Isolation, Culture, and Staining of Single Myofibers.

Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ex vivo by isolation of single myofibers from skeletal muscles and culturing them under suspension conditions. Here, we describe an improved protocol to evaluate ex vivo satellite cells activation through isolation of single myofibers from extensor digitorum longus (EDL) muscle of mice and culturing and staining of myofiber-associated satellite cells with the markers of self-renewal, proliferation, and differentiation.

Regulation of Akt-mTOR, ubiquitin-proteasome and autophagy-lysosome pathways in response to formoterol administration in rat skeletal muscle.

Administration of ß2-agonists triggers skeletal muscle anabolism and hypertrophy. We investigated the time course of the molecular events responsible for rat skeletal muscle hypertrophy in response to 1, 3 and 10 days of formoterol administration (i.p. 2000µg/kg/day). A marked hypertrophy of rat tibialis anterior muscle culminated at day 10. Phosphorylation of Akt, ribosomal protein S6, 4E-BP1 and ERK1/2 was increased at day 3, but returned to control level at day 10. This could lead to a transient increase in protein translation and could explain previous studies that reported increase in protein synthesis following ß2-agonist administration. Formoterol administration was also associated with a significant reduction in MAFbx/atrogin-1 mRNA level (day 3), suggesting that formoterol can also affect protein degradation of MAFbx/atrogin1 targeted substrates, including MyoD and eukaryotic initiation factor-3f (eIF3-f). Surprisingly, mRNA level of autophagy-related genes, light chain 3 beta (LC3b) and gamma-aminobutyric acid receptor-associated protein-like 1 (Gabarapl1), as well as lysosomal hydrolases, cathepsin B and cathepsin L, was significantly and transiently increased after 1 and/or 3 days, suggesting that autophagosome formation would be increased in response to formoterol administration. However, this has to be relativized since the mRNA level of Unc-51-like kinase1 (Ulk1), BCL2/adenovirus E1B interacting protein3 (Bnip3), and transcription factor EB (TFEB), as well as the protein content of Ulk1, Atg13, Atg5-Atg12 complex and p62/Sqstm1 remained unchanged or was even decreased in response to formoterol administration. These results demonstrate that the effects of formoterol are mediated, in part, through the activation of Akt-mTOR pathway and that other signaling pathways become more important in the regulation of skeletal muscle mass with chronic administration of ß2-agonists.

Sirtuin 1 regulates SREBP-1c expression in a LXR-dependent manner in skeletal muscle.

Sirtuin 1 (SIRT1), a NAD(+)-dependent protein deacetylase, has emerged as a main determinant of whole body homeostasis in mammals by regulating a large spectrum of transcriptional regulators in metabolically relevant tissue such as liver, adipose tissue and skeletal muscle. Sterol regulatory element binding protein (SREBP)-1c is a transcription factor that controls the expression of genes related to fatty acid and triglyceride synthesis in tissues with high lipid synthesis rates such as adipose tissue and liver. Previous studies indicate that SIRT1 can regulate the expression and function of SREBP-1c in liver. In the present study, we determined whether SIRT1 regulates SREBP-1c expression in skeletal muscle. SREBP-1c mRNA and protein levels were decreased in the gastrocnemius muscle of mice harboring deletion of the catalytic domain of SIRT1 (SIRT1(Δex4/Δex4) mice). By contrast, adenoviral expression of SIRT1 in human myotubes increased SREBP-1c mRNA and protein levels. Importantly, SREBP-1c promoter transactivation, which was significantly increased in response to SIRT1 overexpression by gene electrotransfer in skeletal muscle, was completely abolished when liver X receptor (LXR) response elements were deleted. Finally, our in vivo data from SIRT1(Δex4/Δex4) mice and in vitro data from human myotubes overexpressing SIRT1 show that SIRT1 regulates LXR acetylation in skeletal muscle cells. This suggests a possible mechanism by which the regulation of SREBP-1c gene expression by SIRT1 may require the deacetylation of LXR transcription factors.