ORIGINAL ARTICLE
Exercise training prevents the attenuation of anesthetic pre-conditioning-mediated cardioprotection in diet-induced obese rats L. Li1, F. Meng2, N. Li3, L. Zhang1, J. Wang4, H. Wang1, D. Li1, X. Zhang1, P. Dong1 and Y. Chen4 1
Department of Anesthesiology, Qilu Hospital, Shandong University, Jinan, Shandong, China Department of Anesthesiology, Jinan Maternity and Childcare Hospital, Jinan, Shandong, China 3 School of Public Health, Jining Medical University, Jinan, Shandong, China 4 Department of Emergency, Qilu Hospital, Shandong University, Jinan, Shandong, China 2
Correspondence Y. Chen, Department of Emergency, Qilu Hospital, Shandong University, Jinan City, Shandong Province, China E-mail: [email protected] Conflicts of interest The authors have no conflict of interest. Funding This study was supported by the National Natural Science Foundation of China (81170136, 81100147, 81300103, 81300219), Specialized Research Fund for the Doctoral Program of Higher Education (20130131110048), the National 973 Basic Research Program of China (2010CB732605), Taishan Scholar Program of Shandong Province, Grant from Department of Science and Technology of Shandong Province (2011GSF11806), Shandong Provincial Outstanding Medical Academic Professional Program, 1020 Program from the Health Department of Shandong Province. Submitted 21 August 2014; accepted 22 August 2014; submission 6 June 2014. Citation Li L, Meng F, Li N, Zhang L, Wang J, Wang H, Li D, Zhang X, Dong P, Chen Y. Exercise training prevents the attenuation of anesthetic pre-conditioning-mediated cardioprotection in diet-induced obese rats. Acta Anaesthesiologica Scandinavica 2014
Background: Obesity abolishes anesthetic pre-conditioninginduced cardioprotection due to impaired reactive oxygen species (ROS)-mediated adenosine monophosphate-activated protein kinase (AMPK) pathway, a consequence of increased basal myocardial oxidative stress. Exercise training has been shown to attenuate obesity-related oxidative stress. Objective: This study tests whether exercise training could normalize ROS-mediated AMPK pathway and prevent the attenuation of anesthetic pre-conditioning-induced cardioprotection in obesity. Methods: Male Sprague–Dawley rats were divided into lean rats fed with control diet and obese rats fed with high-fat diet. After 4 weeks of feeding, lean and obese rats were assigned to sedentary conditions or treadmill exercise for 8 weeks. Results: There was no difference in infarct size between lean sedentary and obese sedentary rats after 25 min of myocardial ischemia followed by 120 min reperfusion. In lean rats, sevoflurane equally reduced infarct size in lean sedentary and lean exercise-trained rats. Molecular studies revealed that AMPK activity, endothelial nitric oxide synthase, and superoxide production measured at the end of ischemia in lean rats were increased in response to sevoflurane. In obese rats, sevoflurane increased the above molecular parameters and reduced infarct size in obese exercise-trained rats but not in obese sedentary rats. Additional study showed that obese exercisetrained rats had decreased basal oxidative stress than obese sedentary rats. Conclusion: The results indicate that exercise training can prevent the attenuation of anesthetic cardioprotection in obesity. Preventing the attenuation of this strategy may be associated with reduced basal oxidative stress and normalized ROS-mediated AMPK pathway, but the causal relationship remains to be determined.
doi: 10.1111/aas.12414
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
bs_bs_banner
1
L. LI ET AL.
Obesity, which is associated with metabolic syndrome, has become an epidemic problem worldwide. It is estimated that more than two thirds of Americans are either overweight or obese. Obesity is often associated with enhanced morbidity and mortality, which is largely due to cardiovascular diseases.1–3 Patients with obesity and metabolic syndrome have a threefold increased risk of coronary heart diseases.4,5 Following an acute myocardial infarction, obese patients experience worse clinical outcomes,6 increased heart failure,7 increased myocardial infarct size, more in-hospital complications,8 and worse myocardial reperfusion following primary percutaneous coronary intervention.9 Thus, using any effective cardioprotective strategy to reduce the consequences of coronary artery disease is important in this population. Volatile anesthetics are reported to produce pharmacological pre-conditioning and reduce ischemia-reperfusion (I/R) injury in the heart in a variety of experimental animal models as well as in humans.10–13 Volatile anesthetics especially in the perioperative period provide a major advantage compared with ischemic pre-conditioning. In normal animals, activation of AMP-activated protein kinase (AMPK), an important sensor and regulator of cellular energy status, is reported to be involved in anesthetic-induced cardioprotective signaling; and this activation requires production of reactive oxygen species (ROS).10,14 However, a recent study demonstrated that anesthetic pre-conditioning against myocardial I/R injury is abolished in diet-induced obesity, and the failure to pre-condition the obese myocardium is probably due to increased basal myocardial oxidative stress, which impairs ROSmediated AMPK activation during anesthetic preconditioning.10 Thus, modulation of obesityrelated oxidative stress may be useful in improving this protective mechanism in the obese heart. Exercise is an important lifestyle modification to prevent cardiovascular diseases. Moderate exercise training has been shown to induce weight loss, improve plasma biomarkers of oxidative status,15 and decrease oxidative stress in the heart and kidney in obese animals and humans.16,17 Exercise training has also been shown to reduce age-related myocardial oxidative stress18 and restore ischemic-pre-conditioning in
the aging heart.19 However, effects of exercise training on anesthetic pre-conditioning in obese heart are unclear. Hence, the aim of the present study was to examine whether exercise training would normalize ROS-mediated AMPK signaling pathway by reducing basal myocardial oxidative stress and prevent the attenuation of anesthetic preconditioning-induced cardioprotection in obesity. For this purpose, the high-fat diet-induced obese rat model was used as a representation of human obesity syndrome.20 Methods Animals and exercise training All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Shandong University (Permit Number: 20120046). The research protocols and procedures were approved by IACUC of Shandong University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. We were planning a study of subjects evenly divided into eight groups with different treatments as described below. Sample size was determined by a power analysis based on the preliminary data. If the effect size was 0.715, five subjects in each group would be needed to be able to reject the null hypothesis that the population means of the eight groups were equal with probability (power) 0.8. The Type I error probability associated with this test of this null hypothesis was 0.05. One hundred thirty-two male Sprague– Dawley rats weighing 82.7 ± 20.7 g (Beijing Laboratory Animal Research Center, Beijing, China) were housed in individual cages in a climatecontrolled environment (22.8 ± 2.0 °C, 45–50% humidity). Following 1 week of acclimation, animals were randomly divided into lean control rats that were fed with a control diet containing 10% of kcal from fat (designated as low fat) and obese rats that were fed with a high-fat diet containing 45% of kcal from fat (Research Diets, New Brunswick, NJ,USA) for 12 consecutive weeks. After 4 weeks of control diet or high-fat diet feeding, lean and obese rats were assigned to sedentary conditions or regular treadmill exercise, Acta Anaesthesiologica Scandinavica (2014)
2
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
respectively. The exercise protocol was adapted from a previously published procedure.16 Briefly, rats were exercised at 0° incline and a speed of 10 m/min for 1 h daily (5-min break every 15 min), 5 days/week. Body weights were measured weekly until the end of the study. After 8 weeks of exercise, the rats were rested for 48 h before any further procedures were performed to eliminate interference from acute effects of exercise. Surgical preparation Rats were anaesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg) and were maintained by sodium pentobarbital (15– 25 mg/kg/min) infusion throughout the experiment. Right femoral vein and artery were cannulated for fluid and drug administration or for arterial blood pressure (BP) measurement. After tracheal intubation, rats were ventilated using a rodent ventilator (Shanghai Alcott Biotech Co., Shanghai, China) with 33% oxygen.
The heart was exposed via a left thoracotomy at the left fifth intercostal space and was suspended in a pericardial cradle. A 6–0 silk suture was placed around the proximal left descending coronary artery in the area immediately below the left atrial appendage. The ends of the suture were threaded through a propylene tube to form a snare. Coronary artery occlusion was produced by clamping the snare onto the epicardial surface of the heart and was confirmed by the appearance of epicardial cyanosis. Reperfusion was achieved by loosening the snare and was verified by observing an epicardial hyperemic response. Hemodynamic data were continuously recorded on a polygraph with Medlab-U/4C501H (Nanjing Mei Yi Technology, Nanjing, China). Experimental protocol I The experimental design for protocol I is illustrated in Fig. 1A. After 12 weeks of control diet or high-fat diet feeding and 8 weeks of exercise training, sedentary rats and exercise-trained rats
Fig. 1. Schematic illustration of the experimental protocol I (A) and II (B). SED, sedentary rats; EXE, exercise-trained rats; SEV or S, sevoflurane pre-conditioning. Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
