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The Role of Oxidative Stress Response Revealed in Preconditioning Heat Stimulation in Skeletal Muscle of Rats
Journal of Surgical Research 176, 108–113 (2012) doi:10.1016/j.jss.2011.09.027
The Role of Oxidative Stress Response Revealed in Preconditioning Heat Stimulation in Skeletal Muscle of Rats Po Jung Pan, M.D., M.S.,*,† Cheng Fong Hsu, M.S.,‡ Jai Jen Tsai, M.D.,†,‡ and Jen Hwey Chiu, M.D., Ph.D.‡,§,1 *Department of Physical Medicine and Rehabilitation, National Yang-Ming University Hospital, I-Lan, Taiwan; †Department of Medicine, National Yang-Ming University, Taipei, Taiwan; ‡Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taiwan; and §Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taiwan Originally submitted June 30, 2011; accepted for publication September 14, 2011
Background. Our previous study showed that preconditioned local somatothermal stimulation (LSTS) protected subsequent ischemia-reperfusion injury of the skeletal muscle. The exact mechanisms of LSTS preconditioning remain unknown. The aim of this study was to test the hypothesis stating that heat stimulation induces free radical production, increases enzymatic scavenging activity, and subsequently enhances the expression of heat shock protein 70 (HSP-70) in skeletal muscles. Materials and Methods. After LSTS was applied onto the left quarter ventral abdomen muscle of male Sprague-Dawley rats, the underling muscles were collected at the intervals of baseline, 5-, 15-, 30-, and 60-min after LSTS. The time-dependent profiles of free radical production and enzymatic scavenging activity were measured. The influence of nitric oxide (NO) on HSP-70 expression was evaluated by pretreatment of an NO synthase inhibitor. Results. The concentrations of reactive oxygen species, NO metabolites, and malondialdehyde increased significantly 5 min after LSTS, whereas the scavenging activity reduced to the lowest level 5 min (dismutase) and 15 min (catalase and glutathione) after LSTS. Expression of HSP-70 was significantly lower in the LSTS with NO synthase inhibitor group than in the LSTS group. Conclusions. LSTS induces oxidative stress and the scavenging response in the underlying skeletal muscle, which might explain the possible mechanisms of LSTS preconditioning-induced muscle plasticity. Ó 2012 Elsevier Inc. All rights reserved.
1 To whom correspondence and reprint requests should be addressed at Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan, No. 155, Section 2, Li-Nong Street, Peitou, Taipei, 112, Taiwan. E-mail: [email protected].
0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.
Key Words: induced hyperthermia; preconditioning; oxidative stress; muscle plasticity; heat shock protein.
INTRODUCTION
Surgical procedures, including organ transplantation, free flap reconstruction, and orthopedic extremity operations, must overcome the ischemic-reperfusion obstacle. Prolonged ischemia and ensuing reperfusion cause a cellular energy crisis and damage skeletal muscles [1]. In 1986, the concept of ‘‘preconditioning’’ was first introduced by Murry et al. [2]. Preconditioning encompasses any sublethal stimulation-induced protection from subsequent critical injury. Previous studies have focused mainly on the ischemic preconditioning of the heart [3]. Magill et al. reported various other prior noxious stimulations including hypoxia, hypothermia, heat stress, and pharmacologic agents [3]. Additional studies have observed the induced-tolerance phenomenon in many other organs or tissues, such as the liver [4], kidney [5], lung, brain [6], and intestine [7]. This has prompted researchers to consider a common mechanism of preconditioning in different tissues, regardless of the stimulus [8]. The mechanisms of ischemic preconditioning in the heart involve an early protection phase, within 2 h of preconditioning, and a late protection phase (‘‘second window of protection’’), 24 to 72 h after preconditioning [3]. Nitric oxide (NO) and other reactive oxygen species (ROS) play vital roles in both phases [9], and de novo synthesis heat shock proteins (HSPs) are significantly involved in protection mechanisms in the late phase [8]. Oxidative stress induces free radicals, including ROS and reactive nitrogen species (RNS), which can damage
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cellular DNA, proteins, and lipids, and cause pathogenesis, especially at high levels [10]. Low to moderate free radical levels can regulate cell signaling for maintenance of redox homeostasis and facilitate cytoprotective HSP synthesis [11]. HSP chaperones are essential cellular machineries induced by heat stimulation or conditions of stress to prevent aggregation and to facilitate the refolding of misfolded proteins [12]. Accumulating evidence has also demonstrated that HSP can attenuate oxidative damage or enhance the repair process in injured muscle tissues [13]. Selsby and Dodd proposed that heat treatment increases heat shock protein 72 expression to protect muscle mass against oxidative stress during atrophy caused by disuse [14]. At a physiological level, Valko et al. also noted that exercise-induced oxidative stress facilitates normal force production in skeletal muscles [11]. This prompted our interest in skeletal muscle cellular response to heat stimulation, especially in preconditioning. This study researched the links among local somatothermal stimulation (LSTS), nitric oxide, and the expression of HSP-70 in skeletal muscles. In addition, this paper hypothesizes that LSTS induces free radical production and subsequent expression of HSP-70. Moreover, the concentration-time profile of cellular response to heat induced oxidative stress is demonstrated. METHODS Animals Male Sprague-Dawley rats weighing 250–300 g were obtained from the Animal Center of National Yang-Ming University (NYMU), Taiwan, Republic of China. They were acclimatized for 3 d before procedures with a standard diet and water, ad libitum, according to the ‘‘Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research’’ (National Academy Press, 2001). The rats were anesthetized with intraperitoneal ketamine (80 mg/kg) during all procedures. The study was approved by the Committee of Experimental Animals of NYMU.
