Effect of eccentric training on mitochondrial function and oxidative stress in the skeletal muscle of rats.

0.34 MB·English

by  Silva, L.A.

BrazilianJournalofMedicalandBiologicalResearch(2013)46:14-20,http://dx.doi.org/10.1590/1414-431X20121956
ISSN1414-431X
Effect of eccentric training on
mitochondrial function and oxidative
stress in the skeletal muscle of rats
L.A. Silva1, K.F. Bom1, C.B. Tromm1, G.L. Rosa1, I. Mariano1, B.G. Pozzi1, T. Tuon1,
E.L. Stresck2, C.T. Souza1 and R.A. Pinho1
1Laborato´riodeFisiologiaeBioquı´micadoExercı´cio,ProgramadePo´s-Graduac¸a˜oemCieˆnciasdaSau´de,
UniversidadedoExtremoSulCatarinense,Criciu´ma,SC,Brasil
2Laborato´riodePatofisiologiaExperimental,ProgramadePo´s-Graduac¸a˜oemCieˆnciasdaSau´de,
UniversidadedoExtremoSulCatarinense,Criciu´ma,SC,Brasil
Abstract
Theobjectiveofthepresentstudywastoinvestigatetheeffectsofeccentrictrainingontheactivityofmitochondrialrespiratory
chainenzymes,oxidativestress,muscledamage,andinflammationofskeletalmuscle.Eighteenmalemice(CF1)weighing
30-35gwererandomlydividedinto3groups(N=6):untrained,trainedeccentricrunning(16˚;TER),andtrainedrunning(0˚)
(TR),andweresubmittedtoan8-weektrainingprogram.TERincreasedmuscleoxidativecapacity(succinatedehydrogenase
and complexes I and II) in a manner similar to TR, and TER did not decrease oxidative damage (xylenol and creatine
phosphate) but increased antioxidant enzyme activity (superoxide dismutase and catalase) similar to TR. Muscle damage
(creatinekinase)andinflammation(myeloperoxidase)werenotreducedbyTER.Inconclusion,wesuggestthatTERimproves
mitochondrial function but does not reduce oxidative stress, muscle damage, or inflammation induced by eccentric
contractions.
Keywords:Eccentrictraining;Mitochondrialenzymeactivity;Oxidativestress;Muscledamage;Inflammation
Introduction
The production of reactive oxygen species (ROS) involving a combination of concentric and eccentric
during physical training depends on the type, intensity, contractions; however, activated skeletal muscles are
and duration of exercise (1). Although regular exercise more likely to be injured by lengthening rather than
training is associated with numerous health benefits, it shortening contractions, and this explains the greater
can beviewed asanintensephysicalstressorleadingto muscle damagesreportedwith eccentricexercise(3,7).
increased oxidative cellular damage, likely due to the Especially in eccentric exercise, the generation of
enhanced production of ROS (2). It appears that ROS- ROShasbeenattributedtoxanthineandNADPHoxidase
mediatedoxidationofproteins,lipids,andnucleicacidsis production,ischemia/reperfusion,prostanoidmetabolism,
not solely dependent on oxygen flux through the mito- phagocyte respiratory burst, disruption of iron-containing
chondria, since oxygen uptake has been shown to differ proteins, and excessive calcium accumulation resulting
drastically between exercise modes. Rather, multiple from high-force eccentric exercise, which usually pro-
factors, including xanthine oxidase, disruption of iron- duces muscleinjury (8-10).
containing proteins, calcium imbalance secondary to Several studies have tested the effects of eccentric
muscle injury, and inflammatory-mediated production of contractions (5,7,8) and intermittent eccentric chronic
ROS and subsequent oxidation of macromolecules after exercise(4)onmuscledamage,inflammation,mitochon-
aerobic, concentric, and eccentric training, may be drial enzyme activity, and oxidative stress markers. On
involved(3-6). the other hand, skeletal muscle injury resulting from
Most of the studies examining oxidative stress with downhill running can be prevented by only 5 days of
acute/chronic exercise were performed using exercises intermittentdownhillrunningatlowspeed(4),suggesting
Correspondence:L.A.Silva,Laborato´riodeFisiologiaeBioquı´micadoExercı´cio,ProgramadePo´s-Graduac¸a˜oemCieˆnciasdaSau´de,
Universidade do Extremo Sul Catarinense, Av. Universita´ria, 1105, 88806-000 Criciu´ma, SC, Brasil. Fax: [phone].
E-mail:[email protected]
ReceivedDecember23,2011.AcceptedSeptember4,2012.FirstpublishedonlineJanuary11,2013.
