Abstract Myocardial infarction (MI) has been associatedwith increases in reactive oxygen species (ROS). Exercisetraining (ET) has been shown to exert positive modulationson vascular function and the purpose of the present studywas to investigate the eVect of moderate ET on the aorticsuperoxide production index, NAD(P)H oxidase activity,superoxide dismutase activity and vasomotor response inMI rats. Aerobic ET was performed during 11 weeks. Myo-cardial infarction signiWcantly diminished maximal exer-cise capacity, and increased vasoconstrictory response tonorepinephrine, which was related to the increased activityof NAD(P)H oxidase and basal superoxide production. Onthe other hand, ET normalized the superoxide productionmostly due to decreased NAD(P)H oxidase activity,although a minor SOD eVect may also be present. Theseadaptations were paralleled by normalization in the vaso-constrictory response to norepinephrine. Thus, diminishedROS production seems to be an important mechanism bywhich ET mediates its beneWcial vascular eVects in the MIcondition.
Keywords Reactive oxygen species · Superoxide dismutase · NAD(P)H oxidase
Introduction
Endothelial tissue exerts a myriad of actions through thevascular control of nitric oxide (NO) bioavailability, whichis an important regulator of the vasodilatory function(Furchgott and Zawadzki 1980). In agreement with thisconcept, several pathologies, such as myocardial infarction,atherosclerosis, hypertension and cardiac heart failure, arerelated with decreased vascular NO bioavailability, whichmay be repercuss in impaired vasodilatory endotheliumdependent function (Bossaller et al. 1997; Kubo et al. 1991;Kojda and Harrison 1999).
Myocardial infarction causes decreased NO bioavailabil-ity and diminished vasodilatory endothelium-dependentfunction may be related to NO degradation. Between degra-dative mechanisms, increased vascular oxidative stress issupported by two observations: (1) vascular superoxidegeneration (which inactivates NO) is increased in myocar-dial infarcted (MI) rats (Bauersachs et al. 1999), (2) treat-ment of isolated blood vessels with antioxidants increasesvascular endothelial dependent function in infarcted rats(Indik et al. 2001; Bauersachs et al. 2001). Together, thesedata corroborate the hypothesis that the diminished vasodi-latory endothelium-dependent function could be a conse-quence of increased vascular reactive oxygen species(ROS) production or diminished antioxidant defense,culminating with decreased NO availability (Munzel andHarrison 1999).
Exercise training improves the vasomotory functionthrough mechanisms dependent on both increased vascularantioxidant defense (Rush et al. 2003; Fukai et al. 2000;
N. E. Zanchi · L. R. G. Bechara · L. Y. Tanaka · T. Bartholomeu · P. R. Ramires (&)Laboratory of Biochemistry of Exercise, School of Physical Education and Sport, University of São Paulo, Av. Prof. Mello Moraes, 65, São Paulo, SP 05508-900, Brazile-mail: [email protected]
Kojda and Hambrecht 2005) and increased NO bioavail-ability (Delp and Laughlin 1997). These actions could bebeneWcial in increased oxidative stress conditions, like thatcaused by MI. Nonetheless, it is unclear how exercise mod-ulates vascular ROS production and NO bioavailability fol-lowing MI. The purpose of this study was to evaluate, ininfarcted rats, the eVects of aerobic exercise training on thesuperoxide production index and vasomotory function.
Methods
Twenty-four male Wistar rats (initial weight, 180–200 g)were obtained from the animal breeding facility of the Fac-ulty of Medicine of the University of São Paulo, and weredistributed in three groups, sham operated (Sham, n = 8),infarcted-sedentary (MI-S, n = 8), and infarcted-trained(MI-T, n = 8). The rats, 4–5 per cage, were housed in a 12 hdark/light cycle with free access to food and water. Theexperimental protocol was approved by the Committee ofEthics in Research of the School of Physical Education andSport of the University of São Paulo.
