Effect of acute and chronic eccentric exercise on FOXO1 mRNA expression as fiber type transition factor in rat skeletal muscles Milad Azad, Neda Khaledi, Mehdi Hedayati PII: DOI: Reference:
S0378-1119(16)30100-7 doi: 10.1016/j.gene.2016.02.033 GENE 41190
To appear in:
Gene
Received date: Revised date: Accepted date:
27 September 2015 18 February 2016 20 February 2016
Please cite this article as: Azad, Milad, Khaledi, Neda, Hedayati, Mehdi, Effect of acute and chronic eccentric exercise on FOXO1 mRNA expression as fiber type transition factor in rat skeletal muscles, Gene (2016), doi: 10.1016/j.gene.2016.02.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Effect of acute and chronic eccentric exercise on FOXO1 mRNA expression as fiber type transition factor in rat skeletal muscles
PT
Milad Azad1, Neda Khaledi1*, Mehdi Hedayati2
RI
1-Faculaty of Physical Education and Sport Science, Kharazmi University, Tehran, Iran. 2-Cellular and Molecular Research Center, Research Institute for Endocrine Sciences, Shahid
SC
Beheshti University of Medical Sciences, Tehran, Iran.
NU
Running title: Acute and chronic eccentric exercise and FOXO1 mRNA in rat skeletal muscles Abbreviations: FoxO, Forkhead box O; MHC, myosin heavy chain; SVL, super vastus lateralis;
MA
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TOS, total oxidative status; MDA, malondialdehyde;
TE
Dr.Neda Khaledi, Ph.D.
D
*Corresponding author
AC CE P
Faculty of Physical Education and Sport Science, Kharazmi University, Tehran, Iran. Phone: +98 2225808
Email: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Skeletal muscle is a highly elastic tissue which can respond to various functional demands by
PT
altering fiber type composition. Exercise effects on muscle fiber phenotype. One of the
RI
transcription factors that induce fiber type transition is forkhead box O1 (FOXO1). Since eccentric contraction considered an essential part of exercise, so we are interesting to see the
SC
effects of eccentric exercise (acute/chronic) on FOXO1 as an important factor of fiber type
NU
transition in rat skeletal muscles. Twenty four Sprague-dawley rats (190-235 g) divided to 3 groups of 8 rats: 1) chronic eccentric exercise (CEE), 2) acute eccentric exercise (AEE) and 3)
MA
control (C). The exercise groups underwent downhill running protocol. CEE was running on treadmill in 3 days of week for 9 weeks, that slope and duration gradually managed from -4° to -
TE
D
16° and 15 min to 90 min, respectively. AEE group was running with 16 m/min on -16° slope for 3 consecutive day that included 18 sets of 5 min with rest interval of 2 min in between. Soleus
AC CE P
and super vastus lateralis (SVL) muscles mRNA were analyzed by real-time RT-PCR. SVL FOXO1 mRNA levels increased by 3.92-fold in AEE and decreased 0.56-fold in CEE group, and was not significant in soleus muscle. In soleus muscle, myosin heavy chain (MHC) IIa, IIx and IIb decreased in AEE group and MHC IIa and IIx decreased in CEE group. In SVL muscle, MHC I, IIa and IIx increased in AEE group and MHC IIa and IIX increased in CEE group. In summary, both acute and chronic eccentric exercise could lead to change in FOXO1 mRNA only in fast SVL muscle of rat and so could induce fiber type transition in both muscles regardless of changes in expression of FOXO1. So, oxidative stress can play important role in change of FOXO1. Key words: Eccentric exercise, Fiber type transition, Skeletal muscle, Gene expression.
2
ACCEPTED MANUSCRIPT 1. Introduction Skeletal muscle is a quite heterogeneous tissue due to different muscle fiber types (Zawadowska
PT
et al., 2004). Because of various parameters, these fiber types classified into several categories,
RI
that the most useful is based on specific myosin profiles(Pette and Staron, 2001). Also, these enables the muscle to adapt to various functional demands(Pette, 1998; Magaudda et al., 2004).