3
L. LI ET AL.
were randomly divided into eight groups (n = 11–13 for each group) for myocardial I/R as follows: (1) L-SED, lean sedentary rats subjected to I/R alone; (2) L-SED + SEV, lean sedentary rats received sevoflurane pre-conditioning before I/R; (3) L-EXE + SEV, lean exercise-trained rats received sevoflurane pre-conditioning before I/R; (4) L-EXE, lean exercise-trained rats subjected to I/R alone; (5) O-SED, obese sedentary rats subjected to I/R alone; (6) O-SED + SEV, obese sedentary rats received sevoflurane pre-conditioning before I/R; (7) O-EXE + SEV, obese exercisetrained rats received sevoflurane pre-conditioning before I/R; and (8) O-EXE, obese exercise-trained rats subjected to I/R alone. The sevoflurane preconditioning and myocardial I/R were performed according to previous studies.10,21 Briefly, after a 30-min stabilization period, rats received 1 minimum alveolar concentration sevoflurane (in rats 2.4 vol%) for three 5-min periods, interspersed with two 5-min washout periods. Ten minutes min after pre-conditioning, rats were subjected to 25 min of regional myocardial ischemia followed by 120 min of reperfusion. At the end of the protocol, the hearts were removed for assessments of area at risk and infarct size. To examine the effects of exercise training on sevoflurane pre-conditioning-induced AMPK activation, nitric oxide (NO) level and superoxide production, some rats from each group (n = 5–6 for each group) were sacrificed at the end of ischemia, and myocardial samples were collected from ischemic left ventricular areas. Part of the fresh heart tissue was then used for measurement of myocardial superoxide production immediately, and the remainder was frozen in liquid nitrogen and stored at −80 °C for Western blot analysis and myocardial NO measurement. We selected this time point to collect tissues based on a previous study14 showing that the sevofluraneinduced AMPK activation occurs during ischemia but not before ischemia. Experimental protocol II To investigate the effects of exercise training on metabolism and basal myocardial oxidative stress, additional sedentary and exercise-trained rats (n = 8 for each group) from lean or obese group without pre-conditioning and I/R were used. The experimental design for protocol II is illustrated
in Fig. 1B. Briefly, after 12 weeks of control diet or high-fat diet feeding and 8 weeks of exercise training, rats were fasted for 12 h prior to blood collection. After blood collection, the animals were sacrificed and hearts were quickly removed. Plasma was separated from whole blood and stored at −80 °C for biochemical assay. Part of the fresh heart tissue was used for measurement of basal myocardial superoxide production immediately, and the remainder was frozen in liquid nitrogen and stored at −80 °C for assessment of basal myocardial oxidative stress by Western blot. Assessment of myocardial infarction At the end of the reperfusion period, the coronary artery was re-occluded, and 0.1% methylene blue was injected intravenously to label the nonischemic, blue-stained, normal area, thereby delineating the risk zone as a non-stained area. The rat was then sacrificed and the heart was removed, weighed, and frozen in a cold chamber at −18 °C. The heart was cut into six crosssectional slices of 2-mm thickness. The slices were incubated at 37 °C for 20 min in 1% 2,3,5triphenyl tetrazolium chloride buffer followed by 10% formaldehyde for 10 min to increase the contrast between stained tissue as a deep red color and non-stained tissue. Slices were then photographed, and the percentages of the at-risk and infarcted areas were calculated using an image analysis program. Western blot analysis of myocardial AMPK and eNOS activity and oxidative stress Myocardial samples were homogenized in icecold cell lysis buffer containing a complete protease inhibitor to isolate total protein. Proteins were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes. Membranes were immunoblotted with antibodies antiphosphorated (Ph)-AMPK at Thr172, anti-total AMPK, anti-Ph-endothelial nitric oxide synthase (eNOS), anti-total eNOS (Cell Signalling), antisuperoxide dismutase (SOD), anti-catalase, antinuclear factor erythroid-derived 2-like 2 (Nrf2) and anti-β-actin (Santa Cruz Biotechnology). Reactive protein was detected by an enhanced chemiluminescence system and analyzed by imaging densitometry. Acta Anaesthesiologica Scandinavica (2014)
4
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
Biochemical assay Plasma glucose, total cholesterol, and triglyceride concentrations were measured with a Hitachi clinical analyzer. Plasma insulin and leptin levels were measured by commercially available rat ELISA kit (Invitrogen, Camarillo, CA). Measurement of myocardial levels of nitrite and nitrate (NOx) The levels of nitrite and nitrate (NOx) were measured in left ventricular tissues using a nitrate/ nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA).
Measurement of myocardial superoxide anion Twelve weeks after respective diet feeding, myocardial superoxide production at the end of ischemia in animals from protocol I and basal myocardial superoxide production in animals from protocol II were measured by a lucigeninenhanced chemiluminescence assay according to previously described method.22 Briefly, Fresh left ventricular tissue was homogenized in a 20-mM sodium phosphate buffer (pH 7.4) containing 0.01 mM EDTA. The homogenate was subjected to low-speed centrifugation to remove nuclei and unbroken cell debris. The pellet was discarded, and the supernatant was obtained immediately for oxygen measurement. After background chemiluminescence in buffer (2 ml) containing lucigenin (5 μM) was measured for 5 min, an aliquot of 100 μl of supernatant was added, and the chemiluminescence was measured for 30 min at
room temperature. O2- levels were expressed as relative light units per second after subtraction of background activity. Statistical analysis Data are expressed as mean ± SD. Differences were analyzed using two-way ANOVA followed by Newman–Keuls multiple-comparison posthoc test. P < 0.05 was considered statistically significant. Results Metabolic effects of exercise training Before exercise training (4 weeks after control diet or high-fat diet feeding), obese rats displayed high body weight compared with lean rats (180 ± 24 vs. 156 ± 16, P < 0.05; obese rats vs. lean rats). At this time point, body weight was similar between lean sedentary and lean exercise-trained rats and also between obese sedentary and obese exercise-trained rats. At the end of the study, obese sedentary rats were markedly heavier than lean sedentary rats (Table 1). In addition, plasma levels of insulin, leptin, and total cholesterol were significantly higher in obese sedentary rats than those in lean sedentary rats. Exercise training significantly reduced these parameters in obese exercise-trained rats but had no effects in lean exercise-trained rats. The levels of blood glucose and plasma triglycerides were similar among groups. Systemic hemodynamic changes There were no significant differences among the eight experimental groups for heart rate (HR) and
Table 1 Effects of exercise training on metabolic parameters.
Body weight (g) Blood glucose (mg/dl) Plasma insulin (ng/ml) Plasma leptin (ng/ml) Plasma triglycerides (mg/dl) Plasma cholesterol (mg/dl)
L-SED
L-EXE
O-SED
O-EXE
342 ± 21 92 ± 21 3.56 ± 1.73 4.19 ± 2.21 115 ± 61 92 ± 41
340 ± 27 90 ± 16 3.52 ± 2.04 4.08 ± 1.75 116 ± 50 90 ± 36
458 ± 33 103 ± 27 8.34 ± 3.03* 7.72 ± 2.72* 124 ± 55 178 ± 66*
407 ± 25*† 101 ± 22 4.22 ± 2.35† 5.59 ± 1.73*† 113 ± 47 135 ± 53*†
*P < 0.05 vs. L-SED; †P < 0.05, O-EXE vs. O-SED. Values are mean ± SD; n = 8 for each group. Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test.
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
5
L. LI ET AL.
Table 2 Hemodynamic parameters in lean (L) and obese (O) rats.
HR (beats/min) L-SED L-SED + SEV L-EXE + SEV L-EXE O-SED O-SED + SEV O-EXE + SEV O-EXE Mean BP (mmHg) L-SED L-SED + SEV L-EXE + SEV L-EXE O-SED O-SED + SEV O-EXE + SEV O-EXE
Baseline
Pre-ischemia
Ischemia 25 min
Reperfusion 120 min
336 ± 26 343 ± 36 340 ± 39 337 ± 44 339 ± 33 338 ± 29 340 ± 31 341 ± 39
335 ± 29 340 ± 39 341 ± 36 337 ± 37 340 ± 39 336 ± 32 339 ± 33 342 ± 25
339 ± 41 342 ± 38 343 ± 39 339 ± 34 343 ± 29 339 ± 41 342 ± 28 345 ± 39
305 ± 23* 310 ± 28* 309 ± 22* 306 ± 30* 307 ± 26* 305 ± 23* 311 ± 25* 308 ± 17*
116 ± 16 111 ± 15 109 ± 31 113 ± 26 110 ± 18 108 ± 31 110 ± 25 109 ± 25
115 ± 23 106 ± 23 103 ± 22 110 ± 31 109 ± 17 109 ± 25 112 ± 21 109 ± 19
82 ± 18* 85 ± 10* 80 ± 17* 83 ± 12* 79 ± 19* 82 ± 13* 86 ± 13* 81 ± 17*
79 ± 14* 80 ± 12* 77 ± 20* 81 ± 20* 78 ± 9* 79 ± 10* 80 ± 10* 75 ± 15*
Baseline: stabilization period; Pre-ischemia: 5 min before ischemia. Values are mean ± SD; n = 6–7 for each group. Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. baseline; HR, heart rate; mean BP, mean blood pressure.
mean BP in the baseline (stabilization period) or in the pre-ischemic period. I/R produced decrease in mean BP in each experimental group. At the end of reperfusion, HR was also significantly decreased in each experimental group as compared with the baseline. These reductions were probably due to surgical stimuli or cardiac dysfunction following acute myocardial infarction. However, there were no significant differences in these parameters across groups. Hemodynamic parameters are shown in Table 2. Effect of sevoflurane pre-conditioning and exercise training on infarct size As shown in Fig. 2, there was no difference in infarct size between lean sedentary and obese sedentary rats following I/R. Sevoflurane preconditioning significantly reduced infarct size in lean sedentary rats, but it failed to protect obese sedentary rats against myocardial infarction. After 8 weeks of exercise training, sevoflurane preconditioning did not further decrease infarct size in lean exercise-trained rats; however, this strategy significantly reduced infarct size in obese exercisetrained rats to the same extent as that observed in lean rats. Exercise training alone did not reduce
infarct size in both lean and obese rats. In addition, the ratio of area at risk following I/R was similar among all groups. Effect of sevoflurane pre-conditioning and exercise training on AMPK activation At the end of ischemia, AMPK activation (phosphorylation of AMPK/total AMPK) did not differ between lean sedentary and obese sedentary rats (Fig. 3). Sevoflurane pre-conditioning similarly increased AMPK activation in lean sedentary and lean exercise-trained rats, whereas it had no significant effect in obese sedentary rats. However, after exercise training, sevoflurane preconditioning significantly increased AMPK activation in obese exercise-trained rats compared with obese sedentary rats. Exercise training alone did not change AMPK activation in either lean rats or obese rats as compared to their sedentary rats. Effect of sevoflurane pre-conditioning and exercise training on endothelial nitric oxide synthase (eNOS) activation and myocardial NOx production Endothelial nitric oxide synthase activation (phosphorylation of eNOS/total eNOS) and NOx Acta Anaesthesiologica Scandinavica (2014)
6
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
Fig. 2. Infarct size (A) and area at risk (B) in lean and obese rats subjected to 25 min of ischemia followed by 120 min of reperfusion in the absence or presence of sevoflurane pre-conditioning. The infarct size was expressed as percent of the area at risk, the area at risk was expressed as percent of left ventricle. Values were presented as mean ± SD (n = 6–7 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. respective lean rats in the same experiment.
level were similar between two sedentary groups (Fig. 4). Sevoflurane pre-conditioning significantly augmented eNOS activation and NOx level in lean sedentary and lean exercise-trained rats but not in obese sedentary rats, whereas eNOS activation and NOx level were enhanced in response to sevoflurane pre-conditioning in obese exercisetrained rats. Neither eNOS activation nor NOx level was altered in lean exercise-trained rats or obese exercise-trained rats without sevoflurane pre-conditioning. Effect of sevoflurane pre-conditioning and exercise training on superoxide anion Myocardial superoxide production, measured at the end of ischemia, was significantly higher in obese sedentary rats than that in lean sedentary rats (Fig. 5). Sevoflurane pre-conditioning augmented superoxide production equally in lean
Fig. 3. Myocardial AMPK activation at the end of ischemia from each group. Representative Western blots are aligned with the matching grouped data. AMPK activation was expressed as the ratio of phosphorylated (Ph-) to total AMPK. Values are presented as means ± SD (n = 5–6 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. respective lean rats in the same experiment.
sedentary and lean exercise-trained rats, but it had no effect in obese sedentary rats. However, superoxide production was increased in obese exercisetrained rats in response to sevoflurane preconditioning. Exercise training alone did not alter superoxide production in lean rats, but it reduced that in obese rats as compared to their sedentary rats.