Myocardium infarction protocol
BrieXy, 22 rats were randomly selected to undergo MIsurgery. Rats were anesthetized by intra-peritonealinjection with a mixture of ketamine (30 mg kg¡1) andxylazine (10 mg kg¡1). The pericardium was opened andthe left coronary artery was distally ligated at 2 mm of itsorigin (polypropylene suture, 5.0, Ethicon, Brazil) (PfeVeret al. 1979). The rats were kept for recovery for a 30-dayperiod, before experimental protocol began (exercisetraining or sedentary). Six rats died early after theoperation (»24 h) while sixteen rats (72.7%) survived theentire 4-week recovery period and were included foranalysis.
Exercise protocol
After a 30-day period of cardiac surgery, the rats from bothSham and MI groups were individually submitted to a pro-gressive exercise test (Gava et al. 1995) to determine theirmaximal exercise capacity, as expressed by the total run-ning distance. Maximal exercise was performed on a tread-mill using an incremental speed protocol (5 m min¡1 every3 min) until exhaustion.
Moderate exercise training protocol
The rats of the MI-T group were submitted to treadmillexercise training, performed Wve times per week, intensityof 55–60% of individual maximal running speed, and
duration of 60 min per session, through an 11-weekperiod. The eYciency of the ET protocol was evidencedby the increase in citrate synthase enzyme in MI-Tcompared to MI-S group (0.56 § 0.06 vs. 0.42 §0.02 mmol min¡1 per mg, respectively, P < 0.05), whichwas measured in the soleus muscle according to Shepherdand Garland (1969).
Animal euthanasia and tissue withdrawal
Training rats were euthanatized in a carbonic gas chamber48 h after the last exercise training session. The heart, thethoracic aorta and the soleus muscle of the right hind limbwere quickly removed. The heart was immediatelyweighted and stored at ¡20°C. The soleus muscle was fro-zen in liquid nitrogen for later analysis. The thoracic aortawas immediately dissected, cleaned of connective and/oradipose tissue and cut into Wve segments of 4 mm lengtheach (rings). Two rings were promptly used for in vitrovasomotor responses, two rings were promptly used forsuperoxide index assay, and one ring was frozen forbiochemistry analysis.
Determination of myocardium infarction area
Hearts were kept in the freezer (¡20°C) for 30 min andthen cut into slices (»2 mm) from the apex to the base. Theheart slices were incubated with triphenil tetrazoliumchloride 1% (in order to discriminate between viable andnonviable myocardium) for 15 min at 37°C, and then theinfarcted area was visually examined (Brandes et al. 1998).In all coronary ligated rats, large infarcted areas werevisible by macroscopic examination.
Study of aorta reactivity in vitro
Aortic rings were carefully submerged in bath organ cham-bers (14 ml) containing oxygenated (95% O2 and 5% CO2)Krebs–Ringer solution (NaCl 115, KCL 4.7, MgSO4 1.2,KH2PO4 1.5, NaHCO3 25, CaCl2 2.5, glucose 11.1 mM;pH 7.4) and were mounted on a force transducer (BIOPAC,USA.), using an initial passive tension of 2.0 g, correspond-ing to the previously determined maximal contractileresponse evoked by norepinephrine (NE). Aortic rings wereallowed to equilibrate for 60 min before the concentration-response curve to agonist was performed.
The acetylcholine (ACh) concentration-response curve(10¡10 to 10¡4 M) was performed in order to investigate theendothelial dependent vasorelaxation response. Initially,rings were precontracted with submaximal NE doses, thensubsequent concentrations of ACh were added to the bathand the maximal response (Emax) and sensitivity (EC50) toACh were determined. After the ACh concentration-
response protocol, bath chambers were washed and therings were allowed for equilibration for a 30-min periodbefore starting a new agonistic protocol.
The sodium nitroprusside (SNP) concentration-responsecurve (10¡10 to 10¡4 M) was carried out to investigateendothelial independent vasorelaxation response. Initially,rings were precontracted with submaximal NE dose, thensubsequent concentrations of SNP were added to the bathand Emax and EC50 to SNP were determined.
