Accepted Article
Received Date : 28-Aug-2014 Revised Date : 24-Oct-2014 Accepted Date : 30-Oct-2014 Article type : Original Manuscript
Cardioprotective effects of melatonin against myocardial injuries induced by chronic intermittent hypoxia in rats
Hang-Mee Yeung1, Ming-Wai Hung1, Chi-Fai Lau1, Man-Lung Fung1,2,*
Department of Physiology1, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
2
Research Centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of
Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
* Corresponding author: Department of Physiology, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong Email: [email protected] Tel: +852 3917 9234 Fax: +852 2855 9730
Running title: Melatonin ameliorates myocardial injury in sleep apnea
Keywords: calcium, intermittent hypoxia, ischemia reperfusion, melatonin, oxidative stress, sarcoplasmic reticulum, sleep apnea
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jpi.12190 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Obstructive sleep apnea (OSA) associated with chronic intermittent hypoxia (CIH) increases the morbidity and mortality of ischemic heart disease in patients. Yet, there is a paucity of preventive measures targeting the pathogenesis of CIH-induced myocardial injury. We examined the cardioprotective effect of melatonin against the inflammation, fibrosis and the deteriorated sarcoplasmic reticulum (SR) Ca2+ homeostasis and ischemia/reperfusion (I/R)induced injury exacerbated by CIH. Adult male Sprague-Dawley rats that had received a daily injection of melatonin (10mg/kg) or vehicle were exposed to CIH treatment mimicking a severe OSA condition for 4 weeks. Systolic pressure, heart weights and malondialdehyde were significantly increased in hypoxic rats but not in the melatonin-treated group, when compared with the normoxic control. Levels of the expression of inflammatory cytokines (TNFα, IL6, COX2) and fibrotic markers (PC1 and TGFβ) were significantly elevated in the hypoxic group but were normalized by melatonin. Additionally, infarct size of isolated hearts with regional I/R was substantial in the hypoxic group treated with vehicle but not in the melatonin-treated group. Moreover, melatonin treatment mitigated the SR-Ca2+ homeostasis in the cardiomyocyte during I/R with (i) Ca2+ overloading, (ii) decreased SR-Ca2+ content, (iii) lowered expression and activity of Ca2+-handling proteins (SERCA2a and NCX1), (iv) decreased expressions of CAMKII and phosphorylated-eNOSser1177. Furthermore, melatonin ameliorated the level of expression of antioxidant enzymes (CAT and MnSOD) and NADPH oxidase (p22 and NOX2). Results support a prophylactic usage of melatonin in OSA patients, which protects against CIH-induced myocardial inflammation and fibrosis with impaired SRCa2+ handling and exacerbated I/R injury.
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Accepted Article
Introduction Obstructive sleep apnea (OSA) is closely associated with cardiovascular diseases, which affects about 5% of the adult population, and increases risks for cardiovascular morbidity and mortality, including systemic hypertension, ischemic heart disease and congestive heart failure with myocardial hypertrophy [1, 2]. Chronic intermittent hypoxia (CIH) caused by repetitive episodes of severe hypoxia and reoxygenation in sleep apnea plays an important role in the pathophysiology of clinical manifestations in OSA patients. The deteriorated effect of CIH on cardiac functions is closely related to oxidative stress. It has been reported that CIH-induced ventricular dysfunction is associated with a decreased activity of myocardial superoxide dismutase of the antioxidant system [3, 4]. In addition, inflammation plays an important role in cardiac remodeling and hypertrophy, which could lead to heart failure. Levels of pro-inflammatory cytokines are significantly elevated in the circulation in OSA patients [5, 6]. It has also been shown that pro-inflammatory cytokines including TNF-alpha (TNFα) and interleukin-6 (IL6) are involved in the adverse myocardial remodeling [7-9]. Furthermore, inflammatory mediators including cycloxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) play pathogenic roles in ischemia/reperfusion (I/R)-induced injury of the heart [10, 11]. Also, transforming growth factor beta-1 (TGFβ1) is one of the cytokines involved in myocardial injury, and it plays a pathogenic role in cardiac fibrosis and increased stiffness of diastolic chamber [12]. Indeed, an increased expression of TGFβ1 is involved in the progression of ventricular hypertrophy and dysfunction [13, 14]. Moreover, reactive oxygen species (ROS) play a key role in impaired sarcoplasmic reticulum (SR)-Ca2+ homeostasis and I/R-induced myocardial injury, which subsequently triggers the inflammatory and fibrotic responses [3, 15]. Mechanistically, changes in the expression and activity of Ca2+-handling proteins lead to altered Ca2+ homeostasis and myocardial function under hypoxic conditions [16, 17]. Calcium-dependent calmodulin kinase II (CAMKII) plays
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a role in the modulation of activities of Ca2+ handling proteins [18, 19]. Also endothelial nitric oxide synthase (eNOS), a phosphorylated target of CAMKII, is involved in the modulation of myocardial contractile function and plays a cardioprotective role against I/R injury [20, 21]. Yet, there is limited information on the involvement of CAMKII and eNOS in the CIH-induced myocardial injury.