Effect of exercise training on basal oxidative stress After 12 weeks of control diet or high-fat diet feeding and 8 weeks of exercise training, the basal level of superoxide production in the heart was greater in obese sedentary rats than that in lean sedentary rats. Superoxide production was unchanged in lean exercise-trained rats, but it was reduced in obese exercise-trained rats (Fig. 6A). Western blot analysis revealed that protein levels of antioxidant enzyme SOD and antioxidant master regulator Nrf2 in the heart were lower in obese sedentary rats than those in lean sedentary rats (Fig. 6B–E). Exercise training reversed protein levels of SOD and Nrf2 in obese
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
7
L. LI ET AL.
Fig. 5. Myocardial superoxide production at the end of ischemia from each group. Values are presented as means ± SD (n = 5–6 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. Respective lean rats in the same experiment.
Fig. 4. Myocardial eNOS activation (A) and nitrite and nitrate (NOx) level (B) at the end of ischemia from each group. Representative Western blots are aligned with the matching grouped data. eNOS activation was expressed as the ratio of phosphorylated (Ph-) to total eNOS. Values are presented as means ± SD (n = 5–6 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. respective lean rats in the same experiment.
exercise-trained rats but did not alter those in lean exercise-trained rats. There was no difference in protein levels of antioxidant enzyme catalase across four groups. Discussion The novel finding of this study is that exercise training prevented the attenuation of anesthetic sevoflurane pre-conditioning-mediated cardioprotection in high-fat diet-induced obese rats. Preventing the attenuation of this protective strategy in obese rats might be related to improvement in basal oxidative stress, normalization of ROSmediated AMPK signaling pathway in response to sevoflurane pre-conditioning, and amelioration of metabolic parameters.
Obesity has become a considerable health problem worldwide over the past 20 years. The increased prevalence of obesity and its associated lifestyle diseases can mostly be attributed to the increased availability and consumption of energy-dense foods and physical inactivity.23,24 A relation has been established for obesity and risk factors for development of cardiovascular diseases in the Framingham Heart Study.25 Obesityrelated disorders are linked to the undesirable outcomes of ischemic heart diseases, although the mechanisms responsible are poorly defined. Therefore, new strategies for cardioprotection are required to improve the clinical outcomes in obese patients with ischemic heart diseases. Anesthetic pre-conditioning is a potential cardioprotective strategy that has been demonstrated to protect against I/R injury, improve functional recovery, decrease post-ischemic myocardial stunning, and attenuate myocardial apoptosis.26 Recent studies demonstrated that activation of AMPK mediates anesthetic pre-conditioninginduced cardioprotection.10,27 Activation of AMPK causes activation of eNOS and release of NOx, which is a crucial step in mediating cardioprotection by sevoflurane pre-conditioning.21 Augmented mitochondrial ROS level in response to pre-conditioning has been proposed as a trigger for activation of this signaling pathway to confer cardioprotection.28,29 However, it has been reported that sevoflurane pre-conditioningActa Anaesthesiologica Scandinavica (2014)
8
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
Fig. 6. Myocardial basal superoxide production (A), protein levels of antioxidant enzymes SOD (B), catalase (C), and antioxidant master regulator Nrf2 (D) in respective sedentary (SED) and exercise-trained (EXE) rats after 12 weeks of control diet (L) or high-fat diet feeding (O). Representative Western blots were shown in figure E. Values are corrected by β-actin and presented as means ± SD (n = 8 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. L-SED rats; †P < 0.05, O-EXE vs. O-SED rats.
induced cardioprotective action was abolished in diet-induced obesity due to a diminished effect of sevoflurane pre-conditioning on activation of the ROS-mediated AMPK signaling pathway.10 In the present study, it was found that sevoflurane pre-conditioning significantly reduced infarct size in lean sedentary rats, but it failed to protect obese sedentary rats against myocardial infarction. Molecular studies revealed that sevoflurane pre-conditioning elevated AMPK activity at the end of ischemia in lean sedentary rats, which was accompanied by increase in eNOS activity and production of myocardial NOx. In addition, myocardial superoxide production after sevoflurane pre-conditioning was augmented. However, these responses were not observed in obese sedentary rats. These results confirm previous findings, indicating that impaired ROSmediated AMPK signaling pathway might lead to the inability to pre-condition the heart against ischemia-reperfusion injury in obesity. Previous studies have suggested that an augmented basal oxidative stress associated with metabolic syndrome is central to the impaired ROS generation
in response to pre-conditioning in obese hearts.10 Augmented oxidative stress at baseline may result in oxidative modification of key mitochondrial enzymes and/or ion channels, leading to failure of enhanced ROS generation in the mitochondria of obese hearts by pre-conditioning.10,29 Thus, it is necessary to examine whether reduction of basal oxidative stress would prevent the attenuation of anesthetic pre-conditioning in obesity. Regular physical exercise has long been recognized as an essential component in the managing obesity, owing to the beneficial effects on weight maintenance/reduction and lowering the risk of developing type 2 diabetes and cardiovascular diseases.30,31 Moderate exercise training can attenuate age or obesity-related myocardial oxidative stress and improve cardiovascular functions.17,18 Previous study has reported that exercise training restores ischemic preconditioning in the aging heart in rats.19 The current study showed that sevoflurane preconditioning significantly reduced infarct size in obese exercise-trained rats but not in obese seden-
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
9
L. LI ET AL.
tary rats. It was also found that sevoflurane preconditioning induced activation of AMPK in obese exercise-trained rats, which was associated with concomitant increases in activation of eNOS, myocardial NOx level, and superoxide production, similar to those observed in lean rats. These results indicate that exercise training may prevent the attenuation of anesthetic cardioprotection in obesity, and preventing the attenuation of this beneficial strategy in obesity might be associated with normalized AMPK–eNOS signaling pathway. Notably, exercise training alone did not reduce infarct size in either lean rats or obese rats. This observation is inconsistent with a recent study showing that regular treadmill exercise reduces myocardial infarct size in both wild-type and obese (ob/ob) mice without changes in metabolic parameters.32 The underlying reasons for this discrepancy remain unclear. Further research is needed to investigate whether this discrepancy is due to differences in animal species, exercise intensity, or changes of metabolic parameters. Since impaired ROS-mediated AMPK signaling pathway is suggested to be a consequence of increased basal oxidative stress associated with metabolic syndrome in obesity, the effects of exercise training on basal oxidative stress and metabolic parameters in additional lean and obese rats were investigated. Twelve weeks of high-fat diet feeding induced metabolic syndrome in obese sedentary rats, including visceral obesity, hyperinsulinemia, hyperleptinemia, and dyslipidemia. The characteristics and metabolic parameters are consistent with previous reports,2,10 indicating that this animal model represents the classical insulin resistance and leptin resistance features of human obesity. Of note, a significant increase in fasting plasma glucose was not observed in highfat diet-induced obese rats in this study, although this animal model has been considered as one of the suitable animal models for type 2 diabetes mellitus. These data are consistent with previous report,2 suggesting that obese rats fed a high-fat diet for 12 weeks might be in the early stages of type 2 diabetes mellitus. However, impaired glucose tolerance has been reported in this obese animal model after 12 weeks of high-fat diet feeding.2 It is possible that exercise did not alter fasting plasma glucose but improved impaired glucose tolerance, although we did not perform an oral glucose tolerance test. Molecular analyses
showed that obese sedentary rats had a higher basal level of superoxide production in the myocardium, accompanied by reduced antioxidant enzyme SOD and antioxidant master regulator Nrf2, suggesting that obesity-related myocardial oxidative stress is associated with diminished antioxidant capacity. More importantly, exercise training was found to ameliorate metabolic syndrome, reduce myocardial oxidative stress, and improve antioxidant systems. These results are supported by a recent study showing that exercise training reduces increased oxidative stress in aging heart by reversing impaired antioxidant systems.18 But the improvement in antioxidant systems may not be the sole reason for the reduction in basal myocardial oxidative stress in obese rats after exercise training. Emerging evidence suggests that obesity is associated with chronic low-grade inflammation in the heart, which has been shown to induce myocardial oxidative stress.33 We could not exclude the possibility that exercise attenuates myocardial oxidative stress by reducing inflammation in the heart of obese rats. Interestingly, in the present study, the basal level of myocardial superoxide production was higher in obese sedentary rats than that in lean sedentary rats, but the infarct size was similar between the two groups. In addition, exercise training alone reduced myocardial superoxide production, but it did not attenuate infarct size following myocardial infarction in obese exercise-trained rats, indicating that myocardial oxidative stress may not be directly related to infarct size in obesity. However, reduction in basal myocardial oxidative stress by exercise training might improve mitochondrial function to produce sufficient ROS in response to sevoflurane pre-conditioning, and normalize AMPK signaling pathway, contributing to preventing the attenuation of anesthetic cardioprotection in obesity. A number of studies have demonstrated that cardioprotective effects of ischemic preconditioning are attenuated in various pathological conditions, including hyperlipidemia34,35 and hyperinsulinemia.36 Hyperlipidemia not only renders the myocardium more susceptible to ischemia and reperfusion-induced injury, but also inhibits ischemic pre-conditioning-mediated cardioprotection by decreasing adenosine release34 or increasing glycogen synthase kinase-3β expression.37 Hyperinsulinemia can suppress Acta Anaesthesiologica Scandinavica (2014)
10
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
ischemic pre-conditioning-mediated cardioprotection by activation of phosphoinositide-3kinase-protein kinase B signaling pathway.36 Thus, we could not exclude the possibility that improved metabolic parameters by exercise prevent the attenuated cardioprotective effects of anesthetic pre-conditioning in obesity through the above mechanisms. Several limitations of the present study should be acknowledged. This study did not demonstrate a causal relationship because no inhibitor or stimulator studies were included in this analysis. In addition, our results suggest that preventing obesity-induced attenuation of anesthetic cardioprotection by exercise may be associated with reduced basal oxidative stress and normalized ROS-mediated AMPK signaling pathway in response to sevoflurane pre-conditioning, but other mechanisms, including the amelioration of metabolic parameters that has been mentioned above, may also directly or indirectly contribute to improved anesthetic cardioprotection. Further studies are required to examine whether an AMPK inhibitor or a ROS stimulator can completely abolish sevoflurane-induced cardioprotection in exercise-trained obese rats and whether an AMPK stimulator or a ROS scavenger can completely prevent the attenuation of anesthetic cardioprotection in obese-sedentary rats. In conclusion, offering anesthetic preconditioning as a cardioprotective technique in patients to attenuate ischemia-reperfusion injury is an important therapeutic target. However, this beneficial strategy is attenuated in obesity. The present study demonstrates that exercise training prevents the attenuation of anesthetic cardioprotection in obesity probably by reducing basal oxidative stress and normalizing AMPK signaling pathway. The current study also indicates that performing regular exercise is an important lifestyle modification to ameliorate metabolic syndrome and in turn reverse impaired signaling pathways that mediate cardioprotective actions in obesity.
