Mioglobin Gen Expression In Soleus Muscle, Gastrocnemius And Cardiac Muscle After Aerobic And Anaerobic Exercise In Wistar Rat Muscle.
MIOGLOBIN GEN EXPRESSION IN SOLEUS MUSCLE,
GASTROCNEMIUS AND CARDIAC MUSCLE
AFTER AEROBIC AND ANAEROBIC EXERCISE
IN WISTAR RAT MUSCLE
Reni Farenia* Roni Lesmana* Ambrosius Purba* Ieva B.Akbar*
Nurhalim Shahib** Noriyuki Koibuchi *** Noriaki Shimokawa***
*Departement of Physiology, Faculty of Medicine, Universitas
Padjadjaran
Bandung, Indonesia
** Departemen of Biochemistry, Faculty of Medicine, Universitas
Padjadjaran
***Departement of Integrative Physiology, Graduate School of
Medicine
Gunma University, Maebashi, Japan
Address for correspondence :
Dr.Reni Farenia ,dr.,M.Kes.,AIF
Bagian Ilmu Faal Fakultas Kedokteran Universitas Padjadjaran
Jl Raya Jatinangor Sumedang Km 21 . Jatinangor
email : [email protected]
0
MYOGLOBIN GENE EXPRESSION OF SOLEUS, GASTROCNEMIUS
AND CARDIAC MUSCLES
AFTER AEROBIC AND ANAEROBIC EXERCISE
IN WISTAR RAT
Reni Farenia*, Roni Lesmana*, Ambrosius Purba* Ieva B.Akbar*
Nurhalim Shahib** Noriyuki Koibuchi ***, Noriaki Shimokawa***
ABSTRACT
Myoglobin (Mb) is a cytoplasmic hemoprotein and have an
important role in oxygen binding, as an oxygen transporter and storage to
maintain physiological process for human body adaptation in hypoxia
condition. The role of exercise in enhancing physical fitness is already well
known. Unfortunately this statement have not any scientific reason in
mRNA myoglobin and its level related to aerobic and anaerobic exercise
and also its function as an hypoxia indicator
Animal experimental study about mRNA Mb of soleus,
gastrocnemius and cardiac muscle was done using 15 Wistar rats (Rattus
Norvegicus) in Laboratorium of Integrative Physiology Graduate school of
Medicine, Gunma University Japan. . The aim of this study was to know
the difference effect between aerobic and anaerobic exercises using
animal treadmill on myoglobin gene (mRNA) expression as an indicator of
tissue hypoxia.. mRNA Mb concentration was examined by AGPC method
and RT- PCR was done to know the difference effect between aerobic and
anaerobic
exercise
on
myoglobin
gene
expression
of
soleus,gastrocnemius and cardiac muscles.
The statistical analysis using Anova on myoglobin gene expression
(mRNAMb) showed very significant (p˂0,01) that aerobic group is higher
than anaerobic group, in soleus muscle (0,9303±0,0,0268 vs
0,7716±0,0250
µgr/ml), gastrocnemius muscle (1,0504±0,0531 vs
0,8760±0,0147µgr/ml) and significant (p≤0,05) in cardiac muscle
(0,7249±0,0600 vs 0,6143±0,0277µgr/ml). Also the RT-PCR showed that
mRNA myoglobin of soleus,gastrocnemius and cardiac muscle was
stronger in aerobic than anaerobic exercise,
In conclusion myoglobin gene expression in soleus,gastrocnemius
and cardiac muscles was higher and stronger gene expression after
aerobic than anaerobic exercise as an indicator of tissue hypoxia.