Melatonin is well known for its antioxidant capacity and anti-inflammatory effects against oxidative stress by scavenging free radicals and activating the antioxidant enzymes and by suppressing the expression of cytokines and inflammatory mediators [22-24]. The cardioprotective effect of melatonin against mitochrondrial oxidative damage and myocardial injuries has been explored in animal models of sepsis and I/R injury [25-28]. In addition, it has been demonstrated that administrations of antioxidant melatonin ameliorates I/R injury, which could lower the level of oxidative stress by scavenging the ROS and also by regulating the expression of antioxidant enzymes [22, 24, 28]. Although the acute effect of melatonin on cardioprotection against I/R injury has been shown [28, 29], the chronic effects of melatonin on the expression and activity of Ca2+ handling proteins and exacerbated I/R injury under CIH conditions are unclear. Also, the CAMKII-eNOS axis could be a potential target of the cardioprotective effect of melatonin. In this study, we aimed to examine the hypotheses that chronic administration of melatonin ameliorates the CIH-induced inflammation and fibrosis caused by oxidative stress, which leads to deteriorated Ca2+ homeostasis in rat ventricular myocytes with altered functions of SR-Ca2+ handling proteins, causing myocardial injury and increased susceptibility to I/R injury in the rat.
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Materials and methods Animals The experimental protocol for this study was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996). Male SpragueDawley rats weighing about 70 g at age 28 days were randomly divided into groups for normoxic control and CIH treatment. Rats were kept in an acrylic chamber for normobaric hypoxia and had free access to water and chow. The oxygen fraction inside the chamber was kept with inspired oxygen alternating from 21 to 5 + 0.5% oxygen per minute for 8 hr/day. The desired oxygen content was established by a mixture of room air and nitrogen that was regulated and monitored by an oxygen analyzer (Vacumetrics Inc., CA, USA). The inspired oxygen level fell to 4-5% (nadir arterial oxygen saturation ca. 70%) for about 15 sec per min, which mimicks the recurrent episodic hypoxemia in OSA patients. For normoxic controls, aged-matched animals were kept in room air in the same housing and maintenance matching the hypoxic groups. The rats were treated for 28 days and were immediately used in the experiments. The animal room was controlled at a constant temperature (22 + 2oC), humidity and light:dark cycle (lights on 07:00-19:00).
Drug preparation Melatonin (Sigma, St. Louis, MO) solution was prepared fresh before injection by dissolving the indoleamine in absolute ethanol and further dilution with normal saline; the final concentration of ethanol was 2%. Melatonin in 10mg/kg body weight [30, 31] or vehicle (2% ethanol in normal saline) was administered intraperitoneally to rats in the melatonin-treated (MIH) and vehicle-treated (VIH) groups respectively each day 30 min before the hypoxic
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treatment.
Blood pressure measurement Systolic pressure was measured by a tail-cuff method. Briefly, the conscious animal was held inside a restraining cage and its tail was placed through the cuff with a photoelectric sensor. Changes in pressure applied to the cuff and arterial pulsation were amplified (IITC, Woodland Hills, CA, USA) and recorded on polygraph tracings. At least four measurements were taken for each animal and the average was calculated as the systolic pressure.
