α-Linolenic Acid Reduces TNF-Induced Apoptosis in C2C12 Myoblasts by Regulating Expression of Apoptotic Proteins

Abstract

Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions such as adjuvant strategy for preventing these conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested as having a positive impact on muscle diseases. We recently demonstrated that a diet enriched with plant derived n3-fatty acid, α-linolenic acid (ALA), exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we here investigate on the expression level of the anti- and pro-apoptotic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of tumor necrosis factor-α (TNF). The results demonstrated that ALA protective effect on C2C12 myoblasts was associated with a decrease in caspase-3 activity and an increase of the Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescuing Bcl-2 expression and to revert Bax translocation to mitochondria both affected in an opposite way by TNF, a major pro-inflammatory cytokine expressed in damaged skeletal muscle. Therefore, ALA counteracts inflammatory signals in the muscle microenvironment and may represent a valuable strategy for ameliorating skeletal muscle pathologies.

Keywords: apoptotic proteins; inflammation; muscle wasting; myoblasts; phytochemicals.

Fig. 1.

ALA reduces caspase-3 activity in C2C12 myoblasts. A) The representative Western bolt shows cleaved caspase-3 expression as assessed in C2C12 cells incubated for 48h in DM (differentiation medium) alone (Mock) or with TNF (tumor necrosis factor-α) or TNF and ALA (α-linolenic acid). α-tubulin was used as a loading control. B) Bands intensities of the western blot signals in (A). Values are expressed as arbitrary units. C) Bars are representative of caspase-3 activity measured by the fluorimetric assay. Fluorescence values, expressed as arbitrary units (AU), were normalized to protein content. Data derived from three separate experiments are presented as the means±SD. * P<0.05 compared with Mock cells; § P<0.05 compared with the TNF group.

Fig. 2.

ALA regulates the expression levels of Bcl-2 family proteins and Bax traslocation. A) Representative Western blots of the protein content of Bcl-2 and Bax. α-tubulin was used as loading control. B) The ratio of Bax/Bcl-2 protein content was estimated on the basis of densitometric measurements of the Western blot signals. C) Western blots of the Bax protein in enriched mitochondrial and cytosolic fractions of C2C12 cells. COX IV and /?/-tubulin were used as mitochondrial and cytosolic marker respectively. D) Relative expression of Bax protein in mitochondria and cytosol. Values are expressed as arbitrary units. Data derived from three separate experiments are presented as the means ±SD. * P<0.05 compared with Mock cells; § P<0.05 compared with the TNF group.