References 1. Heusch G. Obesity – a risk factor or a risk factor for myocardial infarction? Br J Pharmacol 2006; 149: 1–3.
2. Relling DP, Esberg LB, Fang CX, Johnson WT, Murphy EJ, Carlson EC, Saari JT, Ren J. High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of foxo3a transcription factor independent of lipotoxicity and apoptosis. J Hypertens 2006; 24: 549–61. 3. Simpson CR, Buckley BS, McLernon DJ, Sheikh A, Murphy A, Hannaford PC. Five-year prognosis in an incident cohort of people presenting with acute myocardial infarction. PLoS ONE 2011; 6: e26573. 4. Isomaa B, Almgren P, Tuomi T, Forsen B, Lahti K, Nissen M, Taskinen MR, Groop L. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 2001; 24: 683–9. 5. Tabassum F, Batty GD. Are current UK national institute for health and clinical excellence (nice) obesity risk guidelines useful? Cross-sectional associations with cardiovascular disease risk factors in a large, representative English population. PLoS ONE 2013; 8: e67764. 6. Schwartz GG, Olsson AG, Szarek M, Sasiela WJ. Relation of characteristics of metabolic syndrome to short-term prognosis and effects of intensive statin therapy after acute coronary syndrome: an analysis of the myocardial ischemia reduction with aggressive cholesterol lowering (miracle) trial. Diabetes Care 2005; 28: 2508–13. 7. Zeller M, Steg PG, Ravisy J, Laurent Y, Janin-Manificat L, L’Huillier I, Beer JC, Oudot A, Rioufol G, Makki H, Farnier M, Rochette L, Verges B, Cottin Y, Observatoire des Infarctus de Cote-d’Or Survey Working G. Prevalence and impact of metabolic syndrome on hospital outcomes in acute myocardial infarction. Arch Intern Med 2005; 165: 1192–8. 8. Clavijo LC, Pinto TL, Kuchulakanti PK, Torguson R, Chu WW, Satler LF, Kent KM, Suddath WO, Pichard AD, Waksman R. Metabolic syndrome in patients with acute myocardial infarction is associated with increased infarct size and in-hospital complications. Cardiovasc Revasc Med 2006; 7: 7–11. 9. Celik T, Turhan H, Kursaklioglu H, Iyisoy A, Yuksel UC, Ozmen N, Isik E. Impact of metabolic syndrome on myocardial perfusion grade after primary percutaneous coronary intervention in patients with acute st elevation myocardial infarction. Coron Artery Dis 2006; 17: 339–43. 10. Song T, Lv LY, Xu J, Tian ZY, Cui WY, Wang QS, Qu G, Shi XM. Diet-induced obesity suppresses sevoflurane preconditioning against myocardial
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
11
L. LI ET AL.
11.
12.
13.
14.
15.
16.
17.
18.
19.
ischemia-reperfusion injury: role of amp-activated protein kinase pathway. Exp Biol Med (Maywood) 2011; 236: 1427–36. Granfeldt A, Lefer DJ, Vinten-Johansen J. Protective ischaemia in patients: preconditioning and postconditioning. Cardiovasc Res 2009; 83: 234–46. Raphael J, Rivo J, Gozal Y. Isoflurane-induced myocardial preconditioning is dependent on phosphatidylinositol-3-kinase/akt signalling. Br J Anaesth 2005; 95: 756–63. Redel A, Stumpner J, Tischer-Zeitz T, Lange M, Smul TM, Lotz C, Roewer N, Kehl F. Comparison of isoflurane-, sevoflurane-, and desflurane-induced pre- and postconditioning against myocardial infarction in mice in vivo. Exp Biol Med (Maywood) 2009; 234: 1186–91. Lamberts RR, Onderwater G, Hamdani N, Vreden MJ, Steenhuisen J, Eringa EC, Loer SA, Stienen GJ, Bouwman RA. Reactive oxygen species-induced stimulation of 5’amp-activated protein kinase mediates sevoflurane-induced cardioprotection. Circulation 2009; 120: S10–5. Rector RS, Warner SO, Liu Y, Hinton PS, Sun GY, Cox RH, Stump CS, Laughlin MH, Dellsperger KC, Thomas TR. Exercise and diet induced weight loss improves measures of oxidative stress and insulin sensitivity in adults with characteristics of the metabolic syndrome. Am J Physiol Endocrinol Metab 2007; 293: E500–6. Muhammad AB, Lokhandwala MF, Banday AA. Exercise reduces oxidative stress but does not alleviate hyperinsulinemia or renal dopamine d1 receptor dysfunction in obese rats. Am J Physiol Renal Physiol 2011; 300: F98–104. Gao Z, Novick M, Muller MD, Williams RJ, Spilk S, Leuenberger UA, Sinoway LI. Exercise and diet-induced weight loss attenuates oxidative stress related-coronary vasoconstriction in obese adolescents. Eur J Appl Physiol 2013; 113: 519–28. Gounder SS, Kannan S, Devadoss D, Miller CJ, Whitehead KS, Odelberg SJ, Firpo MA, Paine R 3rd, Hoidal JR, Abel ED, Rajasekaran NS. Impaired transcriptional activity of nrf2 in age-related myocardial oxidative stress is reversible by moderate exercise training. PLoS ONE 2012; 7: e45697. Abete P, Calabrese C, Ferrara N, Cioppa A, Pisanelli P, Cacciatore F, Longobardi G, Napoli C, Rengo F. Exercise training restores ischemic preconditioning in the aging heart. J Am Coll Cardiol 2000; 36: 643–50.
20. Madsen AN, Hansen G, Paulsen SJ, Lykkegaard K, Tang-Christensen M, Hansen HS, Levin BE, Larsen PJ, Knudsen LB, Fosgerau K, Vrang N. Long-term characterization of the diet-induced obese and diet-resistant rat model: a polygenetic rat model mimicking the human obesity syndrome. J Endocrinol 2010; 206: 287–96. 21. Fradorf J, Huhn R, Weber NC, Ebel D, Wingert N, Preckel B, Toma O, Schlack W, Hollmann MW. Sevoflurane-induced preconditioning: impact of protocol and aprotinin administration on infarct size and endothelial nitric-oxide synthase phosphorylation in the rat heart in vivo. Anesthesiology 2010; 113: 1289–98. 22. Chen CH, Liu K, Chan JY. Anesthetic preconditioning confers acute cardioprotection via up-regulation of manganese superoxide dismutase and preservation of mitochondrial respiratory enzyme activity. Shock 2008; 29: 300–8. 23. Kahn BB, Flier JS. Obesity and insulin resistance. J Clin Invest 2000; 106: 473–81. 24. Haffner S, Taegtmeyer H. Epidemic obesity and the metabolic syndrome. Circulation 2003; 108: 1541–5. 25. Kenchaiah S, Evans JC, Levy D, Wilson PW, Benjamin EJ, Larson MG, Kannel WB, Vasan RS. Obesity and the risk of heart failure. N Engl J Med 2002; 347: 305–13. 26. Wang C, Xie H, Liu X, Qin Q, Wu X, Liu H, Liu C. Role of nuclear factor-kappab in volatile anaesthetic preconditioning with sevoflurane during myocardial ischaemia/reperfusion. Eur J Anaesthesiol 2010; 27: 747–56. 27. Huffmyer J, Raphael J. Physiology and pharmacology of myocardial preconditioning and postconditioning. Semin Cardiothorac Vasc Anesth 2009; 13: 5–18. 28. Tanaka K, Weihrauch D, Ludwig LM, Kersten JR, Pagel PS, Warltier DC. Mitochondrial adenosine triphosphate-regulated potassium channel opening acts as a trigger for isoflurane-induced preconditioning by generating reactive oxygen species. Anesthesiology 2003; 98: 935–43. 29. Katakam PV, Jordan JE, Snipes JA, Tulbert CD, Miller AW, Busija DW. Myocardial preconditioning against ischemia-reperfusion injury is abolished in Zucker obese rats with insulin resistance. Am J Physiol Regul Integr Comp Physiol 2007; 292: R920–6. 30. Lee-Young RS, Ayala JE, Fueger PT, Mayes WH, Kang L, Wasserman DH. Obesity impairs skeletal muscle AMPK signaling during exercise: role of ampkalpha2 in the regulation of exercise capacity in vivo. Int J Obes (Lond) 2011; 35: 982–9. Acta Anaesthesiologica Scandinavica (2014)
12
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
31. Sigal RJ, Kenny GP, Wasserman DH, Castaneda-Sceppa C, White RD. Physical activity/exercise and type 2 diabetes: a consensus statement from the American diabetes association. Diabetes Care 2006; 29: 1433–8. 32. Pons S, Martin V, Portal L, Zini R, Morin D, Berdeaux A. Ghaleh B. Regular treadmill exercise restores cardioprotective signaling pathways in obese mice independently from improvement in associated co-morbidities. J Mol Cell Cardiol 2013; 54: 82–9. 33. Palomer X, Salvado L, Barroso E, Vazquez-Carrera M. An overview of the crosstalk between inflammatory processes and metabolic dysregulation during diabetic cardiomyopathy. Int J Cardiol 2013; 168: 3160–72. 34. Ueda Y, Kitakaze M, Komamura K, Minamino T, Asanuma H, Sato H, Kuzuya T, Takeda H, Hori M. Pravastatin restored the infarct size-limiting effect
of ischemic preconditioning blunted by hypercholesterolemia in the rabbit model of myocardial infarction. J Am Coll Cardiol 1999; 34: 2120–5. 35. Ferdinandy P, Szilvassy Z, Horvath LI, Csont T, Csonka C, Nagy E, Szentgyorgyi R, Nagy I, Koltai M, Dux L. Loss of pacing-induced preconditioning in rat hearts: role of nitric oxide and cholesterol-enriched diet. J Mol Cell Cardiol 1997; 29: 3321–33. 36. Fullmer TM, Pei S, Zhu Y, Sloan C, Manzanares R, Henrie B, Pires KM, Cox JE, Abel ED, Boudina S. Insulin suppresses ischemic preconditioningmediated cardioprotection through Akt-dependent mechanisms. J Mol Cell Cardiol 2013; 64: 20–9. 37. Yadav HN, Singh M, Sharma PL. Modulation of the cardioprotective effect of ischemic preconditioning in hyperlipidaemic rat heart. Eur J Pharmacol 2010; 643: 78–83.