Histological analysis of Sirius Red staining Freshly isolated perfused rat hearts from normoxic and hypoxic groups were fixed in neutral buffered formalin for at least 72 h. Heart tissues were then processed routinely for dehydration with 70-100% graded alcohol and embedded in blocks with paraffin wax. Serial sections of 5 μm thickness were cut and mounted on silanized slides (DAKO, Denmark). Sections from all rat groups were kept in the oven overnight at 56oC. Quantitative analysis of collagen accumulation in heart tissues from all rat groups was visualized by Sirius Red staining and morphometric analysis. Briefly, tissue sections were stained with 0.1% picroSirius Red (Polysciences Inc., Washington, DC, USA) in saturated aqueous picric acid for 1 h at room temperature followed by differentiation in 0.01% hydrochloric acid. The Sirius Redstained collagen was then quantified by using LEICA Qwin Image Analyzer (Leica Microsystems Ltd., Milton Keynes, UK). All sections from normoxic and hypoxic rat groups were examined by the same person.
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Isolated perfused heart preparation Rats from normoxic and hypoxic groups were decapitated and the hearts were quickly removed and placed in ice-cold Krebs-Henseleit (K-H) perfusion buffer before being mounted on the Langendorff apparatus for perfusion at 37 ºC with K-H buffer at constant pressure (100 cm of H2O) and equilibrated with 95% of O2/5% CO2. The buffer contained (in mM) 118.0 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3 and 11.0 glucose. All the hearts were subjected to regional ischemia described previously [16]. Briefly, a snare was formed by placing a silk suture around the left coronary artery of rat heart. The snare was pulled in order to occlude the coronary artery to produce ischemia. Reperfusion was done by releasing the snare. The isolated hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion which induced myocardial injury.
Measurement of the area of risk To determine the infarct size, the coronary artery was re-occluded at the end of reperfusion and the heart was perfused with 2.5% Evans blue to delineate the area of risk. The hearts were frozen at -70℃, cut into thin slices, which were perpendicular to the septum, from the apex to the base. Then incubated in sodium phosphate buffer containing 1% (w/v) 2,3,5triphenyl-tetrazolium chloride for 10 min in order to visualize the unstained infarct region. The infarct and the risk zone areas were determined by planimetry with software Image/J from the National Institutes of Health. (Bethesda, MD). The infarct size measured was expressed as a percentage of the risk zone.
Determination of myocardial injury by lactate dehydrogenase (LDH) efflux The effluent from each isolated perfused rat heart was collected at 10 min before regional ischemia, 5th min of reperfusion and the LDH was assayed spectrophotometrically by using a
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kit purchased from Sigma-Aldrich (St. Louis, MO), an intra-assay coefficient of variation of less than 10% and an inter-assay coefficient of variation of less than 12%. The LDH activity measured was expressed as units per liter. The limit of detection is about 1 ng/mL.
Measurement of malondialdehyde (MDA) The total lipid peroxides were measured as the amount of MDA using a BIOXYTECH® LPO-586™ kit (OxisResearch, Portland, OR), an intra-assay coefficient of variation of 5.8% and an inter-assay coefficient of variation of 7.2%. The reaction product was measured spectrophotometrically at 586nm. Standard curves were constructed with 1,1,3,3tetraethoxypropane as a standard. The limit of detection is about 1nM. The MDA level of in all rat hearts is expressed as μM, while the MDA concentration (μM) in heart was normalized to wet tissue weight (mg) and expressed as μmol/mg.
Preparation of isolated ventricular myocytes Single ventricular myocytes were isolated from the normoxic and hypoxic rats by using a collagenase method described previously [16]. After isolation, myocytes were allowed to stabilize for at least 30 min before any experiment.