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Eccentrictrainingandoxidativestress 15
that an adaptation takes place in response to the initial storedat-70˚Cforlateranalysis.Serumwasusedforthe
injury as well as a subsequent recovery from eccentric determination of creatine kinase (CK) activity and the
exercise(6). quadriceps muscles (red portion) were used to analyze
However, many questions still remain unanswered succinate dehydrogenase (SDH) and myeloperoxidase
regarding the effects of continuous downhill running at (MPO) activities, mitochondrial respiratory chain enzyme
moderate speed. These include: a) Does continuous activities (complexes I and II), and oxidative stress
downhill running cause a positive or negative change in markers.
theactivityofmitochondrialrespiratorychainenzymes?b)
Doescontinuousdownhillrunningdecreasetheoxidative Biochemicalanalyses
stress, muscle damage, and inflammation induced by Homogenate preparation. Quadriceps muscles were
eccentriccontractions?Thus,theaimofthepresentstudy homogenized(Marcone,Brazil;1:10,w/v)inSETHbuffer
was to determine the effects of moderate continuous (250mM sucrose, 2mM EDTA, 10mM Trizma base,
downhill running on the activity of mitochondrial respira- 50 IU/mL heparin, pH 7.4). The homogenates were
torychainenzymes,oxidativestressparameters,muscle centrifuged at 800 g for 10min, and the supernatant
damage, andinflammation ofskeletal muscle. solutionsweremaintainedat-70˚Cuntilthedetermination
of SDH and mitochondrial respiratory chain enzyme
Material and Methods activities (complexes I and II). The maximum period
between homogenate preparation and enzyme analysis
Animals was ,5days.
The study protocol was reviewed and approved by SDH. Phenazine oxidoreductase (soluble SDH) was
the Ethics Committee of Universidade do Extremo Sul measured by the decrease in absorbance due to the
Catarinense, Criciu´ma, SC, Brazil, according to the reduction of 2,6-dichloroindophenol (DCIP) at 600nm
Guidelines for Animal Care and Experimentation (11). A with 700 nm as the reference wavelength (e =
totalof18malemice(CF1)aged3monthsandweighing 19.1 mM-1?cm-1) in the presence of phenazine
30-35 g were housed in cages with a maximum of 6 methosulfate (PMS). The reaction mixture consisted of
animalspercageandwithwaterandfoodadlibitum.The 40mM potassium phosphate, pH 7.4, 16mM succinate,
animals were kepton a12-hlight/dark cycle at23˚C. and 8mM DCIP. DCIP was preincubated with 40-80mg
homogenate protein at 30˚C for 20min. Subsequently,
Exercise 4mM sodium azide, 7mM rotenone, and 40 mM DCIP
The animals were divided into the following groups wereadded,andthereactionwasinitiatedbytheaddition
(N = 6 each): untrained (UT), trained eccentric running of1mM PMSand monitoredfor 5min(12).
(TER), and trained running (TR). The exercise-training Mitochondrial respiratory chain enzyme activities. On
groups were subjected to running on a motor-driven the day of the assays, the samples were frozen and
treadmill by using a progressive exercise-training regi- defrosted three times in hypotonic assay buffer to fully
men.Allanimalswereaccustomedtotreadmillrunningfor expose the enzymes to the substrates and to achieve
1 week (10 m/min without inclination for 10min/day). maximal activity. NADH dehydrogenase (complex I) was
Aftera1-weekadaptationperiod,thetrainedgroups(TER evaluated by the method described by Cassina and
and TR) were submitted to an 8-week training program. Radi (13) by measuring the rate of NADH-dependent
Thevelocityofthetreadmillwas13 m/minduringthefirst ferricyanidereductionat420nm.Theactivityofsuccinate:
4weeks, and16 m/minduring thelaterweeks. DCIPoxidoreductase(complexII)wasdeterminedbythe
method of Fischer et al. (12). Complex II activity was
Training measuredbymonitoringthedecreaseinabsorbancedue
TER.The TER programwas applied atnight (6:00 to tothereductionof2,6-DCIPat600nm.Theactivitiesofthe
8:00pm)andconsistedofonesessionofdownhillrunning mitochondrialrespiratorychaincomplexesarereportedas
(16˚ decline) for45min/day,5days/weekfor8weeks. nmol?min-1?mgprotein-1(40-80ghomogenateprotein).