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
13
Exercise training prevents the attenuation of anesthetic pre-conditioning-mediated cardioprotection in diet-induced obese rats L. Li1, F. Meng2, N. Li3, L. Zhang1, J. Wang4, H. Wang1, D. Li1, X. Zhang1, P. Dong1 and Y. Chen4 1
Department of Anesthesiology, Qilu Hospital, Shandong University, Jinan, Shandong, China Department of Anesthesiology, Jinan Maternity and Childcare Hospital, Jinan, Shandong, China 3 School of Public Health, Jining Medical University, Jinan, Shandong, China 4 Department of Emergency, Qilu Hospital, Shandong University, Jinan, Shandong, China 2
Correspondence Y. Chen, Department of Emergency, Qilu Hospital, Shandong University, Jinan City, Shandong Province, China E-mail: [email protected] Conflicts of interest The authors have no conflict of interest. Funding This study was supported by the National Natural Science Foundation of China (81170136, 81100147, 81300103, 81300219), Specialized Research Fund for the Doctoral Program of Higher Education (20130131110048), the National 973 Basic Research Program of China (2010CB732605), Taishan Scholar Program of Shandong Province, Grant from Department of Science and Technology of Shandong Province (2011GSF11806), Shandong Provincial Outstanding Medical Academic Professional Program, 1020 Program from the Health Department of Shandong Province. Submitted 21 August 2014; accepted 22 August 2014; submission 6 June 2014. Citation Li L, Meng F, Li N, Zhang L, Wang J, Wang H, Li D, Zhang X, Dong P, Chen Y. Exercise training prevents the attenuation of anesthetic pre-conditioning-mediated cardioprotection in diet-induced obese rats. Acta Anaesthesiologica Scandinavica 2014
Background: Obesity abolishes anesthetic pre-conditioninginduced cardioprotection due to impaired reactive oxygen species (ROS)-mediated adenosine monophosphate-activated protein kinase (AMPK) pathway, a consequence of increased basal myocardial oxidative stress. Exercise training has been shown to attenuate obesity-related oxidative stress. Objective: This study tests whether exercise training could normalize ROS-mediated AMPK pathway and prevent the attenuation of anesthetic pre-conditioning-induced cardioprotection in obesity. Methods: Male Sprague–Dawley rats were divided into lean rats fed with control diet and obese rats fed with high-fat diet. After 4 weeks of feeding, lean and obese rats were assigned to sedentary conditions or treadmill exercise for 8 weeks. Results: There was no difference in infarct size between lean sedentary and obese sedentary rats after 25 min of myocardial ischemia followed by 120 min reperfusion. In lean rats, sevoflurane equally reduced infarct size in lean sedentary and lean exercise-trained rats. Molecular studies revealed that AMPK activity, endothelial nitric oxide synthase, and superoxide production measured at the end of ischemia in lean rats were increased in response to sevoflurane. In obese rats, sevoflurane increased the above molecular parameters and reduced infarct size in obese exercise-trained rats but not in obese sedentary rats. Additional study showed that obese exercisetrained rats had decreased basal oxidative stress than obese sedentary rats. Conclusion: The results indicate that exercise training can prevent the attenuation of anesthetic cardioprotection in obesity. Preventing the attenuation of this strategy may be associated with reduced basal oxidative stress and normalized ROS-mediated AMPK pathway, but the causal relationship remains to be determined.
doi: 10.1111/aas.12414
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
bs_bs_banner
1
L. LI ET AL.
Obesity, which is associated with metabolic syndrome, has become an epidemic problem worldwide. It is estimated that more than two thirds of Americans are either overweight or obese. Obesity is often associated with enhanced morbidity and mortality, which is largely due to cardiovascular diseases.1–3 Patients with obesity and metabolic syndrome have a threefold increased risk of coronary heart diseases.4,5 Following an acute myocardial infarction, obese patients experience worse clinical outcomes,6 increased heart failure,7 increased myocardial infarct size, more in-hospital complications,8 and worse myocardial reperfusion following primary percutaneous coronary intervention.9 Thus, using any effective cardioprotective strategy to reduce the consequences of coronary artery disease is important in this population. Volatile anesthetics are reported to produce pharmacological pre-conditioning and reduce ischemia-reperfusion (I/R) injury in the heart in a variety of experimental animal models as well as in humans.10–13 Volatile anesthetics especially in the perioperative period provide a major advantage compared with ischemic pre-conditioning. In normal animals, activation of AMP-activated protein kinase (AMPK), an important sensor and regulator of cellular energy status, is reported to be involved in anesthetic-induced cardioprotective signaling; and this activation requires production of reactive oxygen species (ROS).10,14 However, a recent study demonstrated that anesthetic pre-conditioning against myocardial I/R injury is abolished in diet-induced obesity, and the failure to pre-condition the obese myocardium is probably due to increased basal myocardial oxidative stress, which impairs ROSmediated AMPK activation during anesthetic preconditioning.10 Thus, modulation of obesityrelated oxidative stress may be useful in improving this protective mechanism in the obese heart. Exercise is an important lifestyle modification to prevent cardiovascular diseases. Moderate exercise training has been shown to induce weight loss, improve plasma biomarkers of oxidative status,15 and decrease oxidative stress in the heart and kidney in obese animals and humans.16,17 Exercise training has also been shown to reduce age-related myocardial oxidative stress18 and restore ischemic-pre-conditioning in
the aging heart.19 However, effects of exercise training on anesthetic pre-conditioning in obese heart are unclear. Hence, the aim of the present study was to examine whether exercise training would normalize ROS-mediated AMPK signaling pathway by reducing basal myocardial oxidative stress and prevent the attenuation of anesthetic preconditioning-induced cardioprotection in obesity. For this purpose, the high-fat diet-induced obese rat model was used as a representation of human obesity syndrome.20 Methods Animals and exercise training All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Shandong University (Permit Number: 20120046). The research protocols and procedures were approved by IACUC of Shandong University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. We were planning a study of subjects evenly divided into eight groups with different treatments as described below. Sample size was determined by a power analysis based on the preliminary data. If the effect size was 0.715, five subjects in each group would be needed to be able to reject the null hypothesis that the population means of the eight groups were equal with probability (power) 0.8. The Type I error probability associated with this test of this null hypothesis was 0.05. One hundred thirty-two male Sprague– Dawley rats weighing 82.7 ± 20.7 g (Beijing Laboratory Animal Research Center, Beijing, China) were housed in individual cages in a climatecontrolled environment (22.8 ± 2.0 °C, 45–50% humidity). Following 1 week of acclimation, animals were randomly divided into lean control rats that were fed with a control diet containing 10% of kcal from fat (designated as low fat) and obese rats that were fed with a high-fat diet containing 45% of kcal from fat (Research Diets, New Brunswick, NJ,USA) for 12 consecutive weeks. After 4 weeks of control diet or high-fat diet feeding, lean and obese rats were assigned to sedentary conditions or regular treadmill exercise, Acta Anaesthesiologica Scandinavica (2014)
2
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
respectively. The exercise protocol was adapted from a previously published procedure.16 Briefly, rats were exercised at 0° incline and a speed of 10 m/min for 1 h daily (5-min break every 15 min), 5 days/week. Body weights were measured weekly until the end of the study. After 8 weeks of exercise, the rats were rested for 48 h before any further procedures were performed to eliminate interference from acute effects of exercise. Surgical preparation Rats were anaesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg) and were maintained by sodium pentobarbital (15– 25 mg/kg/min) infusion throughout the experiment. Right femoral vein and artery were cannulated for fluid and drug administration or for arterial blood pressure (BP) measurement. After tracheal intubation, rats were ventilated using a rodent ventilator (Shanghai Alcott Biotech Co., Shanghai, China) with 33% oxygen.
The heart was exposed via a left thoracotomy at the left fifth intercostal space and was suspended in a pericardial cradle. A 6–0 silk suture was placed around the proximal left descending coronary artery in the area immediately below the left atrial appendage. The ends of the suture were threaded through a propylene tube to form a snare. Coronary artery occlusion was produced by clamping the snare onto the epicardial surface of the heart and was confirmed by the appearance of epicardial cyanosis. Reperfusion was achieved by loosening the snare and was verified by observing an epicardial hyperemic response. Hemodynamic data were continuously recorded on a polygraph with Medlab-U/4C501H (Nanjing Mei Yi Technology, Nanjing, China). Experimental protocol I The experimental design for protocol I is illustrated in Fig. 1A. After 12 weeks of control diet or high-fat diet feeding and 8 weeks of exercise training, sedentary rats and exercise-trained rats
Fig. 1. Schematic illustration of the experimental protocol I (A) and II (B). SED, sedentary rats; EXE, exercise-trained rats; SEV or S, sevoflurane pre-conditioning. Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
