7β-Hydroxycholesterol and 7-ketocholesterol: New oxidative stress biomarkers of sarcopenia inducing cytotoxic effects on myoblasts and myotubes

Abstract

Aging is a complex biological process which can be associated with skeletal muscle degradation leading to sarcopenia. The aim of this study consisted i) to determine the oxidative and inflammatory status of sarcopenic patients and ii) to clarify the impact of oxidative stress on myoblasts and myotubes. To this end, various biomarkers of inflammation (C-reactive protein (CRP), TNF-α, IL-6, IL-8, leukotriene B4 (LTB4)) and oxidative stress (malondialdehyde, conjugated dienes, carbonylated proteins and antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase) as well as oxidized derivatives of cholesterol formed by cholesterol autoxidation (7-ketocholesterol, 7β-hydroxycholesterol), were analyzed. Apelin, a myokine which contributes to muscle strength, was also quantified. To this end, a case-control study was conducted to evaluate the RedOx and inflammatory status in 45 elderly subjects (23 non-sarcopenic; 22 sarcopenic) from 65 years old and higher. SARCopenia-Formular (SARC-F) and Timed Up and Go (TUG) tests were used to distinguish between sarcopenic and non-sarcopenic subjects. By using red blood cells, plasma and/or serum, we observed in sarcopenic patients an increased activity of major antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) associated with lipid peroxidation and protein carbonylation (increased level of malondialdehyde, conjugated dienes and carbonylated proteins). Higher levels of 7-ketocholesterol and 7β-hydroxycholesterol were also observed in the plasma of sarcopenic patients. Significant differences were only observed with 7β-hydroxycholesterol. In sarcopenic patients comparatively to non-sarcopenic subjects, significant increase of CRP, LTB4 and apelin were observed whereas similar levels of TNF-α, IL-6 and IL-8 were found. The increased plasma level of 7-ketocholesterol and 7β-hydroxycholesterol in sarcopenic patients led us to study the cytotoxic effect of these oxysterols on undifferentiated (myoblasts) and differentiated (myotubes) murine C2C12 cells. With the fluorescein diacetate and sulforhodamine 101 assays, an induction of cell death was observed both on undifferentiated and differentiated cells: the cytotoxic effects were less pronounced with 7-ketocholesterol. In addition, IL-6 secretion was never detected whatever the culture conditions, TNF-α secretion was significantly increased on undifferentiated and differentiated C2C12 cells treated with 7-ketocholesterol- and 7β-hydroxycholesterol, and IL-8 secretion was increased on differentiated cells. 7-ketocholesterol- and 7β-hydroxycholesterol-induced cell death was strongly attenuated by α-tocopherol and Pistacia lentiscus L. seed oil both on myoblasts and/or myotubes. TNF-α and/or IL-8 secretions were reduced by α-tocopherol and Pistacia lentiscus L. seed oil. Our data support the hypothesis that the enhancement of oxidative stress observed in sarcopenic patients could contribute, especially via 7β-hydroxycholesterol, to skeletal muscle atrophy and inflammation via cytotoxic effects on myoblasts and myotubes. These data bring new elements to understand the pathophysiology of sarcopenia and open new perspectives for the treatment of this frequent age-related disease.