Key words : : mRNA myoglobin, aerobic,anaerobic
INTRODUCTION
1
Myoglobin(Mb) is a cytoplasmic hemoprotein, expressed in skeletal
muscle and myocardium and reversibly to binds oxygen. The structure of
myoglobin was first delineated by John Kendrew and colleagues over
40·years ago.1,-4. Myoglobin as a transport and Oxygen storage is very
important to mantain physiological process for adaptation in hypoxia
condition Strenuous exercise cause muscle myalgia, fatique and cramp
can decrease atlet performans.5,6 In pathologic condition there are much of
health problem causes by hypoxia . The role of exercise in enhancing
physical fitness is already well known. Unfortunately this statement have
not any scientific research in mRNA myoglobin and its level related to
aerobic and anaerobic exercise and also its function as an hypoxia
indicator
It is nice to know that myoglobin have an important role than
haemoglobin in hypokxia condition. Myoglobin as an oxygen transporter
and storage can maintain physiological process for human body
adaptation in hypoxia condition3,4,,9 The important physiological features of
oxymyoglobin (MbO2) equilibrium curve are that lies from the right to the
left of oxyhaemoglobin (HbO2) equilibrium it has a hyperbolic shape. When
the partiil pressure is low, saturation of MbO 2 is lower than HbO2.1,2
Oxygen storage in myoglobin is important for intermitten exercise. 1,3,4 The
use of gene targetting and molecular biological techniques has revealed
new insight into reulation and myoglobin function,. The aims of the study is
to know mRNA myoglobin changes in skeletal and cardiac muscle after
aerobic and anaerobic exercise. Myoglobin is a cytoplasmic haemoprotein
that expressed in skeletal muscle and cardiac muscle that reversibly binds
O2 by its heme residue a phorphyrin ring from ion complex. 1,3,4 Training in
hypoxia result at the molecular level an upregulation of the regulatory
submit of hypoxia induced factor-1(HIF-1). 17,,19 Possibly as a consequence
of this upregulation of HIF-1 as a transcription factor, the level of mRNA
myoglobin (mRNAMb) increased. Myoglobin are synthesized according to
information in messenger RNA Mb that is synthesized on DNA template by
transcription and it depends on the need of oxygen in the cell. 2,3,5,18 During
aerobic exercise a local hypoxia will stimulate the HIF-1. During anaerobic
exercise , supply of oxygen will decrease until there is no oxygen supply
led to hypoxia . Hypoxia condition will stimulate HIF-1 ,as a transcription
factor to initiate the mRNA production . 17,19,24 So, both aerobic and
anaerobic exercise will increase the mRNA Mb. However ,it has been
reported that that strenous exercise will disrupt the sarcolemna and then
the Mb from the intracellullar enter the circulation. We can found the serum
myoglobin will increase after strenous exercise. 22,23
During aerobic metabolism, oxygen supplied from the storage in
the myoglobin. A cornestone of respiratory regulation stands on the
capasity of Mb to store or fasilitate oxygen transport, In marine mammals
the high consentration of Mb could certainly supply oxygen during a dive
or apnoe,1,3,4 In adaptation to high altitude enhanced Mb expression
2
increases the oxygen depot. The observation agree that with the
correlation between Mb concentration as oxygen supply and oxidative
capasity in different species. 4,6 Without any Mb. neither cardiac muscle
nor skeletal muscle
functions suffers any apparent physiological
impairment.6-10 Muscle strenght and function of muscle correlate with
myoglobin concentration. As we know that muscle strength and function
of myoglobin will increase after 6 week exercise. 4,5 But the physiological
role of Mb have not yet been establish. Recently we can know the
increase of myoglobin from the mRNA Mb.
These studies imply that further investigations are required to
reveal the physiological role of Mb in aerobic and anaerobic exercise.
Increased gene expression during recovery from consecutive aerobic and
anaerobic exercise measured
in mRNA Mb of soleus muscle,
gastrocnemius muscle and cardiac muscle. Soleus muscle and
Gastrocnemius muscle is a skeletal muscle ,but the soleus muscle is a
fast fiber that have rich of myoglobin and gastrocnemius muscle is a slow
twich fiber that less myoglobin. Cardiac muscle is aspecial muscle with
synsitium and rich of myoglobin too.5,6,15
Animal experimental study using Male Wistar Rat, 8 weeks old.The
study conducted with three groups consisted aerobic groups, anaerobic
groups and control groups. Animal treadmill test for aerobic groups 15
m/minute runs for 30 minute and for anaerobic groups groups 25 m/minute
runs for 30 minute, was done during 14 days. 8 RT- PCR was done to
evaluate the difference effect between aerobic and anaerobic physical
activity on myoglobin gene (mRNA) expression on skeletal and
myocardium. . All experimental prosedures were conducted in accordance
with the Japanese Physiological Society Guidelines for care and use of
laboratory animal, as approved by Gunma University Japan, Graduate
School of Medicine Committee on animal care and use.
The objectives of this study is to know the different effect of aerobic
and anaerobic exercise to the mRNA Mb in soleus muscle, gastrocnemius
muscle and cardiac muscle.
METHODS
Animal. Male Wistar rat(N=15), ages 8 weeks, weight 280 – 315 gr,
SPF were used in this study. The animal were kept in standard individual
cages and provided with food and water ad libitum. The room temperature
was kept 23±10 C , and a 12:12 hour light-dark cycle environment was
maintained throughout the experiment. All experimental prosedures were
conducted in accordance with the Japanese Physiological Society
Guidelines for care and use of laboratory animal, as approved by Gunma
University Japan, Graduate School of Medicine Committee on animal care
and use.
Independent variable is aerobic and anaerobic. Dependent variable is
mRNA myoglobin from m .soleus, m gastrocnemius and cardiac muscle.