Measurement of [Ca2+]i A spectrofluorometric method with Fura-2/AM as a Ca2+ indicator was used during the measurement of [Ca2+]i. Ventricular myocytes from either normoxic or hypoxic rats were incubated with 5 μM Fura-2/AM for 35 min. Fluorescent signals obtained at 340 nm (F340) and 380 nm (F380) excitation wave-lengths were recorded and stored in computer for data processing and analysis. The F340/F380 ratio was used to indicate cytosolic [Ca2+]i in the ventricular myocytes. During the measurement of electrically induced [Ca2+]i transients
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(E[Ca2+]i), myocytes were electrically stimulated at 0.2 Hz, whereas measurement of caffeine-induced [Ca2+]i transients (C[Ca2+]i) were done by applying drops of 10 mM caffeine directly to the ventricular myocytes. The amplitude of E[Ca2+]i and C[Ca2+]i were determined as the difference between the resting and the peak [Ca2+]i levels; the time for 50% decay of the transients (T50) was used to represent the decay of both transients. During the measurements the isolated fura-2-loaded cardiomyocytes from each group were incubated for 10 min with non-glucose K-H solution containing 10 mM 2-deoxy-D-glucose and 10 mM sodium dithionite to induce metabolic inhibition and anoxia (MI/A). Reperfusion (R) was followed by incubating the myocytes with normal K-H solution for further 10 min.
Isolation of SR and measurement of 45Ca2+ uptake SR vesicles from all rat groups were obtained by a method described previously [16] with some modifications. Briefly, freshly isolated cardiac myocytes from rats, destroyed by a low temperature (-75o C), were homogenized in extraction medium containing (in mM): 40 imidazole-HCl, 10 NaHCO3, 5 NaN3, 250 sucrose, 1 EDTA (t ~ 2o C; pH 7.0; 5 ml/g tissue), with a Polytron PT 35 homogenizer (Brinkmann, Wesbury, NY) at setting 9 for 10 s each. The homogenate was centrifuged for 5 min at 3000 × g to remove cellular debris. The supernatant was further centrifuged at 48 000 × g for 75 min in Sorvall SM-24 rotor and the supernatant was discarded. The pellet was suspended in 8 ml of a mixture 0.6 mM KCl and 20 mM imidazole-HCl (pH 7.0) and centrifuged at 48 000 × g for 60 min in Sorvall SM-24 rotor. The final pellet was re-homogenized in 1 ml 250 mM sucrose and 40 mM imidazoleHCl, using a Potter-Elvehjem homogenizer with Teflon pestle and stored at – 70o C. All solutions contained three protease inhibitors: soybean trypsin inhibitor (40 μg ml-1), 0.1% phenylmethylsulfonyl (PMSF) and leupeptin (0.5 μg ml-1). The ATP dependent transport Ca2+ to SR was measured at room temperature (22o C). 50 – 100 μg SR protein were added to 1 ml
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of a medium that contained 40 mM imidazole-HCl (pH 7.0), 100 mM KCl, 20 mM NaCl, 5 mM MgCl2, 4 mM ATP-Na2, 5 K-oxalate, 3 μCi 45CaCl2, 5 μM Ru360, an inhibitor of Ca2+ uptake in mitochondria and 5 μM calmidazolium, an inhibitor of Ca2+-ATPase of sarcolemma. A free concentration Ca2+ in this solution (5 μM) was determined by a Ca2+-EGTA buffer. After 10 min, aliquots of 0.9 ml were filtered through Millipore filters (0.45 μm, Bedford, MA, USA). Filters were washed three times with 4 ml cold (2– 4o C) solution containing 40 mM imidazole-HCl (pH 7.0), 100 mM KCl and 0.1 mM EGTA. The
45
Ca
2+
uptake by
SERCA, representing the activity of SERCA, was defined as the difference between the rate of
45
Ca
2+
uptake a K-oxalate containing solution in the presence and absence of 10 μM
cyclopiazonic acid (CPA), a specific inhibitor of SERCA. The difference in uptake in the presence and absence of 50 μM ryanodine, a specific blocker of RyR, was defined as the 45
Ca2+ release via the RyR receptor.