Thermal Modality-LSTS Procedures After the animals were adequately anesthetized, LSTS was performed by applying an electric heating rod (120 W, 110 vV) onto and 0.5 cm above the hair-clipped skin surface of the left quarter area of the ventral abdomen, under which the left external oblique abdominis muscle could be seen (Fig. 1). A fluctuating skin temperature curve (between 37 and 44 C), to prevent adaptation, was achieved using an intermittent power supply with a duty cycle of 4 min on and 5 min off, for three courses during each LSTS dose [15]. Following LSTS applied onto the targeted skin area, the muscle tissues underlying LSTS treatment were time-dependently collected (at 0-, 5-, 15-, 30-, 60-min) to determine free radical production and enzymatic scavenging activity in the first part of this study. The muscle samples harvested at the baseline (time point 0) were used as the non-LSTS control group. The second part of the study analyzed the effects of NO on the production of Hsp70 in four groups. The first group was the normal control group, which did not receive any drugs or LSTS but was anesthetized in the same manner as the other three groups. Rats in the second group, the L-NAME (N-nitro-L-arginine methyl
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ester, a NO synthase inhibitor) control group, were not given LSTS but were treated twice with L-NAME (50 mg/kg, intramuscular injection), 10 min after administering anesthesia. Rats in the third group received two doses of LSTS 12 h apart. The fourth group received pretreated L-NAME and subsequent LSTS in two doses with a 12 h interval.
Measurement of ROS, NO Metabolites, Malondialdehyde (MDA), Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione (GSH) in Skeletal Muscle Tissue The muscles underlying the LSTS application site were removed, homogenized in 5–10 mL of cold buffer (20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose) per g of tissue, and were centrifuged at 1500 3 g for 5 min at 4 C. Dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescence probe for measuring intracellular oxidant production in muscle cells. DCFH-DA was hydrolyzed by esterases to dichlorofluorescein (DCFH), which was trapped within the cell. This non-fluorescent molecule was then oxidized to fluorescent dichlorofluorescein (DCF) by the action of cellular oxidants. DCFH-DA could not appreciably be oxidized to a fluorescent state without prior hydrolysis. Intracellular ROS production was measured using commercially available kits [16]. NO was rapidly converted to nitrite and nitrate (NO2– þ NO3–), which were assayed using a commercially available Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethylene-diamine dihydrochloride in 2.5 % H3PO4) (nitrate/nitrite Colorimetric Assay kit; Cayman Chemical Company, Ann Arbor, MI) [17]. In addition, MDA formation was determined using the thiobarbituric acid reactant substances (TBARS) method [18]. Furthermore, SOD, CAT, and GSH levels were determined using commercially available kits, as described previously [19]. The kits were purchased from the Cayman Chemical Company (Ann Arbor, MI). The SOD assay kit involved utilizing a tetrazolium salt to detect superoxide radicals generated by xanthine oxidase and hypoxanthine. Muscle tissue was homogenized in 5–10 mL of cold buffer (20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose) per g of tissue, and was centrifuged at 1500 3 g for 5 min at 4 C. The reactions were initiated by adding xanthine oxidase and were incubated for 20 min at room temperature. Absorbance at 450 nm was then noted. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% dismutation of the superoxide radical. The CAT assay kit involved utilizing the peroxidatic function of catalase to determine enzyme activity. Muscle tissue was homogenized in 5–10 mL of cold buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA) per g of tissue, and was centrifuged at 10,000 3 g for 15 min at 4 C. The sample was added with accompanying hydrogen peroxide, potassium hydroxide, purpald, and potassium periodate, and the optical density was noted at 540 nm. The activity of CAT was recorded in nmol/min/mL. The GSH assay kit involved utilizing a carefully optimized enzymatic recycling method using glutathione reductase to quantify GSH. Skeletal muscle tissue was homogenized in 5–10 mL of cold buffer (50 mM MES or phosphate, pH 6–7, containing 1 mM EDTA) per g of tissue, and was centrifuged at 10,000 3 g for 15 min at 4 C. This was followed by metaphosphoric acid deproteinization. After adding triethanolamine solution and an assay cocktail [a mixture of MES Buffer (11.25 mL)], reconstituted cofactor mixture (0.45 mL), reconstituted enzyme mixture (2.1 mL), water (2.3 mL), and reconstituted DTNB (0.45 mL), the total GSH in the deproteinated sample was measured at 405 or 414 nm in a spectrophotometer. The GSH concentrations of the samples were determined using the end point method and were expressed as mM.