3
The subject divided into three groups consisted aerobic groups, anaerobic
groups and control groups. Animal treadmill test for aerobic groups 15
m/minute runs and for anaerobic groups 25 m/minute runs for 30 minute
was done during 14 days. 8.At the end of research (day 14) , under
anasthetized ether the subject was sacrifice to get the muscles. To prevent
contamination with haemoglobin, before sacrifice ,perfussion with
DiethylPyrocarbonat 97% (DEPC) was done. After excess connective
tissue and fat were remove, each muscle immediately frozen by liquid
Nitrogen and stored at -800 C.
After all samples were collcted we make RNA extraction using AGPC
method to measured the mRNA concentration and to know the mRNA Mb
gene expression we used RT-PCR.
The myoglobin expression were read by sofware Kodak 2500 under UV
light.
All data reported as mean±SD. An analysis of variance (ANOVA) was used
to determine overall differences and Duncan’s post hoc analyses were
used to compare control , aerobic and anaerobic post exercised data. All
data processed using SPSS 15.0, p˂0,
RESULT :
TABLE 1
The difference of mRNA myoglobin concentration of soleus muscle after
14 days aerobic and anaerobic exercise
control
15m/min 25m/min
Mean of
Intensity
0.931648 0.930314 0.771628
SD
0.039878 0.026858 0.025086
After 14 days aerobic and anaerobics intermitten exercise , the mRNA Mb
of soleus muscle after aerobics exercise is higher than anaerobics
exercise.
Table 2
The difference of mRNA myoglobin concentration of
gastrocnemius muscle after 14 days aerobic and anaerobic exercise
4
Control
Mean of
intensity
SD
15m/min
25m/min
0.869887 1.050406 0.876036
0.044526 0.053192 0.014795
After 14 days aerobic and anaerobics intermitten exercise , the mRNA Mb
of gastrocnemius muscle after aerobics exercise is higher than
anaerobics exercise
Table 3
The difference of mRNA myoglobin concentration of cardiac muscle after
14 days aerobic and anaerobic exercise
Control
Mean of
Intensity
SD
15m/min
25m/min
0.886129 0.724951 0.614331
0.052121 0.060031
0.02772
After 14 days aerobic and anaerobics intermitten exercise , the mRNA Mb
of cardiac muscle after aerobics exercise is higher than anaerobics
exercise
As showed in table 1,2 and 3 , mRNA Mb concentration is increase higher
after 14 days aerobic exercise compare than after anaerobics exercise.
The ANOVA among groups led to be increase in aerobic group higher
than anaerobic group.
As showed in table 1 for soleus muscle( 0.930314 ±
0.053192 vs
0.771628±0.025086 µgr/ml),
as
showed
in
table
2
for
gastrocnemius
muscle(1.050406±0.060031vs0.876036 ± 0.014795 µgr/ml ),
and as showed in table 3 for cardiac muscle
(0.724951±0.060031
µgr/ml vs 0.614331±0.02772 µgr/ml). p≤05
Myoglobin gene expression is increase also showed in the figure 1,2 and
3 the electrophoresis of mRNA Mb . Myoglobin gene expression showed
stronger after aerobic exercise compare to after anaerobic exercise in
soleus muscle, gastrocnemius muscle and cardiac muscle. The mRNA
myoglobin expression showed in 330 bp .
Figure 1
5
Changes in mRNA myoglobin of soleus muscle after aerobic and
anaerobic exercise .
Figure 1. showed mRNA gene expression of soleus muscle, after aerobic
exercise is more stronger mRNA Mb gene expression (330bp) than after
anaerobic exercise. Also the intensity of mRNA Mb of soleus muscle
showed very significant higher after aerobic exercise than anaerobic. (
0.930314 ± 0.053192 vs 0.771628±0.025086 µgr/ml), p≤0,01
Figure 2
Changes in mRNA myoglobin of gastrocnemius muscle after aerobic and
anaerobic exercise
6
Figure 2, Using RT-PCR showed mRNA Mb electrophorese of
gastrocnemius muscle, after aerobic exercise is more stronger mRNA Mb
gene expression (330bp) than after anaerobic exercise. Also the intensity
of mRNA Mb of gastrocnemius muscle showed very significant higher after
aerobic exercise than anaerobic. (1.050406±0.060031 vs 0.876036 ±
0.014795 µgr/ml ), p≤0,01
Figure 3.
Changes in mRNA myoglobin expression of cardiac muscle after aerobic
and anaerobic exercise
7
Figure 3. Using RT-PCR showed mRNA Mb electrophorese of cardiac
muscle, after aerobic exercise is more stronger mRNA Mb gene
expression (330bp) than after anaerobic exercise. Also the intensity of
mRNA Mb of cardiac muscle showed higher after aerobic exercise than
anaerobic.(0.724951±0.060031 µgr/ml vs 0.614331±0.02772 µgr/ml). p≤05
CONCLUSION
As summarized in the Figure 1, showed the mRNA Mb of soleus muscle is
very significant higher after aerobic exercise than after anaerobic exercise.
