/atrogin-1 and MuRF1. ► Antioxidant pretreatment and p38 MAPK inhibition prevented CS induced catabolism.

Introduction

Tobacco smoke is probably the single most significant source of exposure to toxic chemicals in humans. Cigarette mainstream smoke is a complex aerosol consisting of vapor and particulate phases (Smith and Fischer, 2011). Some components, such as aldehydes and nitrogen oxides, are found primarily in the vapor phase, while others such as nicotine predominate in the particulate phase (Smith and Fischer, 2011). Vapor phase CS contains over 10¹⁵ free radicals per puff (Swan and Lessov-Schlaggar, 2007) including various reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Smith and Fischer, 2011).

Smoking is well known to be associated with cardiovascular diseases and is the primary cause of chronic obstructive pulmonary disease (COPD) (Swan and Lessov-Schlaggar, 2007). In addition to the known harmful effects of cigarette smoking, previous studies have revealed smoking associated muscular damage. Skeletal muscle biopsies of heavy smokers presented some structural and metabolic damage in comparison with those of non-smokers. These include atrophy of oxidative muscle fibers (Montes de Oca et al., 2008), impaired synthesis of muscle protein and increased expression of genes associated with impaired muscle maintenance (Petersen et al., 2007). In vivo studies that examined the effects of CS exposure on skeletal muscle have also shown CS induced skeletal muscle damage mediated by increased muscular oxidative stress and systemic inflammation (Barreiro et al., 2012, Rinaldi et al., 2012). Epidemiological studies have identified tobacco use as a risk factor for sarcopenia, the age-related loss of skeletal muscle mass and strength (Castillo et al., 2003, Lee et al., 2007, Szulc et al., 2004). However, the molecular mechanisms by which CS leads to skeletal muscle catabolism and atrophy remain unclear.

Loss of muscle mass is characterized by an imbalance between synthesis and breakdown of muscle proteins, leading to fiber loss and atrophy (Meng and Yu, 2010). The ubiquitin proteasome system (UPS) mediates a large part of the degradation of myofibrillar proteins in skeletal muscle. E3 ubiquitin-ligating enzymes (E3s) are responsible for determining which proteins are targeted for proteasomal degradation. Muscle atrophy F-box protein (MAFbx/atrogin-1) and muscle ring finger-1 protein (MuRF1) are two muscle-specific E3s that are over-expressed in numerous catabolic states (Meng and Yu, 2010). Petersen et al. (2007) have found increased expression of MAFbx/atrogin-1 along with impaired synthesis of muscle protein in skeletal muscle of smokers compared with that of non-smokers and concluded that smoking increases the risk of sarcopenia. However, as far as we are aware, the involvement of MAFbx/atrogin-1 and MuRF1 in CS induced skeletal muscle damage has not been investigated in vitro.

The aim of this study was to investigate the molecular mechanisms by which CS leads to muscle atrophy and degradation of muscle proteins. This was done by exposing C2 myotubes from an in vitro skeletal muscle cell type culture to different levels of whole vapor phase CS. We hypothesized that exposure of cultured myotubes to vapor phase CS will lead to atrophy of myotubes and protein breakdown through increased oxidative stress, activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway and up-regulation of the muscle-specific E3s: MAFbx/atrogin-1 and MuRF1.

Section snippets

Cell culture

The C2 mouse skeletal myoblast cell line was a generous gift from Prof. Bengal (Faculty of Medicine, Technion, Israel). C2 myoblasts were grown in 24 wells, 35 mm and 100 mm plates in growth medium (GM) consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin and 1% (v/v) l-glutamine at 37 °C in humidified 95% air – 5% CO₂ atmosphere. For differentiation of myotubes, myoblasts were plated in 0.1%

CS effect on myotubes viability

In order to examine the effects of vapor phase CS on C2 myotubes without causing cell death, it was essential to determine an appropriate level of negative pressure for CS exposure experiments. Thus, differentiated myotubes were exposed to increasing levels of CS as measured by negative pressure levels. Survival of myotubes following 1 h of incubation with CS was measured by MTT assay and compared with air exposed myotubes. Cell viability remained higher than 80% following 1 h incubation with CS

Discussion

Previous studies have shown various alterations in skeletal muscle of smokers compared with that of non-smokers. These alterations include structural damage (Montes de Oca et al., 2008), impaired protein synthesis and up-regulation of the muscle specific E3 ligase, MAFbx/atrogin-1 (Petersen et al., 2007). In addition, smoking was identified as a risk factor for the loss of muscle mass and strength in old age (Castillo et al., 2003, Lee et al., 2007, Szulc et al., 2004).

In this study, we

Conclusions

Our findings have shown for the first time that exposure of vapor phase CS to cultured skeletal myotubes caused cell atrophy and degradation of MyHC via increased oxidative stress, activation of p38 MAPK and up-regulation of MAFbx/atrogin-1 and MuRF1. Antioxidant pretreatment and inhibition of p38 MAPK blunted over-expression of the above E3s and prevented CS induced catabolism. Although within the limitation of an in vitro study, our findings provide a possible molecular mechanism for the

Acknowledgments

This study was supported by grants from the Rappaport Institute, the Krol Foundation of Barnegat N.J., the Myers-JDC-Brookdale Institute of Gerontology and Human Development, and ESHEL – the association for planning and development of services for the aged in Israel. Special thanks to Dr. Alvira Bromosov for her help in developing the DCF assay.

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