SC
. Presently, it is believed that fiber type transition follow a systematic sequential order: MHCI ↔
NU
MHCIIa ↔ MHCIIx ↔ MHCIIb. (Pette and Staron, 2001). Physical exercise affected on phenotype of muscle fiber (Pette and Staron, 1997; Pagan, 2009). In most type of exercise,
MA
dynamic contractions consists of two distinct phases: concentric and eccentric (Rivera-Brown and Frontera, 2012). Eccentric contractions are considered as an essential part of any movement,
TE
D
especially as a part of exercise (Kerksick et al., 2013), which the most common example is downhill running (Proske and Morgan, 2001).
AC CE P
These transitions become regulated by many varieties of signaling pathways and transcription factors (Spangenburg and Booth, 2003). One of these factors is forkhead box O1 (FOXO1) (Kamei et al., 2004; Yuan et al., 2011; Sanchez et al., 2013) that belongs to the subfamily FoxO (Forkhead box O) transcription factor (Burgering, 2008; Zhang et al., 2011). A few studies have been investigated the mRNA expression levels of FOXO1 in rat. For instance, Yuan and et al showed that endurance training decreased FOXO1 mRNA in both slow and fast muscle in mice. It also caused loss of FOXO1 function that induced transition of fiber type from fast to slow (Yuan et al., 2011). Kamei and et al showed that the expression of genes related to type I fibers in skeletal muscle of FOXO1 transgenic mice decreased that lead to reducing in wheel running activity (Kamei et al., 2004). It has been shown that oxidative stress is effective on FOXO1 changes(Puig and Mattila, 2011).
3
ACCEPTED MANUSCRIPT There is no documented report addressing the effects of acute/or chronic eccentric exercise on FOXO1 mRNA in slow and fast skeletal muscle of rats by itself or as a factor involved in muscle
PT
fiber transition. Since, FOXO1 has many roles in various cellular processes (Salih and Brunet,
RI
2008; Zhang et al., 2011) and so eccentric contractions play important role in most of movements (Kerksick et al., 2013). The aim of this study is to explore the effects of eccentric
SC
exercise (acute/chronic) on FOXO1 as an important factor of fiber type transition in rat skeletal
NU
muscles. In addition, oxidative stress was investigated by measurement of total oxidant status (TOS) and malondialdehyde (MDA) as serum markers of oxidant stress.
MA
2. Materials and Methods 2.1. Animals
TE
D
Twenty four young male Sprague-Dawley rats (4 weeks old), weighing 190-235 g, were purchased from the Razi Vaccine and Serum Research Institute (Karaj, Iran). This experiment
AC CE P
was conducted according to Australian code for the care and use of animals for scientific purposes 8th Edition 2013. Animals were housed in a temperature and humidity controlled room (23-25 °C, 60-65%) with 12:12 h light-dark cycle, and with free access to pallets chow (Razi.In, Iran) and tap water. After rats arrived and were recruited in our laboratory, they were randomly divided into 3 groups of 8, that including: 1) chronic eccentric exercise (CEE) (227g ± 5.48), 2) acute eccentric exercise (AEE) (201.67g ± 7.89), 3) control (C) (205g ± 15.23). All animals were in laboratory for 1 week to become familiar with the environment. At the age of 5 weeks, animals of group 1 started to get familiar with treadmill running for one week then continued to follow the main protocol (Table 1). In order to coordinate the rats age when sacrificed, in the middle of the eleventh week, animals of group 2 (15 weeks old) became familiarized with treadmill running for a duration of 5 minutes at 15 m/min speed, and started to follow the main
4
ACCEPTED MANUSCRIPT protocol for a period of 3 days immediately afterwards. Animals of control group remained until last training session of acute and chronic groups. Finally animals belonging to all experimental
PT
groups were sacrificed forty-eight hours following the last training session.