(p≤0,01)
Also the electrophoresis show stronger mRNA expression in the soleus
muscle after aerobic exercise.
As summarized in the Figure 2, showed the mRNA Mb of gastrocnemius
muscle is significant higher after aerobic exercise than after anaerobic
exercise. (p≤0,01)
Also the electrophoresis show stronger mRNA expression in the
gastrocnemius muscle after aerobic exercise.
As summarized in the Figure 3, showed the mRNA Mb of cardiac muscle
is significant higher after aerobic exercise than after anaerobic exercise.
(p≤0,05)
8
Also the electrophoresis show stronger mRNA expression in the cardiac
muscle after aerobic exercise than after anaerobic exercise.
All of the mRNA Mb in soleus muscle, gastrocnemius muscle and
cardiac muscle after anaerobic exercise is lower than after aerobic
exercise . This showed that streunous exercise can caused disruption of
the cell that led to the myoglobin enter the extracellular or the circulation,
so we can found the myoglobin in the serum.
DISCUSSION
It was previously demonstrated that, when different exercise
aerobic and anaerobic give different effect to the mRNA Mb expression. 1,3,4,
Gas mixtures containing 21-100% oxygen were inhaled during aerobic
exercise , the oxygen content in arterial and venous blood flowing from
working muscles and the time of work without fatique. This accompanied
by a higher oxygen uptake by muscles, Aerobic exercise will increase
oxygen demand lead to relatif hypoxia because the supply is also
increase,2,5 This condition will stimulate the HIF-1 to increase . HIF-1 as a
factor for transcription will initiate to increase the mRNA Mb 1,4,10-12. It is
means the gene expression of myoglobin will increase after aerobic
exercise. In the other hand, during the anaerobic exercise , demand of
oxygen increase but there is no supply , this condition will lead to
hypoxia.22,24 So the HIF-1 also increase. Activation of of HIF-1 appears to
trigger adaptations which diminish the negative effects of chronic exposure
to hypoxia. 23,24
This study showed that aerobic and anaerobic physical activity
increased the mRNA Mb in soleus muscle, gastrocnemius muscle and
cardiac muscle. Whatever the type of exercise can increase mRNA Mb ,
but as we know streunous exercise will lead to anaerobic metabolism . 5,6,12
Anaerobic metabolism will increase lactate production that lead the
decreased pH in the tissue and circulation. 5,6,21 This condition can damage
the sarcolemma anddisrupt the cell. Myoglobin as a macromolecul globin
will enter the extracellular fluid and circulation. 16,17 We can found the
myoglobin in the serum., so the myoglobin content in the muscle tissue will
decrease.
In conclusion myoglobin gene expression in soleus,gastrocnemius
and cardiac muscles was higher and stronger after aerobic than anaerobic
exercise as an indicator of tissue hypoxia. Aerobic exercise is better to
increase the mRNA myoglobin , so it can increase the physical fittness by
maintain supply oxygen to the cells or tissue, even though in hypoxia
condition .
REFERENCES
9
Ordway GA, Garry DJ. Myoglobin an essential hemoprotein in
striated muscle. J Exp Biol.2004;207 : 3441-46
2. Mooren FC, Volker K. Molecular and Cellular Exercise Physiology,
Human kinetics, USA.2005:31-46:55-101
3. Hoppeler H, Vogt M. Muscle tissue adaptions to hypoxia. The Journal
of Experimental Biology 204, 3133-3139 (2001)
4. Wittenberg JB, Wittenberg BA. Myoglobin function reassessed. J Exp
Biol. 2003; 206:2011-20.
5. Foss ML, Keteyian SJ. Physiological basis for exercise and sport,
Mc.Graw Hill New York.2006;59-64:
6. Guyton AC, Hall JE. Textbook of Medical Physiology 11 th edition.
Elsevier Saunders, Philadelphia, Pensylvania.2006;79-82;530;1056-60
7. Grange RW, Meeso A, Chin E, Lau KS, Stull JT, Shelton JM et al.
Functional and molecular adaptation in skeletal muscle of myoglobinmutant mice. AJP Cell Physiol .2001; 281:1487-94.
8. Soya H, Mukai A, Deocaris, Ohiwa N, Nishijima, Fujikawa T et al.
Threshold like pattern of neuronal activation on the hypothalamus
during treadmill running : Estasblishment of minimum running stress
(MRS) rat model. Neuroscience research.2007;58: 341- 8
9. Wilson MT. Reeder BJ.Oxygen binding haem Protein .J Exp Physiol.
2008;93:128-132
10. Brunori, M. Nitric oxide,cytochromme oxidase and myoglobin, Trends
Biochem. scie. 2001; 26: 21-23
11. Brunori, M.. Nitric oxide moves myoglobin centre stage. Trends
Biochem.scie. 2001; 26 : 209-21
12. Wittenberg B.A , Wittenberg JB. Role of Myoglobin in oksigen supply
to red skeletal muscle.J Biol Chem .2005 ;250: 9038-43
13. Ulrike B, Hendgen C, Marc WM, Sruti S, Schmits J, Stefanie B Johan
et al. Nitrite reductase actvity of myoglobin regulates respiration and
cellular viability in myocardial ischemia-reperfusion injury.