Antibodies and Western Blot Analysis for HSP-70 Expression A monoclonal antibody against inducible HSP-70 was purchased commercially (H53220–050; Transduction Lab. Co., Lexington, KY). Tissue homogenates from rat skeletal muscles were obtained in the
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FIG. 1. Thermal modality-local somatothermal stimulation (LSTS) was placed 0.5 cm above the hair-clipped skin surface of the left quarter area of the ventral abdomen (right panel). A fluctuating skin temperature curve (between 37 and 44 C, n ¼ 3) was achieved with an intermittent power and a duty cycle of 4 min on and 5 min off, for three courses during each LSTS dose (left panel).
presence of protease inhibitors on ice, and the homogenate protein was assayed using Bradford’s method [20]. Fifty mg of homogenate was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with skimmed milk and incubated with 10,000-fold diluted MAbs against HSP-70. After washing and incubation with 5000-fold diluted biotin-conjugated secondary antibody (Anti-mouse IgG: HRPO– Horseradish peroxidase, ME5345; Transduction Lab., Lexington, KY), HSP-70 was detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech Inc., Piscataway, NJ) and analyzed using autoradiography. MAb against a-tubulin was used as an internal control. The optical density values were analyzed using image analysis software (Multi-Gauge vers, 2.02; Fuji Photo Film Photo Co., Ltd., Tokyo, Japan).
concentration (Fig. 3, n ¼ 4–5, P < 0.05). Both CAT and GSH consumed to the lowest levels 15 min after LSTS (Fig. 4, n ¼ 3–5 for CAT, n ¼ 5–8 for GSH, P < 0.05). The scavenging ability recovered within 30 min. The influence of NO on the expression of HSP-70 after thermal stimulation is displayed in Fig. 5. The HSP-70/b-actin optical density ratio was significantly lower in the LSTSþL-NAME group than in the LSTS group (n ¼ 3, P < 0.01). DISCUSSION
Statistics The data in each experimental group were analyzed using statistical and graphics software GraphPad Prism 4 and expressed as mean 6 SEM of triplicates. Repeat measures using a one-way ANOVA with Dunnet’s post hoc test for multiple groups and an unpaired t-test for two groups were used in this study. P < 0.05 was determined as statistically significant.
Clinically, various thermal modalities are used on patients suffering from muscular pain or a sequelae of muscle injury. The well-known therapeutic effects
RESULTS
The production and elimination of ROS and NO metabolites is illustrated in a concentration-time profile (Fig. 2). Compared witih the baseline concentrations, the concentrations of ROS and NO metabolites elevated significantly 5 min after thermal stress (n ¼ 4–5, P < 0.05). The concentration of MDA also increased significantly 5 min after thermal stress (Fig. 3, n ¼ 4–5, P < 0.05). As time progressed, the concentrations of ROS and NO metabolites and MDA decreased slowly. The enzymatic antioxidant scavenger system, including SOD, CAT, and GSH, was activated to modulate the oxidative stress. Within 5 min the activity of SOD decreased significantly compared with the baseline
FIG. 2. Concentration-time profiles of reactive oxygen species (ROS) and nitric oxide (NO) metabolites. The concentrations of ROS and NO metabolites elevated significantly 5 min after thermal stress and decreased slowly later. (n ¼ 4 w 5; *, One-way ANOVA test * versus 0 min P < 0.05).
PAN ET AL.: PRECONDITIONING HEAT INDUCED OXIDATIVE STRESS
FIG. 3. Concentration-time profiles of malondialdehyde (MDA) and superoxide dismutase (SOD). The concentrations of MDA elevated significantly 5 min after thermal stress and decreased slowly later. The concentration of SOD reduced to the lowest level 5 min after thermal stimulation and recovered within 30 min. (n ¼ 4 w 5; *, Oneway ANOVA test.* versus 0 min., P < 0.05).
of heat include improving perfusion, increasing flexibility, and decreasing pain. Our study attempted to link heat stimulation and muscle cellular protective mechanisms by activating the enzymatic scavenger system and conducting de novo HSP synthesis later. The findings indicate that heat stimulation can trigger a local muscle tissue reaction and facilitate the ability of muscle cells to protect against subsequent stress or injury. Preconditioning heat stimulation can, therefore, be considered as a type of stimulation that can augment the stress tolerance of muscles. It may have potential use in the maintenance of muscle health. Ischemic preconditioning is the most powerful form of preconditioning stress. However, there are practical and ethical considerations. Previous studies have
FIG. 4. Concentration-time profiles of catalase (CAT) and glutathione (GSH). The enzymatic antioxidant scavenger system, including CAT and GSH, consumed at the lowest level 15 min after local somatothermal stimulation and recovered within 30 min (n ¼ 3–5 for CAT, 5–8 for GSH; *, One-way ANOVA test.* versus 0 min P < 0.05).
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FIG. 5. The influence of nitric oxide on HSP-70 expression after two doses of thermal stimulation. The optical density ratio of heat shock protein 70 (HSP-70)/b-actin was significantly lower in the local somatothermal stimulation and N-nitro-L-arginine methyl ester (LSTSþL-NAME) group than in the LSTS group (n ¼ 3, *unpaired t-test, P < 0.01). PC ¼ positive control.