TR.TRwasperformedatthesametimeasTERand Ferrous oxidation-xylenol orange (xylenol). This
consistedofonesessionofrunning(0˚)for45min/day,5 method detects hydroperoxides (ROOHs) that are the
days/weekfor 8weeks. products of lipoperoxidation (14). The xylenol orange
assay is based on the oxidation of ferrous ions to ferric
Animal sacrifice ions by ROOHs under acidic conditions. Tissues were
Forty-eight hours after the last training session, the homogenized (30 mg/mL), and aliquots (90 mL) were
animals were killed by decapitation. Blood was removed transferred to microcentrifuge vials (1.0mL). Ten micro-
byheartpuncture,centrifugedat1500gfor10minat4˚C, litersof10mMthiaminepyrophosphate inmethanolwas
and serum samples were taken and stored at 2˚C. The addedtothevialstoreduceROOHs.Thevialswerethen
quadriceps muscles (red portion type I fiber) were vortexed and incubated at room temperature for 30min
surgically removed, and the samples were immediately beforetheadditionof900 mLFox2reagent.Aftermixing,
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16 L.A.Silvaetal.
the samples were incubated at room temperature for an observed in the TER (8.4 ± 0.4 nmol?min-1?mg
additional30min.Thevialswerecentrifugedat2400gfor protein-1) and TR (8.3 ± 0.3 nmol?min-1?mg protein-1)
10 min with a swing-out rotor (Hettich Rotanta/RP groups compared to the UT group (4.1 ±
centrifuge, Hettich-Zentrifugen, Germany). Absorbance 0.5 nmol?min-1?mg protein-1)(Figure 1A).
of the supernatant was measured at 560 nm using an Complex I. An increase in complex I activity was
Ultraspec 2000 spectrophotometer (Pharmacia Biotech, observedintheTER(189±23nmol?min-1?mgprotein-1)
Sweden). and TR (196 ± 11 nmol?min-1?mg protein-1) groups
Proteincarbonyls(PCs).Oxidativedamagetoproteins compared to the UT group (84.1 ± 6nmol?min-1?mg
was measured by the determination of carbonyl groups protein-1,P, 0.05) (Figure1B).
based on the reaction with 2,4-dinitrophenylhydrazine Complex II. An increase in complex II activity was
(DNPH)(15).Proteinswereprecipitatedbytheadditionof observedintheTER(1.3±0.07nmol?min-1?mgprotein-1)
20% trichloroacetic acid and reacted with DNPH. The and TR (1.2 ± 0.1 nmol?min-1?mg protein-1) groups
samples were then redissolved in 6 M guanidine compared to the UT group (0.7 ± 0.08nmol?min-1?mg
hydrochloride, and carbonyl contents were determined protein-1,P,0.05)(Figure1C).
by measuring the absorbance at 370 nm with a molar
absorption coefficientof 220,000M-1. Oxidativedamage
Totalsuperoxidedismutase(SOD)andcatalase(CAT) Xylenol.Thelevelofhydroperoxidesinthequadriceps
activities.InordertodetermineCATactivity,tissueportions muscles increased in the TER (32 ± 2.2 nmol/mg
were sonicated in 50 mM phosphate buffer, and the protein) and decreased in the TR (12 ± 1.5 nmol/mg
resulting suspension was centrifuged at 3000 g for protein) groups compared to the UT group (23 ±
10min. The supernatant was used for enzyme assay. 1.8 nmol/mgprotein,P, 0.05)(Figure 2A).
CAT activity was reported as the rate of decrease in PCs.AsshowninFigure2B,therewasanincreasein
hydrogen peroxide (10mM) absorbance at 240nm (16). PCsinthequadricepsmusclesoftheTERgroup(0.79±
SODactivitywasdeterminedbymeasuringtheinhibitionof 0.05nmol/mg protein) and a decrease in the TR group
adrenalineself-oxidationabsorbanceat480nm(17). (0.30±0.04nmol/mgprotein)comparedtotheUTgroup
CK. A specific kit supplied by LABTEST Diagno´stica (0.49 ±0.03nmol/mgprotein,P, 0.05).
S.A.(Brazil)wasused.SerumCKlevelsweredetermined
according tomanufacturer instructions. Antioxidant enzyme activity
MPO activity in skeletal muscle. Tissues were homo- SOD. Figure 3A demonstrates an increase in SOD
genized (50 mg/mL) in 0.5% hexadecyltrimethyl- activity in the quadriceps muscles of the TER (1.6 ±
ammonium bromide and centrifuged at 15,000 g for 0.21U/mg protein) and TR (1.5 ± 0.16U/mg protein,
40 min. The suspension was then sonicated three times P , 0.05) groups compared to the UT group (0.89 ±
for 30 s. An aliquot of the supernatant was mixed with a 0.11U/mg protein).