PNAS.2008;22.
14. Flogel U, Merck M.W,Godecke AL., Decking UKM, Schrader Jurgens,
Myoglobin : A scavenger of Bioactive NO.PNAS. 2001
15. Mc.Comas AJ.,Gardiner PF. Skeletal Muscle form and function. Human
Kinetics., USA,2006:215-220.
16. Akitoshi, Seiyama.Virtual cooperativity in myoglobin oxygen saturation
curve in skeletal muscle in vivo. J. of Dynamic Medicine .2006
17. Kanatous SB, Mammen P, Rosenberg. PB. Martin.CM, Michael DW,
Garry, DJ et al. Hypoxia reprograms calcium signaling and regulates
myoglobin expression. Am J Physiol .2008;296:C393-C402.
18. Shahib.N . Biologi Molekuler Medik I .2005
19. Vogt M, Puntschart A, Geiser J, Zuleger C, Hoppeller. Molecular
adaptation in human skeletal muscle to endurance training under
simulated hypoxic conditions. J appl. Physiol. 2001;91:179-182.
1.
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20. Friedman,B, Kincherf, Borish.S, Richter.G.Effect of low resistance/high
repetition strengh training in hypoxia on muscle structure and gene
expression. Eur.J. Physiol. 2003;446:742-51.
21. Despopoulos. Color Atlas of Physiology. Thieme 2003
22. Zoll J, Ponsot E, Dufour S, Doutreleau S. Exercise training in
normobaric hypoxia in endurance runners. J Appl Physiol 2006. 100:
1258-66.
23. Meyer RA. Aerobic performance and the function of myoglobin in
human skeletal muscle. AJP-regul 2004;287:1304-05
24. Wittenberg BA.Both hypoxia and work are required to enhance
expression of myoglobin in skeletal muscle. AJP Cell
Physiol.2009;296.
11
GASTROCNEMIUS AND CARDIAC MUSCLE
AFTER AEROBIC AND ANAEROBIC EXERCISE
IN WISTAR RAT MUSCLE
Reni Farenia* Roni Lesmana* Ambrosius Purba* Ieva B.Akbar*
Nurhalim Shahib** Noriyuki Koibuchi *** Noriaki Shimokawa***
*Departement of Physiology, Faculty of Medicine, Universitas
Padjadjaran
Bandung, Indonesia
** Departemen of Biochemistry, Faculty of Medicine, Universitas
Padjadjaran
***Departement of Integrative Physiology, Graduate School of
Medicine
Gunma University, Maebashi, Japan
Address for correspondence :
Dr.Reni Farenia ,dr.,M.Kes.,AIF
Bagian Ilmu Faal Fakultas Kedokteran Universitas Padjadjaran
Jl Raya Jatinangor Sumedang Km 21 . Jatinangor
email : [email protected]
0
MYOGLOBIN GENE EXPRESSION OF SOLEUS, GASTROCNEMIUS
AND CARDIAC MUSCLES
AFTER AEROBIC AND ANAEROBIC EXERCISE
IN WISTAR RAT
Reni Farenia*, Roni Lesmana*, Ambrosius Purba* Ieva B.Akbar*
Nurhalim Shahib** Noriyuki Koibuchi ***, Noriaki Shimokawa***
ABSTRACT
Myoglobin (Mb) is a cytoplasmic hemoprotein and have an
important role in oxygen binding, as an oxygen transporter and storage to
maintain physiological process for human body adaptation in hypoxia
condition. The role of exercise in enhancing physical fitness is already well
known. Unfortunately this statement have not any scientific reason in
mRNA myoglobin and its level related to aerobic and anaerobic exercise
and also its function as an hypoxia indicator
Animal experimental study about mRNA Mb of soleus,
gastrocnemius and cardiac muscle was done using 15 Wistar rats (Rattus
Norvegicus) in Laboratorium of Integrative Physiology Graduate school of
Medicine, Gunma University Japan. . The aim of this study was to know
the difference effect between aerobic and anaerobic exercises using
animal treadmill on myoglobin gene (mRNA) expression as an indicator of
tissue hypoxia.. mRNA Mb concentration was examined by AGPC method
and RT- PCR was done to know the difference effect between aerobic and
anaerobic
exercise
on
myoglobin
gene
expression
of
soleus,gastrocnemius and cardiac muscles.