described pharmacologic preconditioning using opioid receptor agonists, NO-releasing agents, adenosine agonists, and K-ATP channel openers [21]; however, these require further development. Thermal preconditioning would provide a more convenient and acceptable method for clinical use, especially in muscles. The role of SOD in injury is to eliminate the superoxide anion (O2-). The O2- was converted to hydrogen peroxide (H2O2) via the action of SOD. In this study, the concentration of SOD decreased significantly 5 min after LSTS. The role of CAT and GSH is to eliminate H2O2, which is changed to hydrogen oxide (H2O). The concentrations of CAT and GSH decreased significantly 15 min after LSTS. Our observations are compatible with those related to the time courses of the expression of free radical scavengers in oxidative stress injury. This indicates that LSTS, in our study, may provoke a pro-oxidant effect with a mechanism distinct from the anti-oxidant effect [22]. The pro-oxidative effect stimulates the production of ROS, which can cause cell damage and protective effects, depending on its concentration. In this study, the concentrations of ROS, NO metabolites, and MDA decreased slowly within 1 h. ROS and NO could be eliminated by the action of SOD, CAT, and GSH. Simultaneously, MDA might be conducted by the blood flow. LSTS may, therefore, provide a safe stimulation method that does not cause permanent injury. The protective effect is, apparently, provoked by the preconditioning stimulation. But, what is the appropriate modality of heat stimulation? Previous investigations have identified characteristics of organ specificity and biphasic dose responses. For example,
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the same heating dosage can be appropriate for muscles but harmful to the brain. A small heat stress can produce a protective effect, but a large one can cause injury in the same tissue [23]. In this study, to mimic the clinical situation of local heat therapy and the convenient positioning of stimulation, the regional muscle of the abdominal wall was designed as the target muscle. Fluctuating heat stimulation, between 37 and 44 C, was applied to prevent adaptation. This was a more comfortable heat stimulation compared with constant heat treatment, such as heat shock, as has been used in previous studies [14, 24]. The temporal separation of heat stimulation can produce stress without accumulating energy and causing severe tissue damage. The duration of heat stimulation was 27 min, which is a value similar to 30 to 40 min, as has been used in other studies [14, 25]. The design of a precise heat stimulation protocol for clinical human use, including the temperature and duration of stimulation, requires further research. The expression of HSP-70 partially decreased when the NO synthase inhibitor was administered. This could be because NO is partially involved in the production of HSP-70. ROS can also provoke other pathways to up-regulate the expression of HSP [26]. Ischemic preconditioning induces NO production within 1 h and provides an early protective effect within 2 to 3 h. Late protection occurs after 1 d and lasts approximately 2 to 3 d because the delayed protective effect is related to the synthesis of HSP [27]. In this study, we made similar observations. The concentration of NO metabolites increased within minutes and de novo HSP-70 expression increased 16 h later. These findings are compatible with the concept of primary and secondary protective windows [3], and provide vital information for determining the appropriate time for clinical heat preconditioning. For surgical operations, such as flap transfers, heat preconditioning could be applied to the donor site 12 to 24 h before the procedure. Preconditioning heat stress could also be applicable for tourniquet-using extremity surgery such as total knee arthroplasty [28]. This study demonstrates the effects of thermal stimulation on cellular responses in muscle tissue, but does not functionally evaluate muscle strength, elasticity, or endurance. A future aim is to seek an appropriate thermal condition that can protect the muscle from injury in humans. Based on this evidence, preconditioning thermal stimulation, as a modality, may potentially be effective in orthopedic application clinically. LSTS can induce oxidative stress and subsequent protective response of the scavenger system in rat skeletal muscles. Synthesis of HSP-70 is influenced by NO. This paper provides evidence of cellular responses to thermal stimulation.
ACKNOWLEDGMENTS This study was supported by grants from National Yang-Ming University Hospital (RD2011–012).
REFERENCES 1. de Groot H, Rauen U. Ischemia-reperfusion injury: Processes in pathogenetic networks: A review. Transplant Proc 2007; 39:481. 2. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation 1986;74:1124. 3. Magill P, Murphy T, Bouchier-Hayes DJ, et al. Preconditioning and its clinical potential. Ir J Med Sci 2009;178:129. 4. Bahde R, Spiegel HU. Hepatic ischaemia-reperfusion injury from bench to bedside. Br J Surg 2010;97:1461. 5. Shihab FS. Preconditioning: From experimental findings to novel therapies in acute kidney injury. Minerva Urol Nefrol 2009;61:143. 6. Durukan A, Tatlisumak T. Preconditioning-induced ischemic tolerance: A window into endogenous gearing for cerebroprotection. Exp Transl Stroke Med 2010;21:2. 7. Wang S, Fan L, Gao K, et al. The protective effect of ischemic preconditioning associated with altered gene expression profiles in intestinal grafts after reperfusion. J Surg Res 2009;153:340. 8. Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: Underlying mechanisms and clinical application. Atherosclerosis 2009;204:334. 9. Miller MJ. Preconditioning for cardioprotection against ischemia reperfusion injury: The roles of nitric oxide, reactive oxygen species, heat shock proteins, reactive hyperemia and antioxidants—a mini review. Can J Cardiol 2001;17:1075. 10. Cataldi A. Cell responses to oxidative stressors. Curr Pharm Des 2010;16:1387. 11. Valko M, Leibfritz D, Moncol J, et al. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44. 12. Bukau B, Horwich AL. The Hsp70 and Hsp60 chaperone machines. Cell 1998;92:351. 13. Kojima A, Goto K, Morioka S, et al. Heat stress facilitates the regeneration of injured skeletal muscle in rats. J Orthop Sci 2007; 12:74. 14. Selsby JT, Dodd SL. Heat treatment reduces oxidative stress and protects muscle mass during immobilization. Am J Physiol Regul Integr Comp Physiol 2005;289:R134. 15. Chiu JH, Tsou MT, Tung HH, et al. Preconditioned somatothermal stimulation on median nerve territory increases myocardial heat shock protein 70 and protects rat hearts against ischemiareperfusion injury. J Thorac Cardiovasc Surg 2003;125:678. 16. Gomes A, Fernandes E, Lima JL. Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 2005;65:45. 17. Fox JB. The determination of nitrite: A critical review. Critical reviews in analytical chemistry. Boca Raton, FL: CRC Press, 1983:283–313. 18. Wasowicz W, Neve J, Peretz A. Optimized steps in fluorometric determination of thiobarbituric acid-reactive substances in serum: Importance of extraction pH and influence of sample preservation and storage. Clin Chem 1993;39:2522. 19. Yu SY, Chiu JH, Yang SD, et al. Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion injury in rats. J Surg Res 2005;128:28. 20. Bradford M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Chem 1976;72:248. 21. Raeburn CD, Cleveland JC Jr, Zimmerman MA, et al. Organ preconditioning. Arch Surg 2001;136:1263.