solutionof1.6 mMtetramethylbenzidineand1mMH O . CAT. A significant increase in CAT activity was
2 2
The activity was measured spectrophotometrically as a observed in the quadriceps muscles of the TER (1.2 ±
change inabsorbance at650 nm at37˚C (18). 0.05U/mg protein) and TR (1.1 ± 0.07U/mg protein)
Protein determination. The amount of protein in the groups compared to the UT group (0.62 ± 0.09U/mg
samples tested for MPO, SDH, complexes I and II protein)(Figure 3B).
enzyme activities, xylenol, PC, CAT, and SOD activities
was determinedbythemethodofLowryetal.(19)using Muscledamageandinflammation
bovine serumalbumin asstandard. CK. A significant increase in serum CK activity was
observed in the TER group (323 ± 19U/L) compared
Statistical analysis with the TR (104 ± 22 U/L) and UT (111 ± 12U/L)
Dataarereportedasmean±SEandwereanalyzed groups (Figure4A).
statisticallybythetwo-wayanalysisofvariance(ANOVA) MPO activity. MPO activity was significantly elevated
followed by the post hoc Tukey HSD test. The level of in the quadriceps muscles of the TER group (341 ± 40
significance was set at 95% (P , 0.05). The software activity/MPO protein) compared to the TR (192 ± 21
used for the analysis of the data was the Statistical activity/MPO protein) and UT (188 ± 20 activity/MPO
PackagefortheSocialSciences(SPSS)version16.0for protein)groups (Figure 4B).
Windows.
Discussion
Results
This study presented data demonstrating that TER is
Enzymeactivitiesof the mitochondrial respiratory not harmful to mitochondrial function, improving the
chain oxidative capacity of muscle (SDH, complexes I and II)
SDH. A significant increase in SDH activity was similar to TR. However, only TR decreases oxidative
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Eccentrictrainingandoxidativestress 17
damage (xylenol and PCs). Antioxidant enzyme activity eccentriccontractions.
(SOD and CAT) increased after TER and TR. Further- Themetabolicresponseofthemusclestoendurance
more, TER did not decrease muscle damage (CK), traininghasbeenestimatedbymeasuringthemarkersof
inflammation (MPO), or oxidative stress induced by oxidative capacity with an increase in the electron
transport chainenzyme activities(5,20). Molnar etal. (3)
demonstrated that intermittent eccentric training leads to
positive adaptations in the mitochondria. However, we
were the first to demonstrate that continuous eccentric
training (TER group) similarly increased the activities of
SDHandmitochondrialcomplexesIandII(Figure 1A-C)
comparedtotheTRgroup.Bothtypesoftraininginvolved
the same volume and intensity. On the basis of these
data, we suggest that eccentric training is not harmful to
mitochondrialfunction.
An important adaptation that accompanies regular
endurance training is a decrease in the level of ROS
generated in the mitochondria (21). Studies have shown
that regular exercise reduces oxidative damage to the
Figure 2. The level of oxidative damage [ferrous oxidation-
Figure 1. Mitochondrial respiratory chain enzyme activities xylenolorange(A)andproteincarbonyls(B)]wasdeterminedin
[succinatedehydrogenase(SDH)(A),complexI(B),andcomplex skeletal muscle (red portion - quadriceps) 48h after the last
II (C)] were determined in skeletal muscle 48h after the last sessionoftraining.Theanimalsweredividedinto3groups(N=
sessionoftraining.Theanimalsweredividedinto3groups(N= 6each):UT=untrained;TER=trainedeccentricrunning(16˚);
6each):UT=untrained;TER=trainedeccentricrunning(16˚); TR= trainedrunning(0˚). Dataare reportedas means±SE.
TR =trainedrunning(0˚).Data are reportedas means± SE. *P,0.05comparedtotheUTgroup;#P,0.05comparedtothe
*P,0.05comparedtotheUTgroup(TukeyHSDtest). UTandTERgroups(TukeyHSDtest).