The statistical analysis using Anova on myoglobin gene expression
(mRNAMb) showed very significant (p˂0,01) that aerobic group is higher
than anaerobic group, in soleus muscle (0,9303±0,0,0268 vs
0,7716±0,0250
µgr/ml), gastrocnemius muscle (1,0504±0,0531 vs
0,8760±0,0147µgr/ml) and significant (p≤0,05) in cardiac muscle
(0,7249±0,0600 vs 0,6143±0,0277µgr/ml). Also the RT-PCR showed that
mRNA myoglobin of soleus,gastrocnemius and cardiac muscle was
stronger in aerobic than anaerobic exercise,
In conclusion myoglobin gene expression in soleus,gastrocnemius
and cardiac muscles was higher and stronger gene expression after
aerobic than anaerobic exercise as an indicator of tissue hypoxia.
Key words : : mRNA myoglobin, aerobic,anaerobic
INTRODUCTION
1
Myoglobin(Mb) is a cytoplasmic hemoprotein, expressed in skeletal
muscle and myocardium and reversibly to binds oxygen. The structure of
myoglobin was first delineated by John Kendrew and colleagues over
40·years ago.1,-4. Myoglobin as a transport and Oxygen storage is very
important to mantain physiological process for adaptation in hypoxia
condition Strenuous exercise cause muscle myalgia, fatique and cramp
can decrease atlet performans.5,6 In pathologic condition there are much of
health problem causes by hypoxia . The role of exercise in enhancing
physical fitness is already well known. Unfortunately this statement have
not any scientific research in mRNA myoglobin and its level related to
aerobic and anaerobic exercise and also its function as an hypoxia
indicator
It is nice to know that myoglobin have an important role than
haemoglobin in hypokxia condition. Myoglobin as an oxygen transporter
and storage can maintain physiological process for human body
adaptation in hypoxia condition3,4,,9 The important physiological features of
oxymyoglobin (MbO2) equilibrium curve are that lies from the right to the
left of oxyhaemoglobin (HbO2) equilibrium it has a hyperbolic shape. When
the partiil pressure is low, saturation of MbO 2 is lower than HbO2.1,2
Oxygen storage in myoglobin is important for intermitten exercise. 1,3,4 The
use of gene targetting and molecular biological techniques has revealed
new insight into reulation and myoglobin function,. The aims of the study is
to know mRNA myoglobin changes in skeletal and cardiac muscle after
aerobic and anaerobic exercise. Myoglobin is a cytoplasmic haemoprotein
that expressed in skeletal muscle and cardiac muscle that reversibly binds
O2 by its heme residue a phorphyrin ring from ion complex. 1,3,4 Training in
hypoxia result at the molecular level an upregulation of the regulatory
submit of hypoxia induced factor-1(HIF-1). 17,,19 Possibly as a consequence
of this upregulation of HIF-1 as a transcription factor, the level of mRNA
myoglobin (mRNAMb) increased. Myoglobin are synthesized according to
information in messenger RNA Mb that is synthesized on DNA template by
transcription and it depends on the need of oxygen in the cell. 2,3,5,18 During
aerobic exercise a local hypoxia will stimulate the HIF-1. During anaerobic
exercise , supply of oxygen will decrease until there is no oxygen supply
led to hypoxia . Hypoxia condition will stimulate HIF-1 ,as a transcription
factor to initiate the mRNA production . 17,19,24 So, both aerobic and
anaerobic exercise will increase the mRNA Mb. However ,it has been
reported that that strenous exercise will disrupt the sarcolemna and then
the Mb from the intracellullar enter the circulation. We can found the serum
myoglobin will increase after strenous exercise. 22,23
During aerobic metabolism, oxygen supplied from the storage in
the myoglobin. A cornestone of respiratory regulation stands on the
capasity of Mb to store or fasilitate oxygen transport, In marine mammals
the high consentration of Mb could certainly supply oxygen during a dive
or apnoe,1,3,4 In adaptation to high altitude enhanced Mb expression
2
increases the oxygen depot. The observation agree that with the
correlation between Mb concentration as oxygen supply and oxidative
capasity in different species. 4,6 Without any Mb. neither cardiac muscle
nor skeletal muscle
functions suffers any apparent physiological
impairment.6-10 Muscle strenght and function of muscle correlate with
myoglobin concentration. As we know that muscle strength and function
of myoglobin will increase after 6 week exercise. 4,5 But the physiological
role of Mb have not yet been establish. Recently we can know the
increase of myoglobin from the mRNA Mb.
These studies imply that further investigations are required to
reveal the physiological role of Mb in aerobic and anaerobic exercise.