PAN ET AL.: PRECONDITIONING HEAT INDUCED OXIDATIVE STRESS 22. Woo AY, Cheng CH, Waye MM. Bacicalein protects rat cardiomyocytes from hypoxia/reoxygenation damage via a prooxidant mechanism. Cardiovasc Res 2005;65:244. 23. Huang YY, Chen AC, Carroll JD, et al. Biphasic dose response in low level light therapy. Dose Response 2009;7:358. 24. Lepore DA, Hurley JV, Stewart AG, et al. Prior heat stress improves survival of ischemic-reperfused skeletal muscle in vivo. Muscle Nerve 2000;23:1847. 25. Carvalho N, Puntel G, Correa P, et al. Protective effects of therapeutic cold and heat against the oxidative damage induced by a muscle strain injury in rats. J Sports Sci 2010;28:923.
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26. Banerjee Mustafi S, Chakraborty PK, Dey RS, Raha S. Heat stress up-regulates chaperone heat shock protein 70 and antioxidant manganese superoxide dismutase through reactive oxygen species (ROS), p38MAPK, and Akt. Cell Stress chaperones 2009; 14:579. 27. Das M, Das DK. Molecular mechanism of preconditioning. IUBMB Life 2008;60:199. 28. Jiang JK, Chiu JH, Lin JK. Local somatothermal stimulation inhibits motility of the internal anal sphincter through nitrergic neural release of nitric oxide. Dis Colon Rectum 2000; 43:381.
The Role of Oxidative Stress Response Revealed in Preconditioning Heat Stimulation in Skeletal Muscle of Rats Po Jung Pan, M.D., M.S.,*,† Cheng Fong Hsu, M.S.,‡ Jai Jen Tsai, M.D.,†,‡ and Jen Hwey Chiu, M.D., Ph.D.‡,§,1 *Department of Physical Medicine and Rehabilitation, National Yang-Ming University Hospital, I-Lan, Taiwan; †Department of Medicine, National Yang-Ming University, Taipei, Taiwan; ‡Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taiwan; and §Division of General Surgery, Department of Surgery, Taipei Veterans General Hospital, Taiwan Originally submitted June 30, 2011; accepted for publication September 14, 2011
Background. Our previous study showed that preconditioned local somatothermal stimulation (LSTS) protected subsequent ischemia-reperfusion injury of the skeletal muscle. The exact mechanisms of LSTS preconditioning remain unknown. The aim of this study was to test the hypothesis stating that heat stimulation induces free radical production, increases enzymatic scavenging activity, and subsequently enhances the expression of heat shock protein 70 (HSP-70) in skeletal muscles. Materials and Methods. After LSTS was applied onto the left quarter ventral abdomen muscle of male Sprague-Dawley rats, the underling muscles were collected at the intervals of baseline, 5-, 15-, 30-, and 60-min after LSTS. The time-dependent profiles of free radical production and enzymatic scavenging activity were measured. The influence of nitric oxide (NO) on HSP-70 expression was evaluated by pretreatment of an NO synthase inhibitor. Results. The concentrations of reactive oxygen species, NO metabolites, and malondialdehyde increased significantly 5 min after LSTS, whereas the scavenging activity reduced to the lowest level 5 min (dismutase) and 15 min (catalase and glutathione) after LSTS. Expression of HSP-70 was significantly lower in the LSTS with NO synthase inhibitor group than in the LSTS group. Conclusions. LSTS induces oxidative stress and the scavenging response in the underlying skeletal muscle, which might explain the possible mechanisms of LSTS preconditioning-induced muscle plasticity. Ó 2012 Elsevier Inc. All rights reserved.
1 To whom correspondence and reprint requests should be addressed at Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan, No. 155, Section 2, Li-Nong Street, Peitou, Taipei, 112, Taiwan. E-mail: [email protected].
0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.
Key Words: induced hyperthermia; preconditioning; oxidative stress; muscle plasticity; heat shock protein.