Plasma membrane purification and NCX assay. For purification of plasma membrane vesicles from all rat groups the procedure described previously [16] was used with some modifications. Briefly, freshly isolated cardiac myocytes from rats, destroyed by low temperature (-75o C), was homogenized in ice-cold buffer containing 0.6 M sucrose and 10 mM imidazole-HCl (pH 7.0). The homogenization consisted of 2 bursts of 7 s each at maximum speed with Polytron PT 35. The homogenate was centrifuged at 1000 × g for 5 min. The supernatant was centrifuged at 12 000 × g for 10 min in Sorvall SM-24 rotor. The 12,000 × g supernatant was diluted in 1.5 volumes of 160 mM NaCl and 20 mM HEPES-Tris (pH 7.4). This vesicle suspension was completed to 30 ml with the same solution supplemented with 0.25 M sucrose. The fraction was then centrifuged 160,000 × g for 70 min (Beckman, L8-M; rotor Ti 50.4). The pellet representing the sarcolemma enriched fraction was dissolved in 0.5 ml of solution (A): 100 mM NaCl, 50 mM
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LiCl, 6 mM KCl, 20 mM HEPES-Tris (pH 7.4) and assayed for Na+/Ca2+ exchange activity. All solutions contained all three protease inhibitors, soybean trypsin inhibitor (40 mg/ml), PMSF (0.1%) and leupeptin (0.5 μg/ml). Na+/Ca2+ exchange was estimated as a specific Na+dependent Ca2+ uptake following the protocol described previously [32] with some modifications. Briefly, 4 μl of the vesicle suspensions were incubated for 50 min at 22o C to load by Na+ via passive diffusion with their suspension medium, i.e. solution A. Afterwards, 15 μl of the vesicle suspension were placed on the side of polystyrene. Eppendorf tube containing 85 μl K-reaction medium: 160 mM KCl, 0.1 mM CaCl2, 100 μCi 45CaCl2 0.2mM EGTA, 2 μM valinomycin, 2 μM Ru360 to prevent the contribution of Na+/Ca2+ exchange of mitochondria and 20 mM HEPES-Tris (pH 7.4). The Ca2+ influx was stopped by diluting the reaction mixture after 2, 5 or 10 seconds with 5 ml of ice-cold termination medium: 160 mM KCl and 2 mM LaCl3. Na+-dependent specific Ca2+ uptake was defined as the total Ca2+ uptake minus unspecific Ca2+ uptake in medium A containing 0.2mM EGTA, 0.1 mM CaCl2, 100 μCi 45CaCl2, 2μM valinomycin, i.e. in solution where no Na+ gradient existed across the membrane. All samples were filtered under through Millipore filters (0.45 µM, Bedford, MA, USA) washed twice with 6 ml of 140 mM KCl and 0.1 mM LaCl2.
Western blot analysis for SR calcium handling proteins, eNOS and CAMKII Total proteins (20 μg per lane) from the homogenate of isolated ventricular myocytes were separated by SDS-PAGE (10% for SERCA, NCX, eNOS and CAMKII; 6% for RyR) and transferred to PDVF membrane. The PVDF membrane was blocked with 5% milk in Trisbuffered saline-Tween and incubated with primary antibody [goat anti-SERCA2 polyclonal antibody, goat anti-eNOS and rabbit anti-p-eNOS (ser-1177) polyclonal antibody (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-RyR2 monoclonal antibody (1:2000 dilution; Affinity BioReagent, Golden, CO), mouse anti-NCX1 monoclonal antibody
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(1:1000 dilution; Abcam, Cambridge, UK) and rabbit anti-CAMKII polyclonal antibody (1:1000 dilution, Stressgen Biotechnologies, Victoria, BC, Canada) at 4℃ overnight. The nitrocellulose membranes were then washed with Tris-buffered saline-Tween (0.05%) solution and incubated with corresponding horseradish peroxidase-conjugated second antibody (1:10000) and mouse anti-β-actin (1:10000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. The reaction was visualized by chemiluminescence with ECL Plus Western Blotting Detection System (Amersham, Buckinghamsire, UK).