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18 L.A.Silvaetal.
brain(22),liver(23),andmuscles (8)ofrats.Ourresults contraction is not harmful to the function of enzymes.
demonstrated a decrease in oxidative damage in the TR Theseresultsshowedthatchroniccontractileactivityalso
group and an increase in the TER group (Figure 2A and appearstoinfluencetheabilityofthemusclestodetoxify
B) compared to the UT group. Several mechanisms can superoxide and hydrogen peroxide with an increase in
help explain the decrease in the oxidative damage skeletal SOD (26) and CAT (25). The increase in the
induced by training (5,24). However, this increase in the enzymescanexplainthereductionofoxidativedamagein
oxidativedamageinducedbyTERisconsistentwiththat theTRgroup.However,intheTERgroup,increasedSOD
reported by Molnar et al. (3) with regard to chronic and CAT activities were not sufficient to protect from
eccentric exercise-enhanced oxidative stress. It is possi- oxidative stress.
ble that eccentric contractions performed daily (5 days a Some studies have reported that repeated eccentric
week),withnotimeforsufficientmusclerecovery,induce exercise-induced adaptations decrease muscle damage
oxidative stress. andinflammation(6,27).Neural,mechanical,andcellular
Various studies have reported increased antioxidant mechanisms are speculated to be causally related to the
defenses under chronic conditions (1,25). Our results developmentofarepeatedbouteffect(28,29).However,
demonstrated increased activity of two enzymes (SOD - this was not confirmed in the present study. We demon-
Figure3A,andCAT-Figure3B)similarlyinducedbyTER strated that eccentric training caused muscle damage
andTE. Bothkindsoftraininginvolvedthesamevolume (increasedserumCK-Figure4A),whichledtoneutrophil
(45 min/day) and intensity (16 m/min). The eccentric invasion (increased muscle MPO - Figure 4B). This can
Figure 3. Antioxidant enzyme levels [A, superoxide dismutase Figure4.Muscledamage(A,creatinekinase)andinflammation
(SOD) and B, catalase (CAT)] were determined in skeletal (B, myeloperoxidase, MPO) levels were determined in skeletal
muscle (red portion - quadriceps) 48h after the last training muscle (red portion - quadriceps) 48h after the last training
session.Theanimalsweredividedinto3groups(N=6each): session.Theanimalsweredividedinto3groups(N=6each):
UT= untrained;TER =trainedeccentricrunning(16˚); TR= UT=untrained;TER =trainedeccentricrunning(16˚);TR=
trained running (0˚). Data are reported as means ± SE. *P , trained running (0˚). Data are reported as means ± SE. *P ,
0.05comparedtotheUTgroup(TukeyHSDtest). 0.05comparedtotheUTgroup(TukeyHSDtest).
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Eccentrictrainingandoxidativestress 19
be explained by the fact that eccentric contractions (38) showed that this heat shock protein response to
activate inflammatory cells to accumulate in skeletal eccentric exercise was concurrently attenuated with
muscle, causing an increase in ROS production and in attenuation of muscle damage when eccentric exercise
transcription factors such as nuclear factor-kappaB wasrepeated8weekslater.Inthepresentstudy,thetime
activation, causing muscle damage and inflammation of recovery of 24 h between training sessions was
(8,26,30,31). insufficient to promote positive adaptations, with conse-
Greater MPO content increases hydroperoxide pro- quent muscle damage, inflammation, and oxidative
duction(xylenol-Figure2A)andcausesproteinoxidation stress. However, the discrepancies in the results may
(carbonyls - Figure 2B). Eccentric exercise induces result from different training models (e.g., treadmill
pathologicchangessuchasfibernecrosisandinflamma- running, bicycle, strength, and swimming); different
torycellinfiltrationthatbecomeapparentafewdaysafter training volumes (15, 30, 45, and 60min), intensities
exercise (32). If this initial triggering damage during (low, moderate, and high), and frequencies (2, 3, and 5)
eccentric contractions were strong enough to induce a per week, and different species (e.g., rats, mice, and
sustained elevation of intracellular Ca2+ concentration humans).
after exercise, then various proteases and phospholi- In addition, the effect of TER may occur on different
pases would be eventually activated (32,33), causing pathways without directly involving the mitochondrial
necrosis. Damaged connective tissue and necrotic fibers metabolism.Muscledamagecanbeinducedbymechan-
would then induce and activate the inflammatory cell ical factors during eccentric contractions (39) such as
infiltration that accompanies ROS production (9,33-35) production of free radicals by cytosolic sources, for
and increasesdamaging oxidants(2,9,10). example,activationofxanthineoxidase,catecholamines,
Therefore, eccentric training has a dual effect on the andprostaglandins(2,40),andactivationofinflammation
muscle: without adequate recovery, the muscle is or transcription factors(31).
damaged in eccentric training, while with recovery, Theresultsofthisstudysuggestthateccentricrunning
eccentric training protects the muscle from this damage. training improves mitochondrial function but does not
Detailedmechanismsunderlyingthisdualeffecthavenot reduce oxidative damage, muscle damage, or the
yetbeenfullystudiedexperimentally(36,37).Vissingetal. inflammationinduced byeccentric contractions.
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