RI
2.2. Exercise Protocol 2.2.1. Chronic exercise
SC
At the age of 6 weeks animals of CEE group underwent eccentric exercise on treadmill (Rodent
NU
treadmill 5 lines, Danesh Salar Iranian Co; Iran) 3 days a week lasting over 9 weeks. Downhill running protocol is used as the eccentric exercise for these animals. During the first week,
MA
animals had been scheduled for running on -4° slope of treadmill for 15 min and they gradually managed to decrease the slope to -16° and increase duration of exercise to 90 min during the
TE
D
final week. Training sessions included 1 set running at the speed of 16 m/min. The training protocol for CEE group has been summarized in Table 1.
AC CE P
2.2.2. Acute exercise
Animals of AEE group, at the age of 15 weeks, began treadmill eccentric exercising concurrent with the final week exercise of CEE group and were running on treadmill for 3 consecutive days. According to eccentric exercise protocol described by Chiang and et al (Chiang et al., 2009), after familiarization, they were immediately exposed to the main protocol that included 18 sets of 5 min with rest interval of 2 min in between. The slope of treadmill was -16° and the running speed was 16 m/min. 2.3. Blood and muscle sample preparation Forty eight hours following the last training session, all animals were anesthetized by ketamine (90 mg/kg)-xaillysin (10 mg/kg) mixture administration(Hau and Schapiro, 2002) and then, blood samples were obtained directly from the left auricle by cardiac puncture and immediately
5
ACCEPTED MANUSCRIPT serum samples were separated by centrifugation at 3000 rpm for 10 min and were stored at -80 °C. Also,
slow-type muscle -soleus- and fast-type muscle -super vastus lateralis (SVL)-
RI
promptly frozen in liquid nitrogen and then stored at -80 °C.
PT
(Luginbuhl et al., 1984) were quickly removed from their bodies; muscles were weighed and
2.4. mRNA extraction
SC
Total RNA was extracted from soleus and SVL samples with RNX-Plus solution (Sina gene,
NU
Cat.no N0RN7713C) according to the manufacturer’s protocol. The purity and concentration of the RNA preparation was displayed by means of a spectrophotometer (Thermo scientific, ND
MA
1000) and using agarose gel electrophoresis. Then total RNA was stored at -80°C in RNase-free water before starting the analysis.
TE
D
2.5. Reverse transcription
Based on data regarding the concentrations obtained, 1-10 microgram of the total RNA was
AC CE P
picked and converted to first-strand cDNA (20 µl) by revertaid first strand cDNA synthesis kit (Thermo scientific, #K1622 ). 2.6. Oligonucleotides
The FOXO1 primers were designed by using the Gene Runner program and the myosin heavy chain (MHC) type I, IIa, IIx and IIb isoforms primers were used as designed previously by Kim and et al (Quadrilatero et al., 2011). Finally, all of them were checked with NCBI (PrimerBLAST). Then the oligonucleotide primers for all studied genes were synthesized applying SinaGene Co (Iran, Tehran). The sequences of primers and details are all described in Table 2. Since the gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH gene) has proved to be the most stable responding gene to exercise in slow- and fast- twitch human muscle fiber (Van Daele, 2010), it was chosen as housekeeping gene in our survey.
6
ACCEPTED MANUSCRIPT 2.7. Real time PCR Initially, amplification efficiency was calculated for each pair of primers. Calculated efficiencies
PT
waved approximately 1 (0.99). A Rotor-Gene Q (QIAGEN, Germany) was used to run the real-
RI
time PCR. The following experimental conditions have been set in Table 2 for 40 cycles. For each well, 2µl of cDNA (20 ng) template was used with the master mix (Thermo scientific)
SC
containing SYBR green in a volume of 25µl, and each sample became duplicated. To calculate
NU
the scope of differences between mRNA expression of control and exercise groups, the 2-ΔΔCT method was used.