Superoxide production index
Fresh intact aortic rings were used to measure the superox-ide production index using a lucigenin (bis-N-methylacridi-nium nitrate)-enhanced chemiluminescence (CL) technique(Janiszewski et al. 2002). The reaction mixture (lucigenindissolved in buVer) without arterial rings did not generate asigniWcant signal. The aortic ring was placed into photo-multiplier tubes and light emissions were detected (basal)using a luminometer (Biolumat LB 9505; Berthold, Wild-bad, Germany). The second aortic ring was exposed for5 min to diphenilodonium (DPI, 20 �M, Alfa Aesar), aninhibitor of NAD(P)H oxidase. The speciWc CL signal wasexpressed as counts per minute minus the average back-ground signal. The results were expressed as cpm mg¡1 dryweight.
Superoxide dismutase enzyme activity
Total SOD activity was determined in aorta homogenates,prepared under liquid nitrogen and centrifuged at5,000 rpm for 5 min. The supernatant was assayed for totalSOD activity by monitoring inhibition rate of xanthine oxi-dase-mediated cytochrome c reduction at pH 7.4 (Leiteet al. 2003), and corrected for protein concentration.
Superoxide dismutase (Cu/Zn) enzyme expression
Aortic sample proteins were separated by 12% SDS-PAGEand transferred to nitrocellulose membranes. The Wlter wasblocked overnight with 20 mM Tris, pH 7.6, containing137 mM NaCl, 0.1% Tween 20 (TBST) and 5% milk, andsubsequently incubated overnight at 22°C with an antibodyagainst SOD Cu/Zn (Oxis, USA). The blots were subse-quently incubated with peroxidase-conjugated secondaryantibody (Oxis, USA) for 1 h, and processed for enhancedchemiluminescence to visualize the immunoreactive bands.The antibodies bound to Western blots were developedwith the chemiluminescent ECL detection system (Amer-sham Corp., USA) using 1:10,000 dilution of goat anti-rab-bit secondary antibody conjugated to horseradishperoxidase (HRP) (Bio-Rad Laboratories, Richmond, CA,USA) on a standard X-ray system.
Data analysis
Results are presented as mean § SEM. Relaxationresponses to ACh and SNP are expressed as the percentageof relaxation relative to the maximal contractile response.Emax and the log of the agonist concentrations producing50% of maximal response (EC50) were estimated by an iter-ative nonlinear regression analysis of each individual con-centration-response curve using GraphPad Prism Software(San Diego, CA, U.S.A.). The data were analyzed by one-or two-way analyses of variance (ANOVA) followed byBonferroni’s post hoc test. P < 0.05 was considered statisti-cally signiWcant.
Results
The body weight of the rats was not inXuenced by either MIsurgery or exercise training protocol (Table 1). However,ventricles weight was higher in MI-S (9.4%) and MI-T(11.2%) groups compared to the Sham group (P < 0.05). Inaddition, the lung weight/body weight ratio was higher inMI-S (23.3%) and MI-T (21%) groups compared to theSham group.
Maximal exercise capacity
Before the exercise training period, maximal exercisecapacity, expressed as total running distance, was dimin-ished in MI-S (19%) and MI-T (16%) groups when com-pared to the Sham group (Fig. 1). On the other hand,exercise training increased total running distance in MI-Tgroup as compared to both Sham (15%) and MI-S (40%)groups.
Aortic endothelial-dependent vasodilatory function
Emax estimated by ACh was reduced in MI-S group as com-pared to Sham and MI-T groups (Fig. 2a). Additionally,
after exercise training period the MI-T was able to normal-ize this response to Sham level.
Aortic endothelial-independent vasodilatory function
Emax estimated by SNP was not diVerent from that betweenSham, MI-S and MI-T groups. However, after exercisetraining, the EC50 to SNP was lower in MI-T group whencompared to both Sham and MI-T groups (Fig. 2b).