3
L. LI ET AL.
were randomly divided into eight groups (n = 11–13 for each group) for myocardial I/R as follows: (1) L-SED, lean sedentary rats subjected to I/R alone; (2) L-SED + SEV, lean sedentary rats received sevoflurane pre-conditioning before I/R; (3) L-EXE + SEV, lean exercise-trained rats received sevoflurane pre-conditioning before I/R; (4) L-EXE, lean exercise-trained rats subjected to I/R alone; (5) O-SED, obese sedentary rats subjected to I/R alone; (6) O-SED + SEV, obese sedentary rats received sevoflurane pre-conditioning before I/R; (7) O-EXE + SEV, obese exercisetrained rats received sevoflurane pre-conditioning before I/R; and (8) O-EXE, obese exercise-trained rats subjected to I/R alone. The sevoflurane preconditioning and myocardial I/R were performed according to previous studies.10,21 Briefly, after a 30-min stabilization period, rats received 1 minimum alveolar concentration sevoflurane (in rats 2.4 vol%) for three 5-min periods, interspersed with two 5-min washout periods. Ten minutes min after pre-conditioning, rats were subjected to 25 min of regional myocardial ischemia followed by 120 min of reperfusion. At the end of the protocol, the hearts were removed for assessments of area at risk and infarct size. To examine the effects of exercise training on sevoflurane pre-conditioning-induced AMPK activation, nitric oxide (NO) level and superoxide production, some rats from each group (n = 5–6 for each group) were sacrificed at the end of ischemia, and myocardial samples were collected from ischemic left ventricular areas. Part of the fresh heart tissue was then used for measurement of myocardial superoxide production immediately, and the remainder was frozen in liquid nitrogen and stored at −80 °C for Western blot analysis and myocardial NO measurement. We selected this time point to collect tissues based on a previous study14 showing that the sevofluraneinduced AMPK activation occurs during ischemia but not before ischemia. Experimental protocol II To investigate the effects of exercise training on metabolism and basal myocardial oxidative stress, additional sedentary and exercise-trained rats (n = 8 for each group) from lean or obese group without pre-conditioning and I/R were used. The experimental design for protocol II is illustrated
in Fig. 1B. Briefly, after 12 weeks of control diet or high-fat diet feeding and 8 weeks of exercise training, rats were fasted for 12 h prior to blood collection. After blood collection, the animals were sacrificed and hearts were quickly removed. Plasma was separated from whole blood and stored at −80 °C for biochemical assay. Part of the fresh heart tissue was used for measurement of basal myocardial superoxide production immediately, and the remainder was frozen in liquid nitrogen and stored at −80 °C for assessment of basal myocardial oxidative stress by Western blot. Assessment of myocardial infarction At the end of the reperfusion period, the coronary artery was re-occluded, and 0.1% methylene blue was injected intravenously to label the nonischemic, blue-stained, normal area, thereby delineating the risk zone as a non-stained area. The rat was then sacrificed and the heart was removed, weighed, and frozen in a cold chamber at −18 °C. The heart was cut into six crosssectional slices of 2-mm thickness. The slices were incubated at 37 °C for 20 min in 1% 2,3,5triphenyl tetrazolium chloride buffer followed by 10% formaldehyde for 10 min to increase the contrast between stained tissue as a deep red color and non-stained tissue. Slices were then photographed, and the percentages of the at-risk and infarcted areas were calculated using an image analysis program. Western blot analysis of myocardial AMPK and eNOS activity and oxidative stress Myocardial samples were homogenized in icecold cell lysis buffer containing a complete protease inhibitor to isolate total protein. Proteins were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes. Membranes were immunoblotted with antibodies antiphosphorated (Ph)-AMPK at Thr172, anti-total AMPK, anti-Ph-endothelial nitric oxide synthase (eNOS), anti-total eNOS (Cell Signalling), antisuperoxide dismutase (SOD), anti-catalase, antinuclear factor erythroid-derived 2-like 2 (Nrf2) and anti-β-actin (Santa Cruz Biotechnology). Reactive protein was detected by an enhanced chemiluminescence system and analyzed by imaging densitometry. Acta Anaesthesiologica Scandinavica (2014)
4
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
Biochemical assay Plasma glucose, total cholesterol, and triglyceride concentrations were measured with a Hitachi clinical analyzer. Plasma insulin and leptin levels were measured by commercially available rat ELISA kit (Invitrogen, Camarillo, CA). Measurement of myocardial levels of nitrite and nitrate (NOx) The levels of nitrite and nitrate (NOx) were measured in left ventricular tissues using a nitrate/ nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA).
Measurement of myocardial superoxide anion Twelve weeks after respective diet feeding, myocardial superoxide production at the end of ischemia in animals from protocol I and basal myocardial superoxide production in animals from protocol II were measured by a lucigeninenhanced chemiluminescence assay according to previously described method.22 Briefly, Fresh left ventricular tissue was homogenized in a 20-mM sodium phosphate buffer (pH 7.4) containing 0.01 mM EDTA. The homogenate was subjected to low-speed centrifugation to remove nuclei and unbroken cell debris. The pellet was discarded, and the supernatant was obtained immediately for oxygen measurement. After background chemiluminescence in buffer (2 ml) containing lucigenin (5 μM) was measured for 5 min, an aliquot of 100 μl of supernatant was added, and the chemiluminescence was measured for 30 min at
room temperature. O2- levels were expressed as relative light units per second after subtraction of background activity. Statistical analysis Data are expressed as mean ± SD. Differences were analyzed using two-way ANOVA followed by Newman–Keuls multiple-comparison posthoc test. P < 0.05 was considered statistically significant. Results Metabolic effects of exercise training Before exercise training (4 weeks after control diet or high-fat diet feeding), obese rats displayed high body weight compared with lean rats (180 ± 24 vs. 156 ± 16, P < 0.05; obese rats vs. lean rats). At this time point, body weight was similar between lean sedentary and lean exercise-trained rats and also between obese sedentary and obese exercise-trained rats. At the end of the study, obese sedentary rats were markedly heavier than lean sedentary rats (Table 1). In addition, plasma levels of insulin, leptin, and total cholesterol were significantly higher in obese sedentary rats than those in lean sedentary rats. Exercise training significantly reduced these parameters in obese exercise-trained rats but had no effects in lean exercise-trained rats. The levels of blood glucose and plasma triglycerides were similar among groups. Systemic hemodynamic changes There were no significant differences among the eight experimental groups for heart rate (HR) and
Table 1 Effects of exercise training on metabolic parameters.
Body weight (g) Blood glucose (mg/dl) Plasma insulin (ng/ml) Plasma leptin (ng/ml) Plasma triglycerides (mg/dl) Plasma cholesterol (mg/dl)
L-SED
L-EXE
O-SED
O-EXE
342 ± 21 92 ± 21 3.56 ± 1.73 4.19 ± 2.21 115 ± 61 92 ± 41
340 ± 27 90 ± 16 3.52 ± 2.04 4.08 ± 1.75 116 ± 50 90 ± 36
458 ± 33 103 ± 27 8.34 ± 3.03* 7.72 ± 2.72* 124 ± 55 178 ± 66*
407 ± 25*† 101 ± 22 4.22 ± 2.35† 5.59 ± 1.73*† 113 ± 47 135 ± 53*†
*P < 0.05 vs. L-SED; †P < 0.05, O-EXE vs. O-SED. Values are mean ± SD; n = 8 for each group. Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test.
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
5
L. LI ET AL.
Table 2 Hemodynamic parameters in lean (L) and obese (O) rats.
HR (beats/min) L-SED L-SED + SEV L-EXE + SEV L-EXE O-SED O-SED + SEV O-EXE + SEV O-EXE Mean BP (mmHg) L-SED L-SED + SEV L-EXE + SEV L-EXE O-SED O-SED + SEV O-EXE + SEV O-EXE
Baseline
Pre-ischemia
Ischemia 25 min
Reperfusion 120 min
336 ± 26 343 ± 36 340 ± 39 337 ± 44 339 ± 33 338 ± 29 340 ± 31 341 ± 39
335 ± 29 340 ± 39 341 ± 36 337 ± 37 340 ± 39 336 ± 32 339 ± 33 342 ± 25
339 ± 41 342 ± 38 343 ± 39 339 ± 34 343 ± 29 339 ± 41 342 ± 28 345 ± 39
305 ± 23* 310 ± 28* 309 ± 22* 306 ± 30* 307 ± 26* 305 ± 23* 311 ± 25* 308 ± 17*
116 ± 16 111 ± 15 109 ± 31 113 ± 26 110 ± 18 108 ± 31 110 ± 25 109 ± 25
115 ± 23 106 ± 23 103 ± 22 110 ± 31 109 ± 17 109 ± 25 112 ± 21 109 ± 19
82 ± 18* 85 ± 10* 80 ± 17* 83 ± 12* 79 ± 19* 82 ± 13* 86 ± 13* 81 ± 17*
79 ± 14* 80 ± 12* 77 ± 20* 81 ± 20* 78 ± 9* 79 ± 10* 80 ± 10* 75 ± 15*
Baseline: stabilization period; Pre-ischemia: 5 min before ischemia. Values are mean ± SD; n = 6–7 for each group. Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. baseline; HR, heart rate; mean BP, mean blood pressure.
mean BP in the baseline (stabilization period) or in the pre-ischemic period. I/R produced decrease in mean BP in each experimental group. At the end of reperfusion, HR was also significantly decreased in each experimental group as compared with the baseline. These reductions were probably due to surgical stimuli or cardiac dysfunction following acute myocardial infarction. However, there were no significant differences in these parameters across groups. Hemodynamic parameters are shown in Table 2. Effect of sevoflurane pre-conditioning and exercise training on infarct size As shown in Fig. 2, there was no difference in infarct size between lean sedentary and obese sedentary rats following I/R. Sevoflurane preconditioning significantly reduced infarct size in lean sedentary rats, but it failed to protect obese sedentary rats against myocardial infarction. After 8 weeks of exercise training, sevoflurane preconditioning did not further decrease infarct size in lean exercise-trained rats; however, this strategy significantly reduced infarct size in obese exercisetrained rats to the same extent as that observed in lean rats. Exercise training alone did not reduce
infarct size in both lean and obese rats. In addition, the ratio of area at risk following I/R was similar among all groups. Effect of sevoflurane pre-conditioning and exercise training on AMPK activation At the end of ischemia, AMPK activation (phosphorylation of AMPK/total AMPK) did not differ between lean sedentary and obese sedentary rats (Fig. 3). Sevoflurane pre-conditioning similarly increased AMPK activation in lean sedentary and lean exercise-trained rats, whereas it had no significant effect in obese sedentary rats. However, after exercise training, sevoflurane preconditioning significantly increased AMPK activation in obese exercise-trained rats compared with obese sedentary rats. Exercise training alone did not change AMPK activation in either lean rats or obese rats as compared to their sedentary rats. Effect of sevoflurane pre-conditioning and exercise training on endothelial nitric oxide synthase (eNOS) activation and myocardial NOx production Endothelial nitric oxide synthase activation (phosphorylation of eNOS/total eNOS) and NOx Acta Anaesthesiologica Scandinavica (2014)
6
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
Fig. 2. Infarct size (A) and area at risk (B) in lean and obese rats subjected to 25 min of ischemia followed by 120 min of reperfusion in the absence or presence of sevoflurane pre-conditioning. The infarct size was expressed as percent of the area at risk, the area at risk was expressed as percent of left ventricle. Values were presented as mean ± SD (n = 6–7 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. respective lean rats in the same experiment.
level were similar between two sedentary groups (Fig. 4). Sevoflurane pre-conditioning significantly augmented eNOS activation and NOx level in lean sedentary and lean exercise-trained rats but not in obese sedentary rats, whereas eNOS activation and NOx level were enhanced in response to sevoflurane pre-conditioning in obese exercisetrained rats. Neither eNOS activation nor NOx level was altered in lean exercise-trained rats or obese exercise-trained rats without sevoflurane pre-conditioning. Effect of sevoflurane pre-conditioning and exercise training on superoxide anion Myocardial superoxide production, measured at the end of ischemia, was significantly higher in obese sedentary rats than that in lean sedentary rats (Fig. 5). Sevoflurane pre-conditioning augmented superoxide production equally in lean
Fig. 3. Myocardial AMPK activation at the end of ischemia from each group. Representative Western blots are aligned with the matching grouped data. AMPK activation was expressed as the ratio of phosphorylated (Ph-) to total AMPK. Values are presented as means ± SD (n = 5–6 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. respective lean rats in the same experiment.
sedentary and lean exercise-trained rats, but it had no effect in obese sedentary rats. However, superoxide production was increased in obese exercisetrained rats in response to sevoflurane preconditioning. Exercise training alone did not alter superoxide production in lean rats, but it reduced that in obese rats as compared to their sedentary rats.