MA
As a primary step we had to, normalize the CT of the target gene to that of the reference (ref) gene, for both the exercise group and control group: ΔCT (exercise) = CT (target, exercise) – CT (ref, exercise),
D
ΔCT (control) = CT (target, control) – CT (ref, control). Second, normalize the ΔCT of the exercise group to the
TE
ΔCT of the control took place: ΔΔCT = ΔCT (exercise) – ΔCT (control). Finally, the expression ratio was
AC CE P
calculated: 2-ΔΔCT (Cho et al., 2010). (For more details about the derivation of the formula describing the exponential amplification of PCR, please refer to User Bulletin 2: ABI PRISM 7700 Sequence Detection System). 2.8. Measurement of TOS and MDA Serum levels of TOS and MDA were measured using a spectrophotometric assay by commercial kits (ZellBio, GmbH, Veltlinerweg, Germany) with sensitivity 0.5μM and 0.1 μM respectively. Intra-assay CVs for TOS and MDA were 4.8% and 4.2% respectively. 2.9. Statistical analysis Statistical analyses were performed through IBM SPSS Statistics version 22. Since the sample size was rather small and also data of real time PCR were not normally detected by Shapiro-Wilk test (Ghasemi and Zahediasl, 2012), we used Wilcoxon two group test to analyze the two pools
7
ACCEPTED MANUSCRIPT of ΔCT values (Yuan et al., 2006). In the present study, we considered 2-ΔΔCt as the point of estimation of ratio (Yuan et al., 2006), and according to User Bulletin 2: ABI PRISM 7700
PT
Sequence Detection System, mRNA expression (fold change) was showed by ratio ± SEM. Data
RI
of TOS and MDA were detected normal by Shapiro-Wilk test, then t-test was used for comparing each groups with control group, so data are expressed as mean ± SEM. The level of statistical
SC
significance was set as P ≤ 0.05.
NU
3. Results 3.1. Muscle FOXO1 mRNA
MA
In soleus muscle, FOXO1 mRNA expression levels had a 27% increase in AEE and a 20% decrease in CEE group comparing with the control group (Fig1-A). In SVL muscle, FoxO1
TE
D
mRNA expression had a 292% increment in AEE and a 44% decrease in CEE group comparing with the control group (Fig1-B). No significant change were observed between the exercise and
AC CE P
the control except the changes in SVL muscle of AEE group (P
S0378-1119(16)30100-7 doi: 10.1016/j.gene.2016.02.033 GENE 41190
To appear in:
Gene
Received date: Revised date: Accepted date:
27 September 2015 18 February 2016 20 February 2016
Please cite this article as: Azad, Milad, Khaledi, Neda, Hedayati, Mehdi, Effect of acute and chronic eccentric exercise on FOXO1 mRNA expression as fiber type transition factor in rat skeletal muscles, Gene (2016), doi: 10.1016/j.gene.2016.02.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Effect of acute and chronic eccentric exercise on FOXO1 mRNA expression as fiber type transition factor in rat skeletal muscles
PT
Milad Azad1, Neda Khaledi1*, Mehdi Hedayati2
RI
1-Faculaty of Physical Education and Sport Science, Kharazmi University, Tehran, Iran. 2-Cellular and Molecular Research Center, Research Institute for Endocrine Sciences, Shahid
SC
Beheshti University of Medical Sciences, Tehran, Iran.
NU
Running title: Acute and chronic eccentric exercise and FOXO1 mRNA in rat skeletal muscles Abbreviations: FoxO, Forkhead box O; MHC, myosin heavy chain; SVL, super vastus lateralis;
MA
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TOS, total oxidative status; MDA, malondialdehyde;
TE
Dr.Neda Khaledi, Ph.D.