INTRODUCTION
Surgical procedures, including organ transplantation, free flap reconstruction, and orthopedic extremity operations, must overcome the ischemic-reperfusion obstacle. Prolonged ischemia and ensuing reperfusion cause a cellular energy crisis and damage skeletal muscles [1]. In 1986, the concept of ‘‘preconditioning’’ was first introduced by Murry et al. [2]. Preconditioning encompasses any sublethal stimulation-induced protection from subsequent critical injury. Previous studies have focused mainly on the ischemic preconditioning of the heart [3]. Magill et al. reported various other prior noxious stimulations including hypoxia, hypothermia, heat stress, and pharmacologic agents [3]. Additional studies have observed the induced-tolerance phenomenon in many other organs or tissues, such as the liver [4], kidney [5], lung, brain [6], and intestine [7]. This has prompted researchers to consider a common mechanism of preconditioning in different tissues, regardless of the stimulus [8]. The mechanisms of ischemic preconditioning in the heart involve an early protection phase, within 2 h of preconditioning, and a late protection phase (‘‘second window of protection’’), 24 to 72 h after preconditioning [3]. Nitric oxide (NO) and other reactive oxygen species (ROS) play vital roles in both phases [9], and de novo synthesis heat shock proteins (HSPs) are significantly involved in protection mechanisms in the late phase [8]. Oxidative stress induces free radicals, including ROS and reactive nitrogen species (RNS), which can damage
108
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cellular DNA, proteins, and lipids, and cause pathogenesis, especially at high levels [10]. Low to moderate free radical levels can regulate cell signaling for maintenance of redox homeostasis and facilitate cytoprotective HSP synthesis [11]. HSP chaperones are essential cellular machineries induced by heat stimulation or conditions of stress to prevent aggregation and to facilitate the refolding of misfolded proteins [12]. Accumulating evidence has also demonstrated that HSP can attenuate oxidative damage or enhance the repair process in injured muscle tissues [13]. Selsby and Dodd proposed that heat treatment increases heat shock protein 72 expression to protect muscle mass against oxidative stress during atrophy caused by disuse [14]. At a physiological level, Valko et al. also noted that exercise-induced oxidative stress facilitates normal force production in skeletal muscles [11]. This prompted our interest in skeletal muscle cellular response to heat stimulation, especially in preconditioning. This study researched the links among local somatothermal stimulation (LSTS), nitric oxide, and the expression of HSP-70 in skeletal muscles. In addition, this paper hypothesizes that LSTS induces free radical production and subsequent expression of HSP-70. Moreover, the concentration-time profile of cellular response to heat induced oxidative stress is demonstrated. METHODS Animals Male Sprague-Dawley rats weighing 250–300 g were obtained from the Animal Center of National Yang-Ming University (NYMU), Taiwan, Republic of China. They were acclimatized for 3 d before procedures with a standard diet and water, ad libitum, according to the ‘‘Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research’’ (National Academy Press, 2001). The rats were anesthetized with intraperitoneal ketamine (80 mg/kg) during all procedures. The study was approved by the Committee of Experimental Animals of NYMU.
Thermal Modality-LSTS Procedures After the animals were adequately anesthetized, LSTS was performed by applying an electric heating rod (120 W, 110 vV) onto and 0.5 cm above the hair-clipped skin surface of the left quarter area of the ventral abdomen, under which the left external oblique abdominis muscle could be seen (Fig. 1). A fluctuating skin temperature curve (between 37 and 44 C), to prevent adaptation, was achieved using an intermittent power supply with a duty cycle of 4 min on and 5 min off, for three courses during each LSTS dose [15]. Following LSTS applied onto the targeted skin area, the muscle tissues underlying LSTS treatment were time-dependently collected (at 0-, 5-, 15-, 30-, 60-min) to determine free radical production and enzymatic scavenging activity in the first part of this study. The muscle samples harvested at the baseline (time point 0) were used as the non-LSTS control group. The second part of the study analyzed the effects of NO on the production of Hsp70 in four groups. The first group was the normal control group, which did not receive any drugs or LSTS but was anesthetized in the same manner as the other three groups. Rats in the second group, the L-NAME (N-nitro-L-arginine methyl
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ester, a NO synthase inhibitor) control group, were not given LSTS but were treated twice with L-NAME (50 mg/kg, intramuscular injection), 10 min after administering anesthesia. Rats in the third group received two doses of LSTS 12 h apart. The fourth group received pretreated L-NAME and subsequent LSTS in two doses with a 12 h interval.
