2.1 Participants
Eighteen healthy men were recruited to this study and randomly divided into eccentric (n= 9) and concentric (n= 9) groups. The experimental protocol consisted of two sessions (familiarization and isokinetic test) described below. Subjects were not involved in resistance training and lower body musculoskeletal conditions for at least six months before beginning the study. All procedures were approved by the University of Tehran ethical committee (Ethic No: IR.UT.SPORT.REC.1397.029).
Table 1
Anthropometric characteristics of participants in two groups
Variables
|
Concentric
|
Eccentric
|
Age (Year)
|
27.25± 2.78
|
25.65± 3.74
|
Height (Cm)
|
175.88± 4.64
|
176.44± 5.57
|
Weight (Kg)
|
74.44± 3.04
|
70.22± 2.81
|
(BMI (kg/m2)
|
24.12± 1.58
|
22.57± 1.58
|
2.2 Familiarization
A week before each resistance exercise, the knee extension Maximal Voluntary Isometric Action (MVIA) of the dominant leg was determined using a standard dynamometer (Biodex, Shirley, NY USA). Leg dominance was identified by asking the individuals the preferred leg to kick a ball (54). Before testing the MVIA all subjects performed a general warm-up consisting of 10-minute cycling without load at the range of 60–80 rpm on the stationary cycle before the MVIA test. Subsequently, each participant underwent a brief familiarization with the isokinetic dynamometer, involving 5–10 submaximal contractions (25% of MVIA) with the dominant leg.
For MVIA, the knee was positioned at a 60-degree knee extension (full extension – 0°). Three trials (5s for each trial) were conducted, with intervals of 3 min between them. Additional trials were conducted if the participants did not achieve at least two trials with similar results. Participants were strongly encouraged during the task.
After MVIA tests, individuals were familiarized with the maximal eccentric and concentric leg extension force was measured. Following a five-minute warm-up on a cycle ergometer, participants received detailed instructions on the eccentric and concentric leg extension exercises, and performed 10 repetitions of each exercise at maximal effort. The maximum force of the knee extensors for eccentric and concentric leg extension was performed with the dominant leg at 60°/s.
2.3 Isokinetic test
The experimental protocol was conducted in a separate day (7 days apart from the familiarization session). Before the isokinetic test each individual performed a general warm-up consisting of five-minute cycling without load, and a specific warm-up using the Biodex isokinetic device at 25% of MVIA (5 concentric or eccentric contractions, according to the group randomization, with an intervals of 30 seconds between each set). The participants then performed one of the isokinetic protocols below with the dominant leg.
Eccentric protocol: Each contraction was performed at 60°/s. Individuals performed 12 sets of 10 repetitions with 30 seconds of rest between each set, for a total of 120 contractions. Movement at the shoulders, hips, and thigh (exercised leg) were restrained with straps to isolate the knee extensors during the protocols and secure the participant to the device. The eccentric contraction was performed at >90% of maximal load eccentric strength and the concentric component was passive (i.e. investigator moved the limb back to the starting position). Visual feedback of the force signal was provided to each individual. Participants were verbally encouraged to maintain the contraction levels during the task. At the end of each set, the rating of perceived exertion (RPE) was determined using the 20-point scale. One participant was not able to maintain the intensity level during the whole task and performed only 10 sets.
Concentric protocol: The protocol was similar to the eccentric one but the individuals performed concentric contractions at 90% of their maximal instead of eccentric ones. The eccentric portion of the movement was passive (i.e. the investigator returned the limb to initial position). Visual feedback was also provided and individuals reported their RPE between sets. The full concentric protocol was performed by all participants.
2.4 Muscle Biopsy
Two samples were collected: 1) four weeks before the eccentric/concentric protocol and 2) immediately after the concentric/eccentric protocol. Tissue biopsies were collected from the mid-portion of the Vastus Lateralis muscle in the morning (11-12 am). The area was locally anesthetized (0.1 ml of lidocaine), and a Bergström needle was used. The samples were cleaned from blood and connective tissue, then frozen immediately in liquid nitrogen and stored at -80 °C until gene expression analysis.