D
*Corresponding author
AC CE P
Faculty of Physical Education and Sport Science, Kharazmi University, Tehran, Iran. Phone: +98 2225808
Email: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Skeletal muscle is a highly elastic tissue which can respond to various functional demands by
PT
altering fiber type composition. Exercise effects on muscle fiber phenotype. One of the
RI
transcription factors that induce fiber type transition is forkhead box O1 (FOXO1). Since eccentric contraction considered an essential part of exercise, so we are interesting to see the
SC
effects of eccentric exercise (acute/chronic) on FOXO1 as an important factor of fiber type
NU
transition in rat skeletal muscles. Twenty four Sprague-dawley rats (190-235 g) divided to 3 groups of 8 rats: 1) chronic eccentric exercise (CEE), 2) acute eccentric exercise (AEE) and 3)
MA
control (C). The exercise groups underwent downhill running protocol. CEE was running on treadmill in 3 days of week for 9 weeks, that slope and duration gradually managed from -4° to -
TE
D
16° and 15 min to 90 min, respectively. AEE group was running with 16 m/min on -16° slope for 3 consecutive day that included 18 sets of 5 min with rest interval of 2 min in between. Soleus
AC CE P
and super vastus lateralis (SVL) muscles mRNA were analyzed by real-time RT-PCR. SVL FOXO1 mRNA levels increased by 3.92-fold in AEE and decreased 0.56-fold in CEE group, and was not significant in soleus muscle. In soleus muscle, myosin heavy chain (MHC) IIa, IIx and IIb decreased in AEE group and MHC IIa and IIx decreased in CEE group. In SVL muscle, MHC I, IIa and IIx increased in AEE group and MHC IIa and IIX increased in CEE group. In summary, both acute and chronic eccentric exercise could lead to change in FOXO1 mRNA only in fast SVL muscle of rat and so could induce fiber type transition in both muscles regardless of changes in expression of FOXO1. So, oxidative stress can play important role in change of FOXO1. Key words: Eccentric exercise, Fiber type transition, Skeletal muscle, Gene expression.
2
ACCEPTED MANUSCRIPT 1. Introduction Skeletal muscle is a quite heterogeneous tissue due to different muscle fiber types (Zawadowska
PT
et al., 2004). Because of various parameters, these fiber types classified into several categories,
RI
that the most useful is based on specific myosin profiles(Pette and Staron, 2001). Also, these enables the muscle to adapt to various functional demands(Pette, 1998; Magaudda et al., 2004).
SC
. Presently, it is believed that fiber type transition follow a systematic sequential order: MHCI ↔
NU
MHCIIa ↔ MHCIIx ↔ MHCIIb. (Pette and Staron, 2001). Physical exercise affected on phenotype of muscle fiber (Pette and Staron, 1997; Pagan, 2009). In most type of exercise,
MA
dynamic contractions consists of two distinct phases: concentric and eccentric (Rivera-Brown and Frontera, 2012). Eccentric contractions are considered as an essential part of any movement,
TE
D
especially as a part of exercise (Kerksick et al., 2013), which the most common example is downhill running (Proske and Morgan, 2001).
AC CE P
These transitions become regulated by many varieties of signaling pathways and transcription factors (Spangenburg and Booth, 2003). One of these factors is forkhead box O1 (FOXO1) (Kamei et al., 2004; Yuan et al., 2011; Sanchez et al., 2013) that belongs to the subfamily FoxO (Forkhead box O) transcription factor (Burgering, 2008; Zhang et al., 2011). A few studies have been investigated the mRNA expression levels of FOXO1 in rat. For instance, Yuan and et al showed that endurance training decreased FOXO1 mRNA in both slow and fast muscle in mice. It also caused loss of FOXO1 function that induced transition of fiber type from fast to slow (Yuan et al., 2011). Kamei and et al showed that the expression of genes related to type I fibers in skeletal muscle of FOXO1 transgenic mice decreased that lead to reducing in wheel running activity (Kamei et al., 2004). It has been shown that oxidative stress is effective on FOXO1 changes(Puig and Mattila, 2011).
3
ACCEPTED MANUSCRIPT There is no documented report addressing the effects of acute/or chronic eccentric exercise on FOXO1 mRNA in slow and fast skeletal muscle of rats by itself or as a factor involved in muscle
PT
fiber transition. Since, FOXO1 has many roles in various cellular processes (Salih and Brunet,
RI
2008; Zhang et al., 2011) and so eccentric contractions play important role in most of movements (Kerksick et al., 2013). The aim of this study is to explore the effects of eccentric
SC
exercise (acute/chronic) on FOXO1 as an important factor of fiber type transition in rat skeletal
NU
muscles. In addition, oxidative stress was investigated by measurement of total oxidant status (TOS) and malondialdehyde (MDA) as serum markers of oxidant stress.