Aortic vasoconstrictory response to norepinephrine
The aortic vasoconstrictor response to NE (10¡7 M) washigher in MI-S (61%) group as compared to the Shamgroup (Fig. 3a). On the other hand, after exercise trainingperiod, the aortic vasoconstrictor response to NE was simi-lar between MI-T and Sham groups.
Aortic vascular superoxide production index
Figure 3b illustrates the aortic superoxide production indexmeasured by lucigenin chemiluminescence in the absence(Basal) or presence of diphenyliodonium (DPI), an inhibi-tor of NAD(P)H oxidase enzyme activity. In basal state,MI-S group showed higher values in vascular superoxidelevels than in those from Sham and MI-T groups. Impor-tantly, the pre-incubation with DPI in this preparationdecreased the superoxide production in all groups, but thepercent of diminishment was greater in the MI-S group(46%) as compared to the Sham (25%) and MI-T (25%)groups, thus normalizing the superoxide production to basallevels observed in Sham group.
Superoxide dismutase enzyme activity
MI-S group did not show decreases in SOD activity whencompared to Sham group, which suggests that the increasedsuperoxide production in MI-S rats is not a result ofdecreased SOD antioxidant defense. On the other hand, MI-T group showed increased SOD activity (Fig. 3c).
Superoxide dismutase enzyme expression
Superoxide expression of Cu–Zn SOD was assessedthrough immunoblotting techniques. Both MI and exercisetraining did not modify aortic SOD expression (Data notshown).
Discussion
The main Wnding was that an important biochemical mech-anism controlling the NO bioavailability, namely vascularsuperoxide production was increased in aortic ring form
Fig. 1 Exercise capacity, expressed as total running distance, fromrats of Sham, MI-S (infarcted-sedentary) and MI-T (infarcted-trained)groups. Data are mean § SEM. * P < 0.05 compared to Sham group,# P < 0.05 compared to pre-ET from MI-T group
Fig. 2 Response curves to acetylcholine (ACh, a) and sodium nitroprusside (SNP, b) in aortic rings from Sham (Wlled square), infarcted-sedentary (Wlled circle) and infarcted-trained (open circle) groups. Data are mean § SEM. MI-S Infarcted-sedentary, MI-T infarcted-trained, Emax maximal response, EC50 is represented as ¡Log of the molar concentration to produce 50% of the maximal response. * P < 0.05 compared to Sham and MI-T groups, # P < 0.05 compared to Sham and IM-S groups
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Eur J Appl Physiol (2008) 104:1045–1052 1049
myocardial infarcted rats, concomitantly with an increasedvasoconstrictory response to norepineprhine. On the otherhand, exercise training evoked a marked diminishment inthe aortic superoxide production and increased superoxidedismutase activity, thus modifying positively the complexvascular biochemistry regulation impaired by myocardialinfarctation. In addition, these beneWcial exercises trainingadaptation on the aortic superoxide production and SODactivity may be contributed to the decreased aortic constric-tory response to norepinephrine and decreased aortic
vasodilatory response to sodium nitroprusside in myocar-dial infarcted rats, representing two important biologicalresponses in the context of vascular regulation.
Superoxide is continuously produced under basal condi-tion and constitutes an important regulatory mechanism,Wnely controlled in vascular cells. However, increased vas-cular superoxide production has been observed in severalpathological conditions (Bauersachs et al. 1999, 2001) andcontributes to increased degradation of NO. The mainmechanisms involved in the increased vascular superoxideproduction are activation of the enzymatic vascular com-plex NAD(P)H oxidase, decoupling of the enzyme endothe-lial NO synthase (eNOS), increases in cytochrome P450activity and xantine-oxidase activity, besides decreases inlocal antioxidant defense (Kojda and Harrison 1999).
The MI-S group had higher superoxide production indexthan the Sham and MI-T groups. In addition, the chemicalinhibition of the vascular enzyme NAD(P)H oxidase withDPI decreased the superoxide production in all groups, butthe percent of diminishment was greater in the MI-S group,thus normalizing the superoxide production to basal levelsobserved in SHAM group. The increased vascularNAD(P)H oxidase enzyme activity may be important forincreased superoxide production of the aortic ring of MIrats. In addition, vascular antioxidant SOD activity andexpression, which represents the Wrst defense against super-oxide anions, was not impaired in the sedentary MI groupwhen compared to the Sham group.
