Belt electrode-tetanus muscle stimulation prevents denervation-induced atrophy of rat multiple skeletal muscle groups via ribosome biogenesis
Belt electrode-tetanus muscle stimulation prevents denervation-induced atrophy of rat multiple skeletal muscle groups via ribosome biogenesis
They were divided into three groups: control (CONT), denervation (DEN), and denervation + electrical stimulation (DEN + ES), and chronic response analysis was performed. For electrical stimulation, chronic responses were performed under isoflurane anesthesia (1 set of 5 min, once every 2 days for 7 days), and 24 h after the last stimulation, the gastrocnemius muscle was collected Ten-week-old male Sprague-Dawley rats were used.The rats were housed in cages at 23 °C with a 12 h/12 h light/dark cycle (dark time 18:00–06:00). All rats received a standard solid diet (CE-7; CLEA Japan) and water ad libitum.All rats were acclimatized by rearing in the environment described above for 1 week prior to the experiment. The right and left ankle joints were shaved under isoflurane anesthesia (anesthetic aspiration rate: 250–300 mL/min, concentration: 2.0–2.2%). The rats were placed on their backs on a table, and belt-type electrodes were attached to both ankle joints. The muscles of the lower extremities were stimulated simultaneously on both sides using electrical stimulation at 60 Hz, eliciting continuous tetanus for 5 min. Stimulation intensity was preliminarily tested with a belt electrode and set to 3.0 mA, which is the minimum current run value at which maximum torque was obtained with 60 Hz stimulation. During contraction, the lower limb was not immobilized, and the exercise was performed in a natural extension position. Data were analyzed using iDEP. Gene expression difference analysis was performed using DEseq2 [false discovery rate (FDR) cutoff: 0.1, minimum fold change: 2]. Pathway analysis was performed on the GO Biological Process gene set using Generally Applicable Gene Set Enrichment for pathway analysis (GAGE; FDR cutoff:0.10). Muscle was homogenized with TRIzol reagent. Chloroform was added, mixed, and allowed to stand for 15 minutes, and the muscle samples were centrifuged at 4°C, 12,000 x g for 15 minutes. The supernatant was collected, ethanol was added and mixed, and total RNA was extracted using RNeasy kit (74106; QIAGEN, Hilden, Germany).  Sequencing libraries were prepared using the QuantSeq 30 mRNA-seq LibraryPrep Kit FWD for Illumina (Lexogen, Inc.). After removal of the RNA template, a second strand was synthesized using random primers. The double-stranded cDNA library was purified using magnetic beads after washing, and the library was amplified by PCR using primers containing adapters and i7 index sequences. Libraries were purified with magnetic beads, quantified using an Affluorometer (Qubit 4, Thermo Fisher Scientific), and pooled to equimolar concentrations.