MA
2. Materials and Methods 2.1. Animals
TE
D
Twenty four young male Sprague-Dawley rats (4 weeks old), weighing 190-235 g, were purchased from the Razi Vaccine and Serum Research Institute (Karaj, Iran). This experiment
AC CE P
was conducted according to Australian code for the care and use of animals for scientific purposes 8th Edition 2013. Animals were housed in a temperature and humidity controlled room (23-25 °C, 60-65%) with 12:12 h light-dark cycle, and with free access to pallets chow (Razi.In, Iran) and tap water. After rats arrived and were recruited in our laboratory, they were randomly divided into 3 groups of 8, that including: 1) chronic eccentric exercise (CEE) (227g ± 5.48), 2) acute eccentric exercise (AEE) (201.67g ± 7.89), 3) control (C) (205g ± 15.23). All animals were in laboratory for 1 week to become familiar with the environment. At the age of 5 weeks, animals of group 1 started to get familiar with treadmill running for one week then continued to follow the main protocol (Table 1). In order to coordinate the rats age when sacrificed, in the middle of the eleventh week, animals of group 2 (15 weeks old) became familiarized with treadmill running for a duration of 5 minutes at 15 m/min speed, and started to follow the main
4
ACCEPTED MANUSCRIPT protocol for a period of 3 days immediately afterwards. Animals of control group remained until last training session of acute and chronic groups. Finally animals belonging to all experimental
PT
groups were sacrificed forty-eight hours following the last training session.
RI
2.2. Exercise Protocol 2.2.1. Chronic exercise
SC
At the age of 6 weeks animals of CEE group underwent eccentric exercise on treadmill (Rodent
NU
treadmill 5 lines, Danesh Salar Iranian Co; Iran) 3 days a week lasting over 9 weeks. Downhill running protocol is used as the eccentric exercise for these animals. During the first week,
MA
animals had been scheduled for running on -4° slope of treadmill for 15 min and they gradually managed to decrease the slope to -16° and increase duration of exercise to 90 min during the
TE
D
final week. Training sessions included 1 set running at the speed of 16 m/min. The training protocol for CEE group has been summarized in Table 1.
AC CE P
2.2.2. Acute exercise
Animals of AEE group, at the age of 15 weeks, began treadmill eccentric exercising concurrent with the final week exercise of CEE group and were running on treadmill for 3 consecutive days. According to eccentric exercise protocol described by Chiang and et al (Chiang et al., 2009), after familiarization, they were immediately exposed to the main protocol that included 18 sets of 5 min with rest interval of 2 min in between. The slope of treadmill was -16° and the running speed was 16 m/min. 2.3. Blood and muscle sample preparation Forty eight hours following the last training session, all animals were anesthetized by ketamine (90 mg/kg)-xaillysin (10 mg/kg) mixture administration(Hau and Schapiro, 2002) and then, blood samples were obtained directly from the left auricle by cardiac puncture and immediately
5
ACCEPTED MANUSCRIPT serum samples were separated by centrifugation at 3000 rpm for 10 min and were stored at -80 °C. Also,
slow-type muscle -soleus- and fast-type muscle -super vastus lateralis (SVL)-
RI
promptly frozen in liquid nitrogen and then stored at -80 °C.
PT
(Luginbuhl et al., 1984) were quickly removed from their bodies; muscles were weighed and
2.4. mRNA extraction
SC
Total RNA was extracted from soleus and SVL samples with RNX-Plus solution (Sina gene,
NU
Cat.no N0RN7713C) according to the manufacturer’s protocol. The purity and concentration of the RNA preparation was displayed by means of a spectrophotometer (Thermo scientific, ND
MA
1000) and using agarose gel electrophoresis. Then total RNA was stored at -80°C in RNase-free water before starting the analysis.