Effect of exercise training on basal oxidative stress After 12 weeks of control diet or high-fat diet feeding and 8 weeks of exercise training, the basal level of superoxide production in the heart was greater in obese sedentary rats than that in lean sedentary rats. Superoxide production was unchanged in lean exercise-trained rats, but it was reduced in obese exercise-trained rats (Fig. 6A). Western blot analysis revealed that protein levels of antioxidant enzyme SOD and antioxidant master regulator Nrf2 in the heart were lower in obese sedentary rats than those in lean sedentary rats (Fig. 6B–E). Exercise training reversed protein levels of SOD and Nrf2 in obese
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
7
L. LI ET AL.
Fig. 5. Myocardial superoxide production at the end of ischemia from each group. Values are presented as means ± SD (n = 5–6 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. Respective lean rats in the same experiment.
Fig. 4. Myocardial eNOS activation (A) and nitrite and nitrate (NOx) level (B) at the end of ischemia from each group. Representative Western blots are aligned with the matching grouped data. eNOS activation was expressed as the ratio of phosphorylated (Ph-) to total eNOS. Values are presented as means ± SD (n = 5–6 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. respective SED rats; †P < 0.05 vs. respective lean rats in the same experiment.
exercise-trained rats but did not alter those in lean exercise-trained rats. There was no difference in protein levels of antioxidant enzyme catalase across four groups. Discussion The novel finding of this study is that exercise training prevented the attenuation of anesthetic sevoflurane pre-conditioning-mediated cardioprotection in high-fat diet-induced obese rats. Preventing the attenuation of this protective strategy in obese rats might be related to improvement in basal oxidative stress, normalization of ROSmediated AMPK signaling pathway in response to sevoflurane pre-conditioning, and amelioration of metabolic parameters.
Obesity has become a considerable health problem worldwide over the past 20 years. The increased prevalence of obesity and its associated lifestyle diseases can mostly be attributed to the increased availability and consumption of energy-dense foods and physical inactivity.23,24 A relation has been established for obesity and risk factors for development of cardiovascular diseases in the Framingham Heart Study.25 Obesityrelated disorders are linked to the undesirable outcomes of ischemic heart diseases, although the mechanisms responsible are poorly defined. Therefore, new strategies for cardioprotection are required to improve the clinical outcomes in obese patients with ischemic heart diseases. Anesthetic pre-conditioning is a potential cardioprotective strategy that has been demonstrated to protect against I/R injury, improve functional recovery, decrease post-ischemic myocardial stunning, and attenuate myocardial apoptosis.26 Recent studies demonstrated that activation of AMPK mediates anesthetic pre-conditioninginduced cardioprotection.10,27 Activation of AMPK causes activation of eNOS and release of NOx, which is a crucial step in mediating cardioprotection by sevoflurane pre-conditioning.21 Augmented mitochondrial ROS level in response to pre-conditioning has been proposed as a trigger for activation of this signaling pathway to confer cardioprotection.28,29 However, it has been reported that sevoflurane pre-conditioningActa Anaesthesiologica Scandinavica (2014)
8
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
Fig. 6. Myocardial basal superoxide production (A), protein levels of antioxidant enzymes SOD (B), catalase (C), and antioxidant master regulator Nrf2 (D) in respective sedentary (SED) and exercise-trained (EXE) rats after 12 weeks of control diet (L) or high-fat diet feeding (O). Representative Western blots were shown in figure E. Values are corrected by β-actin and presented as means ± SD (n = 8 for each group). Statistics were performed using two-way ANOVA followed by Newman–Keuls post-hoc test. *P < 0.05 vs. L-SED rats; †P < 0.05, O-EXE vs. O-SED rats.
induced cardioprotective action was abolished in diet-induced obesity due to a diminished effect of sevoflurane pre-conditioning on activation of the ROS-mediated AMPK signaling pathway.10 In the present study, it was found that sevoflurane pre-conditioning significantly reduced infarct size in lean sedentary rats, but it failed to protect obese sedentary rats against myocardial infarction. Molecular studies revealed that sevoflurane pre-conditioning elevated AMPK activity at the end of ischemia in lean sedentary rats, which was accompanied by increase in eNOS activity and production of myocardial NOx. In addition, myocardial superoxide production after sevoflurane pre-conditioning was augmented. However, these responses were not observed in obese sedentary rats. These results confirm previous findings, indicating that impaired ROSmediated AMPK signaling pathway might lead to the inability to pre-condition the heart against ischemia-reperfusion injury in obesity. Previous studies have suggested that an augmented basal oxidative stress associated with metabolic syndrome is central to the impaired ROS generation
in response to pre-conditioning in obese hearts.10 Augmented oxidative stress at baseline may result in oxidative modification of key mitochondrial enzymes and/or ion channels, leading to failure of enhanced ROS generation in the mitochondria of obese hearts by pre-conditioning.10,29 Thus, it is necessary to examine whether reduction of basal oxidative stress would prevent the attenuation of anesthetic pre-conditioning in obesity. Regular physical exercise has long been recognized as an essential component in the managing obesity, owing to the beneficial effects on weight maintenance/reduction and lowering the risk of developing type 2 diabetes and cardiovascular diseases.30,31 Moderate exercise training can attenuate age or obesity-related myocardial oxidative stress and improve cardiovascular functions.17,18 Previous study has reported that exercise training restores ischemic preconditioning in the aging heart in rats.19 The current study showed that sevoflurane preconditioning significantly reduced infarct size in obese exercise-trained rats but not in obese seden-
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
9
L. LI ET AL.
tary rats. It was also found that sevoflurane preconditioning induced activation of AMPK in obese exercise-trained rats, which was associated with concomitant increases in activation of eNOS, myocardial NOx level, and superoxide production, similar to those observed in lean rats. These results indicate that exercise training may prevent the attenuation of anesthetic cardioprotection in obesity, and preventing the attenuation of this beneficial strategy in obesity might be associated with normalized AMPK–eNOS signaling pathway. Notably, exercise training alone did not reduce infarct size in either lean rats or obese rats. This observation is inconsistent with a recent study showing that regular treadmill exercise reduces myocardial infarct size in both wild-type and obese (ob/ob) mice without changes in metabolic parameters.32 The underlying reasons for this discrepancy remain unclear. Further research is needed to investigate whether this discrepancy is due to differences in animal species, exercise intensity, or changes of metabolic parameters. Since impaired ROS-mediated AMPK signaling pathway is suggested to be a consequence of increased basal oxidative stress associated with metabolic syndrome in obesity, the effects of exercise training on basal oxidative stress and metabolic parameters in additional lean and obese rats were investigated. Twelve weeks of high-fat diet feeding induced metabolic syndrome in obese sedentary rats, including visceral obesity, hyperinsulinemia, hyperleptinemia, and dyslipidemia. The characteristics and metabolic parameters are consistent with previous reports,2,10 indicating that this animal model represents the classical insulin resistance and leptin resistance features of human obesity. Of note, a significant increase in fasting plasma glucose was not observed in highfat diet-induced obese rats in this study, although this animal model has been considered as one of the suitable animal models for type 2 diabetes mellitus. These data are consistent with previous report,2 suggesting that obese rats fed a high-fat diet for 12 weeks might be in the early stages of type 2 diabetes mellitus. However, impaired glucose tolerance has been reported in this obese animal model after 12 weeks of high-fat diet feeding.2 It is possible that exercise did not alter fasting plasma glucose but improved impaired glucose tolerance, although we did not perform an oral glucose tolerance test. Molecular analyses
showed that obese sedentary rats had a higher basal level of superoxide production in the myocardium, accompanied by reduced antioxidant enzyme SOD and antioxidant master regulator Nrf2, suggesting that obesity-related myocardial oxidative stress is associated with diminished antioxidant capacity. More importantly, exercise training was found to ameliorate metabolic syndrome, reduce myocardial oxidative stress, and improve antioxidant systems. These results are supported by a recent study showing that exercise training reduces increased oxidative stress in aging heart by reversing impaired antioxidant systems.18 But the improvement in antioxidant systems may not be the sole reason for the reduction in basal myocardial oxidative stress in obese rats after exercise training. Emerging evidence suggests that obesity is associated with chronic low-grade inflammation in the heart, which has been shown to induce myocardial oxidative stress.33 We could not exclude the possibility that exercise attenuates myocardial oxidative stress by reducing inflammation in the heart of obese rats. Interestingly, in the present study, the basal level of myocardial superoxide production was higher in obese sedentary rats than that in lean sedentary rats, but the infarct size was similar between the two groups. In addition, exercise training alone reduced myocardial superoxide production, but it did not attenuate infarct size following myocardial infarction in obese exercise-trained rats, indicating that myocardial oxidative stress may not be directly related to infarct size in obesity. However, reduction in basal myocardial oxidative stress by exercise training might improve mitochondrial function to produce sufficient ROS in response to sevoflurane pre-conditioning, and normalize AMPK signaling pathway, contributing to preventing the attenuation of anesthetic cardioprotection in obesity. A number of studies have demonstrated that cardioprotective effects of ischemic preconditioning are attenuated in various pathological conditions, including hyperlipidemia34,35 and hyperinsulinemia.36 Hyperlipidemia not only renders the myocardium more susceptible to ischemia and reperfusion-induced injury, but also inhibits ischemic pre-conditioning-mediated cardioprotection by decreasing adenosine release34 or increasing glycogen synthase kinase-3β expression.37 Hyperinsulinemia can suppress Acta Anaesthesiologica Scandinavica (2014)
10
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
ischemic pre-conditioning-mediated cardioprotection by activation of phosphoinositide-3kinase-protein kinase B signaling pathway.36 Thus, we could not exclude the possibility that improved metabolic parameters by exercise prevent the attenuated cardioprotective effects of anesthetic pre-conditioning in obesity through the above mechanisms. Several limitations of the present study should be acknowledged. This study did not demonstrate a causal relationship because no inhibitor or stimulator studies were included in this analysis. In addition, our results suggest that preventing obesity-induced attenuation of anesthetic cardioprotection by exercise may be associated with reduced basal oxidative stress and normalized ROS-mediated AMPK signaling pathway in response to sevoflurane pre-conditioning, but other mechanisms, including the amelioration of metabolic parameters that has been mentioned above, may also directly or indirectly contribute to improved anesthetic cardioprotection. Further studies are required to examine whether an AMPK inhibitor or a ROS stimulator can completely abolish sevoflurane-induced cardioprotection in exercise-trained obese rats and whether an AMPK stimulator or a ROS scavenger can completely prevent the attenuation of anesthetic cardioprotection in obese-sedentary rats. In conclusion, offering anesthetic preconditioning as a cardioprotective technique in patients to attenuate ischemia-reperfusion injury is an important therapeutic target. However, this beneficial strategy is attenuated in obesity. The present study demonstrates that exercise training prevents the attenuation of anesthetic cardioprotection in obesity probably by reducing basal oxidative stress and normalizing AMPK signaling pathway. The current study also indicates that performing regular exercise is an important lifestyle modification to ameliorate metabolic syndrome and in turn reverse impaired signaling pathways that mediate cardioprotective actions in obesity.