2.5 Gene expression
Expression of Myosin Heavy Chain (MHC) I, IIA, IIX, Myo-D, MRF4, Murf, Atrogin-1, Decorin, Myonection, and IL-15 was identified with real-time Polymerase Chain Reaction (PCR). Total RNA was extracted based on the Cinna Gen protocol using kiazol solution (Cinnacolon, Iran). To ensure no contamination with genomic DNA, the samples were first exposed to DNAse (DNase I Fermentas). The quality of extracted RNAs was evaluated with a spectrophotometric device (DPI-1, Kiagen). For reverse-transcribed of RNAs to cDNA used Strand cDNA Synthesis Kit (Oligo dt MWG-Biotech, Germany). The PCR reaction was conducted using the PCR master mix (Applied Biosystems) and Syber Green in the ABI Step One (Applied Biosystems, Sequences Detection Systems, Foster City, CA). For each Real-Time PCR cycle, a total of 40 cycles were considered, and the temperatures for each cycle were set at 94 ° C for 20 s, 60-58 ° C for the 30s, and 72 ° C for 30 s, respectively. The primer sequence is shown in Table.2. The expression of GAPDH confirmed that PCR conditions were optimized. Melting diagrams were performed to check the accuracy of PCR reactions. They were evaluated for each gene individually and at each reaction time, along with negative control diagrams to check for contamination in each reaction. Relative expression of the gene was analyzed using Prism software after manually setting the baseline and threshold. The RT-PCR data were analyzed using the delta-delta cycle threshold (DDCt) method. All Real-time PCR procedures were performed in triplicate.
Table 2
Target genes and sequence of primers
Gene
|
Forward/Reverse
|
Primer (5’ → 3’)
|
MyoD
|
F
|
GGTGGGGGATAGTGGGTGGG
|
R
|
TGTGGCAAAGGAGGAGAGAGAG
|
MRF4
|
F
|
GATAACGGGTAAGGAAGGAGGAG
|
R
|
AAGGATTAGGTCAGGAAGGGT
|
IL-15
|
F
|
GGAGGTATTGTGGATGGATGG
|
R
|
ACAGAAGTAGCACTGGATGGA
|
Myonectin
|
F
|
AGGTGGTGGATGAGAGATGGT
|
R
|
TACTGTGGGGAAAATGTGGGA
|
Decorin
|
F
|
TGAAGGGAGAAGACATTGG
|
R
|
GGAAGATAGGGAGAGGTAGA
|
MHC I
|
F
|
CATTGAGGAGCTGGAGGAGGA
|
R
|
TGGCTTCATGCTGTAGGGTGG
|
MHC IIA
|
F
|
AGCAGAGAAGGAGGAAAAGTGA
|
R
|
CACTTGTAGGGGTTGACAGTGA
|
MHC IIX
|
F
|
GCAGGAGGAGTACAAGAAGGAA
|
R
|
TTGGGGTTCTGGAAGTTGTTGG
|
Atrogin-1
|
F
|
ACTCCACACCCTCTACACATCC
|
R
|
TCTCCATCCCATACACCCACA
|
MuRF-1
|
F
|
TGTCCAGACCATCATCACTCA
|
R
|
AACTCACTTCTCTTCTCATCCA
|
GAPDH
|
F
|
GCA GGG ATG ATGTTC TGG
|
R
|
CTT TGG TAT CGT GGA AGG AC
|
2.6 Statistical Analysis
Statistical analysis was conducted using SPSS software (version 21; IBM, Chicago, IL) and reported as mean ± SD. The normality of the data and equality of variances were tested using the Shapiro–Wilk and Levine test, respectively. Mixed-design repeated measures analysis of variance (ANOVA) was run to analyses differences across time (baseline vs. follow-up) and groups (eccentric vs. concentric) in all variables (Myo-D, MRF4, IL-15, Myonection, Decorin, MHC I, IIA, IIX, Atrogin-1, and Murf mRNA expression). Where an interaction effect was observed, independent samples t test was performed to compare variables at baseline and follow-up change between eccentric and concentric groups. In addition, changes over time (baseline vs. follow-up) in each group were assessed using paired t-test. Partial eta squared effect size was reported for group ×time interaction. For all statistical analyses an alpha of p≤0.05 was considered statistically significant for all comparisons.