RT-PCR Total RNAs from the isolated ventricular myocytes of normoxic and CIH rats were extracted by using the Illustra RNAspin mini RNA Isolation Kit (GE Health Care, Piscataway, NJ, USA). 1μg of RNA was reverse transcribed into cDNA with SuperscriptTM first strand synthesis system (Invitrogen Life Technologies, Carlsbad, CA, USA) for RT-PCR. PCR was done in RoboCycler (Stratagene, La Jolla, CA, USA) with Taq polymerase, AmpliTaq GoldTM (Roche Moelcular Systems, Pleasanton, CA, USA). Target genes for amplification, primer sets and thermal conditions were listed in Table 1.
Statistical analysis Values were expressed as means+/-SEM. One-way ANOVA (Newman-Keuls Multiple comparison test) and unpaired t-test were used to determine the differences among the multiple groups. The significance level was set at P
Received Date : 28-Aug-2014 Revised Date : 24-Oct-2014 Accepted Date : 30-Oct-2014 Article type : Original Manuscript
Cardioprotective effects of melatonin against myocardial injuries induced by chronic intermittent hypoxia in rats
Hang-Mee Yeung1, Ming-Wai Hung1, Chi-Fai Lau1, Man-Lung Fung1,2,*
Department of Physiology1, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
2
Research Centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of
Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China
* Corresponding author: Department of Physiology, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong Email: [email protected] Tel: +852 3917 9234 Fax: +852 2855 9730
Running title: Melatonin ameliorates myocardial injury in sleep apnea
Keywords: calcium, intermittent hypoxia, ischemia reperfusion, melatonin, oxidative stress, sarcoplasmic reticulum, sleep apnea
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jpi.12190 This article is protected by copyright. All rights reserved.
Accepted Article
Abstract Obstructive sleep apnea (OSA) associated with chronic intermittent hypoxia (CIH) increases the morbidity and mortality of ischemic heart disease in patients. Yet, there is a paucity of preventive measures targeting the pathogenesis of CIH-induced myocardial injury. We examined the cardioprotective effect of melatonin against the inflammation, fibrosis and the deteriorated sarcoplasmic reticulum (SR) Ca2+ homeostasis and ischemia/reperfusion (I/R)induced injury exacerbated by CIH. Adult male Sprague-Dawley rats that had received a daily injection of melatonin (10mg/kg) or vehicle were exposed to CIH treatment mimicking a severe OSA condition for 4 weeks. Systolic pressure, heart weights and malondialdehyde were significantly increased in hypoxic rats but not in the melatonin-treated group, when compared with the normoxic control. Levels of the expression of inflammatory cytokines (TNFα, IL6, COX2) and fibrotic markers (PC1 and TGFβ) were significantly elevated in the hypoxic group but were normalized by melatonin. Additionally, infarct size of isolated hearts with regional I/R was substantial in the hypoxic group treated with vehicle but not in the melatonin-treated group. Moreover, melatonin treatment mitigated the SR-Ca2+ homeostasis in the cardiomyocyte during I/R with (i) Ca2+ overloading, (ii) decreased SR-Ca2+ content, (iii) lowered expression and activity of Ca2+-handling proteins (SERCA2a and NCX1), (iv) decreased expressions of CAMKII and phosphorylated-eNOSser1177. Furthermore, melatonin ameliorated the level of expression of antioxidant enzymes (CAT and MnSOD) and NADPH oxidase (p22 and NOX2). Results support a prophylactic usage of melatonin in OSA patients, which protects against CIH-induced myocardial inflammation and fibrosis with impaired SRCa2+ handling and exacerbated I/R injury.
This article is protected by copyright. All rights reserved.
