Endocrine Abstracts

Cushing’s Syndrome (CS) is associated with a severe myopathy mainly affecting type 2 muscle fibres, with a higher prevalence in male subjects. The excess of glucocorticoids (GC) levels represents the most common cause of myopathy due to an alteration of protein metabolism. The non-coding post-transcriptional gene regulators muscle-specific microRNAs (miRNA) control muscle development and differentiation but when dysregulated lead to skeletal muscle (SM) disorder. The aim of the current study was to investigate circulating miRNAs expression profile during SM wasting in mouse differentiated C2C12 cell line, in a condition of cortisol excess. To mimic a CS condition in vitro, and particularly a condition resembling a pituitary-dependent CS with increased ACTH and cortisol, mouse differentiated C2C12 cells have been treated with ACTH at 2.2*10⁻¹¹M (corresponding to 100 pg/mL patients’ serum levels), hydrocortisone (HC) at 1.4*10⁻⁶M (corresponding to 300 ng/mL) and in combination for 12 h. In order to confirm that 12h of drug treatment were able to induce SM wasting, gene and protein expression of Atrogin and Murf were evaluated by RT-qPCR and Western Blot, respectively. Circulating miRNAs expression profile miScript miRNA was evaluated by PCR arrays. The results of the study demonstrated that 12h of HC treatment at 1.1*10⁻⁶M was sufficient to induce a condition resembling muscle wasting in C2C12 cell line prompting a significant increase of Atrogin gene (P=0.01) and ATROGIN protein expression compared to the control and compared to ACTH treatment (P=0.001) and a non-significant increase of Murf gene (P=0.07) and MURF protein expression. Circulating miRNA microarray analysis demonstrated that in C2C12 treated for 12 h with HC, miR 133a-3p (P=0.006), miR 122-3p (P=0.018) and miR 200b-3p (P=0.044) were significantly up-regulated compared to the control. Conversely, C2C12 treated for 12 h with ACTH did not show any change in miRNA expression profile. The preliminary results of the current study demonstrated that 12h of treatment with HC is sufficient to induce a condition resembling muscle wasting in C2C12 by means of an up-regulation of main miRNAs known to be involved in the development of myopathies. In conclusion, the up-regulation of specific miRNAs might potentially be used as marker or predictive factors for the development of GC-induced myopathy in patients with CS.