References 1. Heusch G. Obesity – a risk factor or a risk factor for myocardial infarction? Br J Pharmacol 2006; 149: 1–3.
2. Relling DP, Esberg LB, Fang CX, Johnson WT, Murphy EJ, Carlson EC, Saari JT, Ren J. High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of foxo3a transcription factor independent of lipotoxicity and apoptosis. J Hypertens 2006; 24: 549–61. 3. Simpson CR, Buckley BS, McLernon DJ, Sheikh A, Murphy A, Hannaford PC. Five-year prognosis in an incident cohort of people presenting with acute myocardial infarction. PLoS ONE 2011; 6: e26573. 4. Isomaa B, Almgren P, Tuomi T, Forsen B, Lahti K, Nissen M, Taskinen MR, Groop L. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 2001; 24: 683–9. 5. Tabassum F, Batty GD. Are current UK national institute for health and clinical excellence (nice) obesity risk guidelines useful? Cross-sectional associations with cardiovascular disease risk factors in a large, representative English population. PLoS ONE 2013; 8: e67764. 6. Schwartz GG, Olsson AG, Szarek M, Sasiela WJ. Relation of characteristics of metabolic syndrome to short-term prognosis and effects of intensive statin therapy after acute coronary syndrome: an analysis of the myocardial ischemia reduction with aggressive cholesterol lowering (miracle) trial. Diabetes Care 2005; 28: 2508–13. 7. Zeller M, Steg PG, Ravisy J, Laurent Y, Janin-Manificat L, L’Huillier I, Beer JC, Oudot A, Rioufol G, Makki H, Farnier M, Rochette L, Verges B, Cottin Y, Observatoire des Infarctus de Cote-d’Or Survey Working G. Prevalence and impact of metabolic syndrome on hospital outcomes in acute myocardial infarction. Arch Intern Med 2005; 165: 1192–8. 8. Clavijo LC, Pinto TL, Kuchulakanti PK, Torguson R, Chu WW, Satler LF, Kent KM, Suddath WO, Pichard AD, Waksman R. Metabolic syndrome in patients with acute myocardial infarction is associated with increased infarct size and in-hospital complications. Cardiovasc Revasc Med 2006; 7: 7–11. 9. Celik T, Turhan H, Kursaklioglu H, Iyisoy A, Yuksel UC, Ozmen N, Isik E. Impact of metabolic syndrome on myocardial perfusion grade after primary percutaneous coronary intervention in patients with acute st elevation myocardial infarction. Coron Artery Dis 2006; 17: 339–43. 10. Song T, Lv LY, Xu J, Tian ZY, Cui WY, Wang QS, Qu G, Shi XM. Diet-induced obesity suppresses sevoflurane preconditioning against myocardial
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
11
L. LI ET AL.
11.
12.
13.
14.
15.
16.
17.
18.
19.
ischemia-reperfusion injury: role of amp-activated protein kinase pathway. Exp Biol Med (Maywood) 2011; 236: 1427–36. Granfeldt A, Lefer DJ, Vinten-Johansen J. Protective ischaemia in patients: preconditioning and postconditioning. Cardiovasc Res 2009; 83: 234–46. Raphael J, Rivo J, Gozal Y. Isoflurane-induced myocardial preconditioning is dependent on phosphatidylinositol-3-kinase/akt signalling. Br J Anaesth 2005; 95: 756–63. Redel A, Stumpner J, Tischer-Zeitz T, Lange M, Smul TM, Lotz C, Roewer N, Kehl F. Comparison of isoflurane-, sevoflurane-, and desflurane-induced pre- and postconditioning against myocardial infarction in mice in vivo. Exp Biol Med (Maywood) 2009; 234: 1186–91. Lamberts RR, Onderwater G, Hamdani N, Vreden MJ, Steenhuisen J, Eringa EC, Loer SA, Stienen GJ, Bouwman RA. Reactive oxygen species-induced stimulation of 5’amp-activated protein kinase mediates sevoflurane-induced cardioprotection. Circulation 2009; 120: S10–5. Rector RS, Warner SO, Liu Y, Hinton PS, Sun GY, Cox RH, Stump CS, Laughlin MH, Dellsperger KC, Thomas TR. Exercise and diet induced weight loss improves measures of oxidative stress and insulin sensitivity in adults with characteristics of the metabolic syndrome. Am J Physiol Endocrinol Metab 2007; 293: E500–6. Muhammad AB, Lokhandwala MF, Banday AA. Exercise reduces oxidative stress but does not alleviate hyperinsulinemia or renal dopamine d1 receptor dysfunction in obese rats. Am J Physiol Renal Physiol 2011; 300: F98–104. Gao Z, Novick M, Muller MD, Williams RJ, Spilk S, Leuenberger UA, Sinoway LI. Exercise and diet-induced weight loss attenuates oxidative stress related-coronary vasoconstriction in obese adolescents. Eur J Appl Physiol 2013; 113: 519–28. Gounder SS, Kannan S, Devadoss D, Miller CJ, Whitehead KS, Odelberg SJ, Firpo MA, Paine R 3rd, Hoidal JR, Abel ED, Rajasekaran NS. Impaired transcriptional activity of nrf2 in age-related myocardial oxidative stress is reversible by moderate exercise training. PLoS ONE 2012; 7: e45697. Abete P, Calabrese C, Ferrara N, Cioppa A, Pisanelli P, Cacciatore F, Longobardi G, Napoli C, Rengo F. Exercise training restores ischemic preconditioning in the aging heart. J Am Coll Cardiol 2000; 36: 643–50.
20. Madsen AN, Hansen G, Paulsen SJ, Lykkegaard K, Tang-Christensen M, Hansen HS, Levin BE, Larsen PJ, Knudsen LB, Fosgerau K, Vrang N. Long-term characterization of the diet-induced obese and diet-resistant rat model: a polygenetic rat model mimicking the human obesity syndrome. J Endocrinol 2010; 206: 287–96. 21. Fradorf J, Huhn R, Weber NC, Ebel D, Wingert N, Preckel B, Toma O, Schlack W, Hollmann MW. Sevoflurane-induced preconditioning: impact of protocol and aprotinin administration on infarct size and endothelial nitric-oxide synthase phosphorylation in the rat heart in vivo. Anesthesiology 2010; 113: 1289–98. 22. Chen CH, Liu K, Chan JY. Anesthetic preconditioning confers acute cardioprotection via up-regulation of manganese superoxide dismutase and preservation of mitochondrial respiratory enzyme activity. Shock 2008; 29: 300–8. 23. Kahn BB, Flier JS. Obesity and insulin resistance. J Clin Invest 2000; 106: 473–81. 24. Haffner S, Taegtmeyer H. Epidemic obesity and the metabolic syndrome. Circulation 2003; 108: 1541–5. 25. Kenchaiah S, Evans JC, Levy D, Wilson PW, Benjamin EJ, Larson MG, Kannel WB, Vasan RS. Obesity and the risk of heart failure. N Engl J Med 2002; 347: 305–13. 26. Wang C, Xie H, Liu X, Qin Q, Wu X, Liu H, Liu C. Role of nuclear factor-kappab in volatile anaesthetic preconditioning with sevoflurane during myocardial ischaemia/reperfusion. Eur J Anaesthesiol 2010; 27: 747–56. 27. Huffmyer J, Raphael J. Physiology and pharmacology of myocardial preconditioning and postconditioning. Semin Cardiothorac Vasc Anesth 2009; 13: 5–18. 28. Tanaka K, Weihrauch D, Ludwig LM, Kersten JR, Pagel PS, Warltier DC. Mitochondrial adenosine triphosphate-regulated potassium channel opening acts as a trigger for isoflurane-induced preconditioning by generating reactive oxygen species. Anesthesiology 2003; 98: 935–43. 29. Katakam PV, Jordan JE, Snipes JA, Tulbert CD, Miller AW, Busija DW. Myocardial preconditioning against ischemia-reperfusion injury is abolished in Zucker obese rats with insulin resistance. Am J Physiol Regul Integr Comp Physiol 2007; 292: R920–6. 30. Lee-Young RS, Ayala JE, Fueger PT, Mayes WH, Kang L, Wasserman DH. Obesity impairs skeletal muscle AMPK signaling during exercise: role of ampkalpha2 in the regulation of exercise capacity in vivo. Int J Obes (Lond) 2011; 35: 982–9. Acta Anaesthesiologica Scandinavica (2014)
12
© 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
EXERCISE AND ANESTHETIC CARDIOPROTECTION
31. Sigal RJ, Kenny GP, Wasserman DH, Castaneda-Sceppa C, White RD. Physical activity/exercise and type 2 diabetes: a consensus statement from the American diabetes association. Diabetes Care 2006; 29: 1433–8. 32. Pons S, Martin V, Portal L, Zini R, Morin D, Berdeaux A. Ghaleh B. Regular treadmill exercise restores cardioprotective signaling pathways in obese mice independently from improvement in associated co-morbidities. J Mol Cell Cardiol 2013; 54: 82–9. 33. Palomer X, Salvado L, Barroso E, Vazquez-Carrera M. An overview of the crosstalk between inflammatory processes and metabolic dysregulation during diabetic cardiomyopathy. Int J Cardiol 2013; 168: 3160–72. 34. Ueda Y, Kitakaze M, Komamura K, Minamino T, Asanuma H, Sato H, Kuzuya T, Takeda H, Hori M. Pravastatin restored the infarct size-limiting effect
of ischemic preconditioning blunted by hypercholesterolemia in the rabbit model of myocardial infarction. J Am Coll Cardiol 1999; 34: 2120–5. 35. Ferdinandy P, Szilvassy Z, Horvath LI, Csont T, Csonka C, Nagy E, Szentgyorgyi R, Nagy I, Koltai M, Dux L. Loss of pacing-induced preconditioning in rat hearts: role of nitric oxide and cholesterol-enriched diet. J Mol Cell Cardiol 1997; 29: 3321–33. 36. Fullmer TM, Pei S, Zhu Y, Sloan C, Manzanares R, Henrie B, Pires KM, Cox JE, Abel ED, Boudina S. Insulin suppresses ischemic preconditioningmediated cardioprotection through Akt-dependent mechanisms. J Mol Cell Cardiol 2013; 64: 20–9. 37. Yadav HN, Singh M, Sharma PL. Modulation of the cardioprotective effect of ischemic preconditioning in hyperlipidaemic rat heart. Eur J Pharmacol 2010; 643: 78–83.
Acta Anaesthesiologica Scandinavica (2014) © 2014 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd
13