These results conWrm our hypothesis that MI increasesaortic ROS production, an event potentially deleterious tothe vascular function especially when chronically presentbecause it can cause oxidation of proteins, lipids, and DNA(Darley-Usmar et al. 1995). However, a deWcit in the anti-oxidant defense, specially SOD activity and expression wasnot the primary cause. Thus, our data suggest that increasedvascular NAD(P)H enzyme activity was mainly responsiblefor the increased superoxide production seemed in thisstudy.
In humans or experimental animals, not suVering fromendothelial dysfunction, exercise training beneWts endothe-lial function (Higashi et al. 1999). These positive eVects ofexercise training are partly due to the improved removaland/or decreased production of ROS in several tissues, suchas myocardium (Ramires and Ji 2001) and skeletal muscle(Leeuwenburgh et al. 1994). However, it is not understoodhow blood vessels adapt to the increase in blood Xowinduced by exercise training.
A current hypothesis is that the acute exposition of bloodvessels to increased blood Xow increases temporarily thevascular ROS production (Laurindo et al. 1994). Thisincreased blood Xow associated with increased vascularROS production when chronically performed, seems tostimulate increases in the vascular antioxidant defense,
Fig. 3 a Developed tension to norepinephrine (10–7 M) in aorticrings. b Superoxide production index in fresh aortic rings under basalcondition and exposed for 5 min to diphenilodonium (DPI). c Super-oxide dismutase activity in aorta homogenate from Sham, MI-S (inf-arcted-sedentary) and MI-T (infarcted-trained) groups. Data aremean § SEM. * P < 0.05 compared to other groups
diminishing the oxidative stress (Rush et al. 2003), aphenomenon also observed in skeletal muscle (Ji 2008).However, in the presence of a pathological state like MI,these modiWcations elicited by exercise training are notknown.
The eVects of exercise training on SOD activity/expres-sion are debated. In skeletal muscles, exercise training elic-iting increases in the total SOD activity is strictlydependent of exercise intensity and the adaptation responsevaries according to the skeletal muscle studied. Weobserved that the MI-T group showed increased vascularSOD activity (but not increased SOD expression, data notshown) when compared to Sham and MI-S groups. Theresult of increased SOD activity are in accordance withmoderate exercise protocols (Rush et al. 2000, 2003) andpossibly indicate steady-state adaptations modulated byexercise training. Importantly, such increases in the antiox-idant defense results in less oxidative damage during oxida-tive challenges in both skeletal muscle and vascular tissues(Powers et al. 1999; Rush et al. 2003). However, we couldnot observe an increase in SOD-1 (Cu/Zn) protein expres-sion. This discrepancy could be a debt in the exercisetraining duration employed, which was 11 weeks versus16–19 weeks in the supra-cited studies. Another factorresponsible for such discrepancies could be the diVerencesin the animal species employed (rats vs. pigs). Increasedvascular SOD-1 activity possibly indicates a potentialmechanism by which exercise training leads to a reducedoxidative stress. In fact, vascular superoxide productionwas normalized in MI-T group when compared to the othergroups, an eVect apparently dependent of increased vascu-lar SOD enzyme activity. However, diminished NAD(P)Hoxidase enzyme activity was the main additional responsi-bility to the vascular superoxide decreases mediated byexercise training, because in MI-T group, DPI inhibitionshowed minor eVects on superoxide production as com-pared to MI-S group. Thus, besides increased antioxidantdefense, an important additional mechanism of exercisetraining mediating improvements in vasomotor functionseems to be the diminished superoxide production throughdecreased vascular NAD(P)H oxidase enzyme activity. Themechanisms involved in the diminished activity or expres-sion of NAD(P)H oxidase are highly complex. A dimin-ished angiotensin II concentration, an alteration in the ratiobetween angiotensin receptors in the smooth muscle mem-brane (with a diminished population in the AT-1 receptorsand increased population in the AT-2 receptor), and adiminished expression on the mRNA coding the subunit ofgp91 phox of NAD(P)H oxidase which correlates with itsdiminished protein concentration are included among thesemechanisms (Adams et al. 2005; Graham and Rush 2004).
