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350 Aminizadeh et al. | Journal of Clinical and Translational Research 2024; 10(6): 348-356
study, we hypothesize that MitoQ supplementation, both as an for 8 weeks, 5 days/week, 50 min/day (speed at the last week:
isolated treatment and combined treatment with ET and HIIT, 65 – 70% of V max ). The iso-distance method was used for two
will significantly influence mitochondrial dynamics within protocols to unify two types of the training protocol. Forty-eight
the gastrocnemius muscle of male rats. Moreover, we propose hours after the end of the final training session, animals were
that the distinct characteristics of each training modality may anesthetized and sacrificed. The gastrocnemius muscle was
differentially impact mitochondrial dynamic when paired with extracted and washed with cold normal saline, frozen by liquid
MitoQ supplementation. This study aims to provide deeper nitrogen, and stored at −80°C for real-time polymerase chain
insights into how these interventions can enhance mitochondrial reaction (PCR) and Western blotting.
function.
2.4. Western blotting
2. Materials and Methods
The protein levels of DRP1 and MFN1 were measured by
2.1. Animals means of Western blotting. Initially, 20 mg of skeletal muscle
tissue was homogenized in cold RIPA (radioimmunoprecipitation
Forty-two male Wistar rats, each weighing 200 ± 10 g and
aged 8 weeks, were obtained from the Physiology Research assay) buffer containing 0.5% sodium deoxycholate, 150 mM
Center (Karman, Iran) and maintained under standard conditions sodium chloride (NaCl), 1.0 mM EDTA, 0.1%, 50 mM Tris-
as follows: 12-h light/dark cycle; 23 ± 2°C; and humidity of HCl, sodium dodecyl sulfate (SDS), and protease inhibitor with
55 ± 10%. Animals were randomly divided into 6 groups (n = 7): a pH of 7.4. After homogenization, the mixture was centrifuged
(i) Untreated control (CTL); (ii) MitoQ, which received 250 µM (15000 rpm, 20 min, 4°C) and the supernatant was collected.
MitoQ in drinking water for 8 weeks; (iii) ET, which performed Protein levels in the supernatant were determined using the
ET; (iv) ET + MitoQ; (v) HIIT, which performed high-intensity Bradford method. Equal volumes of sample buffer (composed
interval training; and (vi) HIIT + MitoQ. In the ET groups, of 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.005%
the rats were trained on a treadmill for 8 weeks, 5 days/week, bromophenol blue, and 5% β-mercaptoethanol) were mixed
50 min/day. Animals in the HIIT groups performed training with the protein sample from the supernatant. The mixture was
(80 – 90% of V max in 2-min intervals) 5 days/week for 8 weeks by incubated at 95°C for 5 min to denature the proteins, after which
running on a treadmill. The animal procedure was approved by each sample was loaded into the appropriate well of a 10%
the Animal Care Committee of the Ethics Committee of Kerman SDS-PAGE gel (running buffer: 25 mM Tris, 192 mM glycine,
University of Medical Sciences (IR.KMU.REC.1400.292). All 0.1% SDS, pH 8.3). Following electrophoresis, proteins were
methods were performed in adherence to the ARRIVE (Animal transferred onto a polyvinylidene fluoride (PVDF) membrane
Research: Reporting of In Vivo Experiments) guidelines 2.0. using a transfer buffer (20% methanol, 25 mM Tris, 192 mM
glycine, pH 8.3) under the following conditions: 200 mA for
2.2. MitoQ treatment 60 min at 4°C. The blocking step was performed with a blocking

MitoQ (MitoQ Ltd, New Zealand) was given to animals at a solution consisting of nonfat dried milk (5% w/v) diluted in Tris-
dose of 250 µM in drinking water [25]. To assess the targeting buffered saline with Tween 20 (TBST; 20 mM Tris, 150 mM
of MitoQ in tissues, the concentration of MitoQ was measured NaCl, 0.1% Tween 20, pH 7.6). After blocking, the membrane
in the whole lysate tissues by means of high-performance was washed five times for 5 min each with TBST. For antibody
liquid chromatography-mass spectrometry (HPLC-MS) [26]. detection, the PVDF membrane was incubated with primary
For HPLC-MS calibration, we used the MitoQ internal antibodies: anti-DRP1 (sc-271583, Santa Cruz Biotechnology)
standard (MRC Mitochondrial Biology Unit and Department and anti-MFN1 (sc-166644, Santa Cruz Biotechnology).
of Medicine, University of Cambridge), and the tissue levels Following five washes (5 min each) with TBST, the blots were
of MitoQ in the CTL group and MitoQ group were 0 and incubated with a peroxidase-conjugated secondary antibody.
5.68 ± 0.81 pmol/100 mg protein, respectively. After additional washing, the blots were visualized using a
chemiluminescent detection system. The amount of protein
2.3. Training protocol was measured by quantitative density analysis and compared

The rats’ familiarization to adapt to the training on treadmill to β-actin (detected with anti-β-actin; sc-47778, Santa Cruz
lasted 2 weeks, with each session set at a speed of 15 m/min Biotechnology) as a control by Image J software [30].
for 15 min. The intensity of exercise training was calculated 2.5. Total RNA extraction and quantitative realtime PCR
by measuring V max . For calculation of the intensity, the speed
test started with a warm-up of 10 m/min and then increased The real-time PCR method was used to determine the
(3 m/min) till exhaustion [27]. Blood lactate levels were relative expression of the genes. Total RNA was extracted
measured by a lactometer (Lactate Scout Company/Code: 37, from the skeletal muscle tissues using EZ-10 Spin Column
Germany) directly after incremental test, and levels above Total RNA Mini-preps Kit (Bio Basic, Canada, Cat number,
6 mmol/L were considered high intensity [28]. For HIIT, each BS1361-SK8655). The process of RNA extraction typically
session consisted of ten 2-min work bouts/day at 80 – 90% involves the destruction of tissue in a chemical environment that
of V max and separated by 2-min rest periods, 5 days/week for simultaneously inactivates ribonucleases and the subsequent
8 weeks [29]. In the ET groups, rats were trained on a treadmill capture of RNA molecules using columns while leaving other

DOI: http://doi.org/10.36922/jctr.24.00044