Increased gene expression during recovery from consecutive aerobic and
anaerobic exercise measured
in mRNA Mb of soleus muscle,
gastrocnemius muscle and cardiac muscle. Soleus muscle and
Gastrocnemius muscle is a skeletal muscle ,but the soleus muscle is a
fast fiber that have rich of myoglobin and gastrocnemius muscle is a slow
twich fiber that less myoglobin. Cardiac muscle is aspecial muscle with
synsitium and rich of myoglobin too.5,6,15
Animal experimental study using Male Wistar Rat, 8 weeks old.The
study conducted with three groups consisted aerobic groups, anaerobic
groups and control groups. Animal treadmill test for aerobic groups 15
m/minute runs for 30 minute and for anaerobic groups groups 25 m/minute
runs for 30 minute, was done during 14 days. 8 RT- PCR was done to
evaluate the difference effect between aerobic and anaerobic physical
activity on myoglobin gene (mRNA) expression on skeletal and
myocardium. . All experimental prosedures were conducted in accordance
with the Japanese Physiological Society Guidelines for care and use of
laboratory animal, as approved by Gunma University Japan, Graduate
School of Medicine Committee on animal care and use.
The objectives of this study is to know the different effect of aerobic
and anaerobic exercise to the mRNA Mb in soleus muscle, gastrocnemius
muscle and cardiac muscle.
METHODS
Animal. Male Wistar rat(N=15), ages 8 weeks, weight 280 – 315 gr,
SPF were used in this study. The animal were kept in standard individual
cages and provided with food and water ad libitum. The room temperature
was kept 23±10 C , and a 12:12 hour light-dark cycle environment was
maintained throughout the experiment. All experimental prosedures were
conducted in accordance with the Japanese Physiological Society
Guidelines for care and use of laboratory animal, as approved by Gunma
University Japan, Graduate School of Medicine Committee on animal care
and use.
Independent variable is aerobic and anaerobic. Dependent variable is
mRNA myoglobin from m .soleus, m gastrocnemius and cardiac muscle.
3
The subject divided into three groups consisted aerobic groups, anaerobic
groups and control groups. Animal treadmill test for aerobic groups 15
m/minute runs and for anaerobic groups 25 m/minute runs for 30 minute
was done during 14 days. 8.At the end of research (day 14) , under
anasthetized ether the subject was sacrifice to get the muscles. To prevent
contamination with haemoglobin, before sacrifice ,perfussion with
DiethylPyrocarbonat 97% (DEPC) was done. After excess connective
tissue and fat were remove, each muscle immediately frozen by liquid
Nitrogen and stored at -800 C.
After all samples were collcted we make RNA extraction using AGPC
method to measured the mRNA concentration and to know the mRNA Mb
gene expression we used RT-PCR.
The myoglobin expression were read by sofware Kodak 2500 under UV
light.
All data reported as mean±SD. An analysis of variance (ANOVA) was used
to determine overall differences and Duncan’s post hoc analyses were
used to compare control , aerobic and anaerobic post exercised data. All
data processed using SPSS 15.0, p˂0,
RESULT :
TABLE 1
The difference of mRNA myoglobin concentration of soleus muscle after
14 days aerobic and anaerobic exercise
control
15m/min 25m/min
Mean of
Intensity
0.931648 0.930314 0.771628
SD
0.039878 0.026858 0.025086
After 14 days aerobic and anaerobics intermitten exercise , the mRNA Mb
of soleus muscle after aerobics exercise is higher than anaerobics
exercise.
Table 2
The difference of mRNA myoglobin concentration of
gastrocnemius muscle after 14 days aerobic and anaerobic exercise
4
Control
Mean of
intensity
SD
15m/min
25m/min
0.869887 1.050406 0.876036
0.044526 0.053192 0.014795
After 14 days aerobic and anaerobics intermitten exercise , the mRNA Mb
of gastrocnemius muscle after aerobics exercise is higher than
anaerobics exercise
Table 3
The difference of mRNA myoglobin concentration of cardiac muscle after
14 days aerobic and anaerobic exercise
Control
Mean of
Intensity
SD
15m/min
25m/min
0.886129 0.724951 0.614331
0.052121 0.060031
0.02772
After 14 days aerobic and anaerobics intermitten exercise , the mRNA Mb
of cardiac muscle after aerobics exercise is higher than anaerobics
exercise
As showed in table 1,2 and 3 , mRNA Mb concentration is increase higher
after 14 days aerobic exercise compare than after anaerobics exercise.
The ANOVA among groups led to be increase in aerobic group higher
than anaerobic group.
As showed in table 1 for soleus muscle( 0.930314 ±
0.053192 vs
0.771628±0.025086 µgr/ml),
as
showed
in
table
2
for
gastrocnemius
muscle(1.050406±0.060031vs0.876036 ± 0.014795 µgr/ml ),
and as showed in table 3 for cardiac muscle
(0.724951±0.060031
µgr/ml vs 0.614331±0.02772 µgr/ml). p≤05
Myoglobin gene expression is increase also showed in the figure 1,2 and
3 the electrophoresis of mRNA Mb . Myoglobin gene expression showed
stronger after aerobic exercise compare to after anaerobic exercise in
soleus muscle, gastrocnemius muscle and cardiac muscle. The mRNA
myoglobin expression showed in 330 bp .