MI was also capable of bringing about an increased vas-cular constrictory response to NE. The increased vascular
constrictory response may have several explanations. Teer-link et al. (1994) attributed the increased vascular constric-tory response to a diminished basal NO production ininfarcted rats. The mechanisms contributing to the dimin-ished basal NO response may be dependent on both adecreased NO synthesis through diminished eNOS (endo-thelial NO synthase) expression/activity, or on an increasedNO degradation, mediated through increased vascular ROSproduction. However, the increased eNOS expression mod-ulated by exercise training appears to be temporary elicitingmore stable vascular modiWcations, like alterations in thevascular caliber. According to this concept, we measured theeNOS expression in some animals and no alterations wereveriWed after the exercise protocol (unpublished data). Onthe other hand, our results suggest that the increased vaso-constrictory response to NE was paralleled by an increasedvascular ROS production, because the normalized vasocon-strictory response to NE in the MI-T group was related to anormalized superoxide production and to an increased vas-cular SOD enzyme activity. Thus, in addition to a possibleincreased basal NO bioavailability, a direct eVect mediatedthrough the decreased superoxide production in the MI-Tgroup may have contributed to the response, in accordancewith that superoxide anion can act as a contracting factor, asproposed by Katusic and Vanhoutte (1989).
Exercise training also diminished the aortic sensitivity tosodium nitroprusside in MI rats. Since MI-T group pre-sented a decrease on superoxide production parallel to anincreased SOD activity and exercise training is able toincrease eNOS activity (Hambrecht et al. 2003; IndolWet al. 2002), the changed SNP response might be related toan increased NO bioavailability after the exercise training.Transgenic mice overexpressing eNOS in the endotheliumpresent a reduced vascular reactivity to sodium nitroprus-side (Yamashita et al. 2000; Ohashi et al. 1998). In addi-tion, in conditions of stimulated NO release the vasodilatorresponses to SNP are attenuated in vessels with intact endo-thelium (Pohl and Busse 1987).
MI size has been used to access the cardiac impairmentproduced by the myocardial infarcted (Thuillez et al. 1995;PfeVer et al. 1979). However, it has been described bothlarge impaired and unaltered endothelial vasodilatory func-tion caused by MI of similar size (»30% of left ventriclearea) (Pereira et al. 2005; Brandes et al. 1998). Weobserved only a slight impairment of the maximal vasodila-tory response to ACh in MI rats, despite decreased exercisecapacity (in the absence of impairments of muscle citratesynthase activity, suggesting cardiac impairments),increased lung and ventricle weight and large infarct sizearea in the MI group. Thus, the impairments of the cardiachemodynamic status seem to be the sine qua non conditionto induce robust endothelial dysfunction, which does notappear to be reached in this study.
In conclusion, myocardial infarction increased the vaso-constrictory response to norepinephrine, which was possi-bly related to the increased response of NAD(P)H oxidaseenzyme activity and basal vascular superoxide production.On the other hand, exercise training normalized the super-oxide production, mostly due to decreased vascularNAD(P)H oxidase enzyme activity, although a minor SODeVect may also be present. These adaptations were paral-leled by normalization in the vasoconstrictory response tonorepinephrine. Thus, diminished vascular ROS productionseems to be an important mechanism by which exercisetraining mediates its beneWcial vascular eVects after MIcondition.
Acknowledgments We gratefully acknowledge the scientiWc andtechnical support provided by Dr. Francisco Rafael Martins Laurindoand his research group (Laboratory of Vascular Biology, InCor-HCF-MUSP).
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