Measurement of ROS, NO Metabolites, Malondialdehyde (MDA), Superoxide Dismutase (SOD), Catalase (CAT), and Glutathione (GSH) in Skeletal Muscle Tissue The muscles underlying the LSTS application site were removed, homogenized in 5–10 mL of cold buffer (20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose) per g of tissue, and were centrifuged at 1500 3 g for 5 min at 4 C. Dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescence probe for measuring intracellular oxidant production in muscle cells. DCFH-DA was hydrolyzed by esterases to dichlorofluorescein (DCFH), which was trapped within the cell. This non-fluorescent molecule was then oxidized to fluorescent dichlorofluorescein (DCF) by the action of cellular oxidants. DCFH-DA could not appreciably be oxidized to a fluorescent state without prior hydrolysis. Intracellular ROS production was measured using commercially available kits [16]. NO was rapidly converted to nitrite and nitrate (NO2– þ NO3–), which were assayed using a commercially available Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl) ethylene-diamine dihydrochloride in 2.5 % H3PO4) (nitrate/nitrite Colorimetric Assay kit; Cayman Chemical Company, Ann Arbor, MI) [17]. In addition, MDA formation was determined using the thiobarbituric acid reactant substances (TBARS) method [18]. Furthermore, SOD, CAT, and GSH levels were determined using commercially available kits, as described previously [19]. The kits were purchased from the Cayman Chemical Company (Ann Arbor, MI). The SOD assay kit involved utilizing a tetrazolium salt to detect superoxide radicals generated by xanthine oxidase and hypoxanthine. Muscle tissue was homogenized in 5–10 mL of cold buffer (20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, and 70 mM sucrose) per g of tissue, and was centrifuged at 1500 3 g for 5 min at 4 C. The reactions were initiated by adding xanthine oxidase and were incubated for 20 min at room temperature. Absorbance at 450 nm was then noted. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% dismutation of the superoxide radical. The CAT assay kit involved utilizing the peroxidatic function of catalase to determine enzyme activity. Muscle tissue was homogenized in 5–10 mL of cold buffer (50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA) per g of tissue, and was centrifuged at 10,000 3 g for 15 min at 4 C. The sample was added with accompanying hydrogen peroxide, potassium hydroxide, purpald, and potassium periodate, and the optical density was noted at 540 nm. The activity of CAT was recorded in nmol/min/mL. The GSH assay kit involved utilizing a carefully optimized enzymatic recycling method using glutathione reductase to quantify GSH. Skeletal muscle tissue was homogenized in 5–10 mL of cold buffer (50 mM MES or phosphate, pH 6–7, containing 1 mM EDTA) per g of tissue, and was centrifuged at 10,000 3 g for 15 min at 4 C. This was followed by metaphosphoric acid deproteinization. After adding triethanolamine solution and an assay cocktail [a mixture of MES Buffer (11.25 mL)], reconstituted cofactor mixture (0.45 mL), reconstituted enzyme mixture (2.1 mL), water (2.3 mL), and reconstituted DTNB (0.45 mL), the total GSH in the deproteinated sample was measured at 405 or 414 nm in a spectrophotometer. The GSH concentrations of the samples were determined using the end point method and were expressed as mM.
Antibodies and Western Blot Analysis for HSP-70 Expression A monoclonal antibody against inducible HSP-70 was purchased commercially (H53220–050; Transduction Lab. Co., Lexington, KY). Tissue homogenates from rat skeletal muscles were obtained in the
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FIG. 1. Thermal modality-local somatothermal stimulation (LSTS) was placed 0.5 cm above the hair-clipped skin surface of the left quarter area of the ventral abdomen (right panel). A fluctuating skin temperature curve (between 37 and 44 C, n ¼ 3) was achieved with an intermittent power and a duty cycle of 4 min on and 5 min off, for three courses during each LSTS dose (left panel).
presence of protease inhibitors on ice, and the homogenate protein was assayed using Bradford’s method [20]. Fifty mg of homogenate was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with skimmed milk and incubated with 10,000-fold diluted MAbs against HSP-70. After washing and incubation with 5000-fold diluted biotin-conjugated secondary antibody (Anti-mouse IgG: HRPO– Horseradish peroxidase, ME5345; Transduction Lab., Lexington, KY), HSP-70 was detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech Inc., Piscataway, NJ) and analyzed using autoradiography. MAb against a-tubulin was used as an internal control. The optical density values were analyzed using image analysis software (Multi-Gauge vers, 2.02; Fuji Photo Film Photo Co., Ltd., Tokyo, Japan).
concentration (Fig. 3, n ¼ 4–5, P < 0.05). Both CAT and GSH consumed to the lowest levels 15 min after LSTS (Fig. 4, n ¼ 3–5 for CAT, n ¼ 5–8 for GSH, P < 0.05). The scavenging ability recovered within 30 min. The influence of NO on the expression of HSP-70 after thermal stimulation is displayed in Fig. 5. The HSP-70/b-actin optical density ratio was significantly lower in the LSTSþL-NAME group than in the LSTS group (n ¼ 3, P < 0.01). DISCUSSION
Statistics The data in each experimental group were analyzed using statistical and graphics software GraphPad Prism 4 and expressed as mean 6 SEM of triplicates. Repeat measures using a one-way ANOVA with Dunnet’s post hoc test for multiple groups and an unpaired t-test for two groups were used in this study. P < 0.05 was determined as statistically significant.
Clinically, various thermal modalities are used on patients suffering from muscular pain or a sequelae of muscle injury. The well-known therapeutic effects
RESULTS
The production and elimination of ROS and NO metabolites is illustrated in a concentration-time profile (Fig. 2). Compared witih the baseline concentrations, the concentrations of ROS and NO metabolites elevated significantly 5 min after thermal stress (n ¼ 4–5, P < 0.05). The concentration of MDA also increased significantly 5 min after thermal stress (Fig. 3, n ¼ 4–5, P < 0.05). As time progressed, the concentrations of ROS and NO metabolites and MDA decreased slowly. The enzymatic antioxidant scavenger system, including SOD, CAT, and GSH, was activated to modulate the oxidative stress. Within 5 min the activity of SOD decreased significantly compared with the baseline
FIG. 2. Concentration-time profiles of reactive oxygen species (ROS) and nitric oxide (NO) metabolites. The concentrations of ROS and NO metabolites elevated significantly 5 min after thermal stress and decreased slowly later. (n ¼ 4 w 5; *, One-way ANOVA test * versus 0 min P < 0.05).