Figure 1
5
Changes in mRNA myoglobin of soleus muscle after aerobic and
anaerobic exercise .
Figure 1. showed mRNA gene expression of soleus muscle, after aerobic
exercise is more stronger mRNA Mb gene expression (330bp) than after
anaerobic exercise. Also the intensity of mRNA Mb of soleus muscle
showed very significant higher after aerobic exercise than anaerobic. (
0.930314 ± 0.053192 vs 0.771628±0.025086 µgr/ml), p≤0,01
Figure 2
Changes in mRNA myoglobin of gastrocnemius muscle after aerobic and
anaerobic exercise
6
Figure 2, Using RT-PCR showed mRNA Mb electrophorese of
gastrocnemius muscle, after aerobic exercise is more stronger mRNA Mb
gene expression (330bp) than after anaerobic exercise. Also the intensity
of mRNA Mb of gastrocnemius muscle showed very significant higher after
aerobic exercise than anaerobic. (1.050406±0.060031 vs 0.876036 ±
0.014795 µgr/ml ), p≤0,01
Figure 3.
Changes in mRNA myoglobin expression of cardiac muscle after aerobic
and anaerobic exercise
7
Figure 3. Using RT-PCR showed mRNA Mb electrophorese of cardiac
muscle, after aerobic exercise is more stronger mRNA Mb gene
expression (330bp) than after anaerobic exercise. Also the intensity of
mRNA Mb of cardiac muscle showed higher after aerobic exercise than
anaerobic.(0.724951±0.060031 µgr/ml vs 0.614331±0.02772 µgr/ml). p≤05
CONCLUSION
As summarized in the Figure 1, showed the mRNA Mb of soleus muscle is
very significant higher after aerobic exercise than after anaerobic exercise.
(p≤0,01)
Also the electrophoresis show stronger mRNA expression in the soleus
muscle after aerobic exercise.
As summarized in the Figure 2, showed the mRNA Mb of gastrocnemius
muscle is significant higher after aerobic exercise than after anaerobic
exercise. (p≤0,01)
Also the electrophoresis show stronger mRNA expression in the
gastrocnemius muscle after aerobic exercise.
As summarized in the Figure 3, showed the mRNA Mb of cardiac muscle
is significant higher after aerobic exercise than after anaerobic exercise.
(p≤0,05)
8
Also the electrophoresis show stronger mRNA expression in the cardiac
muscle after aerobic exercise than after anaerobic exercise.
All of the mRNA Mb in soleus muscle, gastrocnemius muscle and
cardiac muscle after anaerobic exercise is lower than after aerobic
exercise . This showed that streunous exercise can caused disruption of
the cell that led to the myoglobin enter the extracellular or the circulation,
so we can found the myoglobin in the serum.
DISCUSSION
It was previously demonstrated that, when different exercise
aerobic and anaerobic give different effect to the mRNA Mb expression. 1,3,4,
Gas mixtures containing 21-100% oxygen were inhaled during aerobic
exercise , the oxygen content in arterial and venous blood flowing from
working muscles and the time of work without fatique. This accompanied
by a higher oxygen uptake by muscles, Aerobic exercise will increase
oxygen demand lead to relatif hypoxia because the supply is also
increase,2,5 This condition will stimulate the HIF-1 to increase . HIF-1 as a
factor for transcription will initiate to increase the mRNA Mb 1,4,10-12. It is
means the gene expression of myoglobin will increase after aerobic
exercise. In the other hand, during the anaerobic exercise , demand of
oxygen increase but there is no supply , this condition will lead to
hypoxia.22,24 So the HIF-1 also increase. Activation of of HIF-1 appears to
trigger adaptations which diminish the negative effects of chronic exposure
to hypoxia. 23,24
This study showed that aerobic and anaerobic physical activity
increased the mRNA Mb in soleus muscle, gastrocnemius muscle and
cardiac muscle. Whatever the type of exercise can increase mRNA Mb ,
but as we know streunous exercise will lead to anaerobic metabolism . 5,6,12
Anaerobic metabolism will increase lactate production that lead the
decreased pH in the tissue and circulation. 5,6,21 This condition can damage
the sarcolemma anddisrupt the cell. Myoglobin as a macromolecul globin
will enter the extracellular fluid and circulation. 16,17 We can found the
myoglobin in the serum., so the myoglobin content in the muscle tissue will
decrease.
In conclusion myoglobin gene expression in soleus,gastrocnemius
and cardiac muscles was higher and stronger after aerobic than anaerobic
exercise as an indicator of tissue hypoxia. Aerobic exercise is better to
increase the mRNA myoglobin , so it can increase the physical fittness by
maintain supply oxygen to the cells or tissue, even though in hypoxia
condition .
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