Total RNA was extracted from gastrocnemius muscles using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to previous studies (9 (link),26 (link),35 (link),61 (link)). The RNA concentration and quality were determined using a CFX96™ Real-Time PCR Detection system using iTaq™ SYBR-Green (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA). The samples were treated with recombinant DNase I (DNA-free DNA removal kit; Ambion, Austin, TX, USA) to remove possible DNA contamination. RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manu facturer’s protocol. The PCR cycling conditions were as follows: Initial pre-denaturation of 95°C for 1 min, denaturation for 15 sec, annealing of 55–65°C for 20 sec and extension of 72°C for 30 sec. A total of 50 cycles were performed. 18S ribosomal RNA was used as an internal control. PCR primer sequences are listed in Table I. For quantitative analysis, the intact control muscle tissue was used as the control, and the relative expression of Atrogin-1, MuRF 1, PI3K p85α, Akt1, Adenosine A1R, TRPV4, Myostatin and SIRT1 was calculated using the 2−ΔΔCt method (62 (link)).