PAN ET AL.: PRECONDITIONING HEAT INDUCED OXIDATIVE STRESS
FIG. 3. Concentration-time profiles of malondialdehyde (MDA) and superoxide dismutase (SOD). The concentrations of MDA elevated significantly 5 min after thermal stress and decreased slowly later. The concentration of SOD reduced to the lowest level 5 min after thermal stimulation and recovered within 30 min. (n ¼ 4 w 5; *, Oneway ANOVA test.* versus 0 min., P < 0.05).
of heat include improving perfusion, increasing flexibility, and decreasing pain. Our study attempted to link heat stimulation and muscle cellular protective mechanisms by activating the enzymatic scavenger system and conducting de novo HSP synthesis later. The findings indicate that heat stimulation can trigger a local muscle tissue reaction and facilitate the ability of muscle cells to protect against subsequent stress or injury. Preconditioning heat stimulation can, therefore, be considered as a type of stimulation that can augment the stress tolerance of muscles. It may have potential use in the maintenance of muscle health. Ischemic preconditioning is the most powerful form of preconditioning stress. However, there are practical and ethical considerations. Previous studies have
FIG. 4. Concentration-time profiles of catalase (CAT) and glutathione (GSH). The enzymatic antioxidant scavenger system, including CAT and GSH, consumed at the lowest level 15 min after local somatothermal stimulation and recovered within 30 min (n ¼ 3–5 for CAT, 5–8 for GSH; *, One-way ANOVA test.* versus 0 min P < 0.05).
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FIG. 5. The influence of nitric oxide on HSP-70 expression after two doses of thermal stimulation. The optical density ratio of heat shock protein 70 (HSP-70)/b-actin was significantly lower in the local somatothermal stimulation and N-nitro-L-arginine methyl ester (LSTSþL-NAME) group than in the LSTS group (n ¼ 3, *unpaired t-test, P < 0.01). PC ¼ positive control.
described pharmacologic preconditioning using opioid receptor agonists, NO-releasing agents, adenosine agonists, and K-ATP channel openers [21]; however, these require further development. Thermal preconditioning would provide a more convenient and acceptable method for clinical use, especially in muscles. The role of SOD in injury is to eliminate the superoxide anion (O2-). The O2- was converted to hydrogen peroxide (H2O2) via the action of SOD. In this study, the concentration of SOD decreased significantly 5 min after LSTS. The role of CAT and GSH is to eliminate H2O2, which is changed to hydrogen oxide (H2O). The concentrations of CAT and GSH decreased significantly 15 min after LSTS. Our observations are compatible with those related to the time courses of the expression of free radical scavengers in oxidative stress injury. This indicates that LSTS, in our study, may provoke a pro-oxidant effect with a mechanism distinct from the anti-oxidant effect [22]. The pro-oxidative effect stimulates the production of ROS, which can cause cell damage and protective effects, depending on its concentration. In this study, the concentrations of ROS, NO metabolites, and MDA decreased slowly within 1 h. ROS and NO could be eliminated by the action of SOD, CAT, and GSH. Simultaneously, MDA might be conducted by the blood flow. LSTS may, therefore, provide a safe stimulation method that does not cause permanent injury. The protective effect is, apparently, provoked by the preconditioning stimulation. But, what is the appropriate modality of heat stimulation? Previous investigations have identified characteristics of organ specificity and biphasic dose responses. For example,
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the same heating dosage can be appropriate for muscles but harmful to the brain. A small heat stress can produce a protective effect, but a large one can cause injury in the same tissue [23]. In this study, to mimic the clinical situation of local heat therapy and the convenient positioning of stimulation, the regional muscle of the abdominal wall was designed as the target muscle. Fluctuating heat stimulation, between 37 and 44 C, was applied to prevent adaptation. This was a more comfortable heat stimulation compared with constant heat treatment, such as heat shock, as has been used in previous studies [14, 24]. The temporal separation of heat stimulation can produce stress without accumulating energy and causing severe tissue damage. The duration of heat stimulation was 27 min, which is a value similar to 30 to 40 min, as has been used in other studies [14, 25]. The design of a precise heat stimulation protocol for clinical human use, including the temperature and duration of stimulation, requires further research. The expression of HSP-70 partially decreased when the NO synthase inhibitor was administered. This could be because NO is partially involved in the production of HSP-70. ROS can also provoke other pathways to up-regulate the expression of HSP [26]. Ischemic preconditioning induces NO production within 1 h and provides an early protective effect within 2 to 3 h. Late protection occurs after 1 d and lasts approximately 2 to 3 d because the delayed protective effect is related to the synthesis of HSP [27]. In this study, we made similar observations. The concentration of NO metabolites increased within minutes and de novo HSP-70 expression increased 16 h later. These findings are compatible with the concept of primary and secondary protective windows [3], and provide vital information for determining the appropriate time for clinical heat preconditioning. For surgical operations, such as flap transfers, heat preconditioning could be applied to the donor site 12 to 24 h before the procedure. Preconditioning heat stress could also be applicable for tourniquet-using extremity surgery such as total knee arthroplasty [28]. This study demonstrates the effects of thermal stimulation on cellular responses in muscle tissue, but does not functionally evaluate muscle strength, elasticity, or endurance. A future aim is to seek an appropriate thermal condition that can protect the muscle from injury in humans. Based on this evidence, preconditioning thermal stimulation, as a modality, may potentially be effective in orthopedic application clinically. LSTS can induce oxidative stress and subsequent protective response of the scavenger system in rat skeletal muscles. Synthesis of HSP-70 is influenced by NO. This paper provides evidence of cellular responses to thermal stimulation.
ACKNOWLEDGMENTS This study was supported by grants from National Yang-Ming University Hospital (RD2011–012).
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