TE
D
2.5. Reverse transcription
Based on data regarding the concentrations obtained, 1-10 microgram of the total RNA was
AC CE P
picked and converted to first-strand cDNA (20 µl) by revertaid first strand cDNA synthesis kit (Thermo scientific, #K1622 ). 2.6. Oligonucleotides
The FOXO1 primers were designed by using the Gene Runner program and the myosin heavy chain (MHC) type I, IIa, IIx and IIb isoforms primers were used as designed previously by Kim and et al (Quadrilatero et al., 2011). Finally, all of them were checked with NCBI (PrimerBLAST). Then the oligonucleotide primers for all studied genes were synthesized applying SinaGene Co (Iran, Tehran). The sequences of primers and details are all described in Table 2. Since the gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH gene) has proved to be the most stable responding gene to exercise in slow- and fast- twitch human muscle fiber (Van Daele, 2010), it was chosen as housekeeping gene in our survey.
6
ACCEPTED MANUSCRIPT 2.7. Real time PCR Initially, amplification efficiency was calculated for each pair of primers. Calculated efficiencies
PT
waved approximately 1 (0.99). A Rotor-Gene Q (QIAGEN, Germany) was used to run the real-
RI
time PCR. The following experimental conditions have been set in Table 2 for 40 cycles. For each well, 2µl of cDNA (20 ng) template was used with the master mix (Thermo scientific)
SC
containing SYBR green in a volume of 25µl, and each sample became duplicated. To calculate
NU
the scope of differences between mRNA expression of control and exercise groups, the 2-ΔΔCT method was used.
MA
As a primary step we had to, normalize the CT of the target gene to that of the reference (ref) gene, for both the exercise group and control group: ΔCT (exercise) = CT (target, exercise) – CT (ref, exercise),
D
ΔCT (control) = CT (target, control) – CT (ref, control). Second, normalize the ΔCT of the exercise group to the
TE
ΔCT of the control took place: ΔΔCT = ΔCT (exercise) – ΔCT (control). Finally, the expression ratio was
AC CE P
calculated: 2-ΔΔCT (Cho et al., 2010). (For more details about the derivation of the formula describing the exponential amplification of PCR, please refer to User Bulletin 2: ABI PRISM 7700 Sequence Detection System). 2.8. Measurement of TOS and MDA Serum levels of TOS and MDA were measured using a spectrophotometric assay by commercial kits (ZellBio, GmbH, Veltlinerweg, Germany) with sensitivity 0.5μM and 0.1 μM respectively. Intra-assay CVs for TOS and MDA were 4.8% and 4.2% respectively. 2.9. Statistical analysis Statistical analyses were performed through IBM SPSS Statistics version 22. Since the sample size was rather small and also data of real time PCR were not normally detected by Shapiro-Wilk test (Ghasemi and Zahediasl, 2012), we used Wilcoxon two group test to analyze the two pools
7
ACCEPTED MANUSCRIPT of ΔCT values (Yuan et al., 2006). In the present study, we considered 2-ΔΔCt as the point of estimation of ratio (Yuan et al., 2006), and according to User Bulletin 2: ABI PRISM 7700
PT
Sequence Detection System, mRNA expression (fold change) was showed by ratio ± SEM. Data
RI
of TOS and MDA were detected normal by Shapiro-Wilk test, then t-test was used for comparing each groups with control group, so data are expressed as mean ± SEM. The level of statistical
SC
significance was set as P ≤ 0.05.
NU
3. Results 3.1. Muscle FOXO1 mRNA
MA
In soleus muscle, FOXO1 mRNA expression levels had a 27% increase in AEE and a 20% decrease in CEE group comparing with the control group (Fig1-A). In SVL muscle, FoxO1
TE
D
mRNA expression had a 292% increment in AEE and a 44% decrease in CEE group comparing with the control group (Fig1-B). No significant change were observed between the exercise and
AC CE P
the control except the changes in SVL muscle of AEE group (P