Accepted Article
Introduction Obstructive sleep apnea (OSA) is closely associated with cardiovascular diseases, which affects about 5% of the adult population, and increases risks for cardiovascular morbidity and mortality, including systemic hypertension, ischemic heart disease and congestive heart failure with myocardial hypertrophy [1, 2]. Chronic intermittent hypoxia (CIH) caused by repetitive episodes of severe hypoxia and reoxygenation in sleep apnea plays an important role in the pathophysiology of clinical manifestations in OSA patients. The deteriorated effect of CIH on cardiac functions is closely related to oxidative stress. It has been reported that CIH-induced ventricular dysfunction is associated with a decreased activity of myocardial superoxide dismutase of the antioxidant system [3, 4]. In addition, inflammation plays an important role in cardiac remodeling and hypertrophy, which could lead to heart failure. Levels of pro-inflammatory cytokines are significantly elevated in the circulation in OSA patients [5, 6]. It has also been shown that pro-inflammatory cytokines including TNF-alpha (TNFα) and interleukin-6 (IL6) are involved in the adverse myocardial remodeling [7-9]. Furthermore, inflammatory mediators including cycloxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) play pathogenic roles in ischemia/reperfusion (I/R)-induced injury of the heart [10, 11]. Also, transforming growth factor beta-1 (TGFβ1) is one of the cytokines involved in myocardial injury, and it plays a pathogenic role in cardiac fibrosis and increased stiffness of diastolic chamber [12]. Indeed, an increased expression of TGFβ1 is involved in the progression of ventricular hypertrophy and dysfunction [13, 14]. Moreover, reactive oxygen species (ROS) play a key role in impaired sarcoplasmic reticulum (SR)-Ca2+ homeostasis and I/R-induced myocardial injury, which subsequently triggers the inflammatory and fibrotic responses [3, 15]. Mechanistically, changes in the expression and activity of Ca2+-handling proteins lead to altered Ca2+ homeostasis and myocardial function under hypoxic conditions [16, 17]. Calcium-dependent calmodulin kinase II (CAMKII) plays
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a role in the modulation of activities of Ca2+ handling proteins [18, 19]. Also endothelial nitric oxide synthase (eNOS), a phosphorylated target of CAMKII, is involved in the modulation of myocardial contractile function and plays a cardioprotective role against I/R injury [20, 21]. Yet, there is limited information on the involvement of CAMKII and eNOS in the CIH-induced myocardial injury.
Melatonin is well known for its antioxidant capacity and anti-inflammatory effects against oxidative stress by scavenging free radicals and activating the antioxidant enzymes and by suppressing the expression of cytokines and inflammatory mediators [22-24]. The cardioprotective effect of melatonin against mitochrondrial oxidative damage and myocardial injuries has been explored in animal models of sepsis and I/R injury [25-28]. In addition, it has been demonstrated that administrations of antioxidant melatonin ameliorates I/R injury, which could lower the level of oxidative stress by scavenging the ROS and also by regulating the expression of antioxidant enzymes [22, 24, 28]. Although the acute effect of melatonin on cardioprotection against I/R injury has been shown [28, 29], the chronic effects of melatonin on the expression and activity of Ca2+ handling proteins and exacerbated I/R injury under CIH conditions are unclear. Also, the CAMKII-eNOS axis could be a potential target of the cardioprotective effect of melatonin. In this study, we aimed to examine the hypotheses that chronic administration of melatonin ameliorates the CIH-induced inflammation and fibrosis caused by oxidative stress, which leads to deteriorated Ca2+ homeostasis in rat ventricular myocytes with altered functions of SR-Ca2+ handling proteins, causing myocardial injury and increased susceptibility to I/R injury in the rat.
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Accepted Article
Materials and methods Animals The experimental protocol for this study was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85-23, revised 1996). Male SpragueDawley rats weighing about 70 g at age 28 days were randomly divided into groups for normoxic control and CIH treatment. Rats were kept in an acrylic chamber for normobaric hypoxia and had free access to water and chow. The oxygen fraction inside the chamber was kept with inspired oxygen alternating from 21 to 5 + 0.5% oxygen per minute for 8 hr/day. The desired oxygen content was established by a mixture of room air and nitrogen that was regulated and monitored by an oxygen analyzer (Vacumetrics Inc., CA, USA). The inspired oxygen level fell to 4-5% (nadir arterial oxygen saturation ca. 70%) for about 15 sec per min, which mimicks the recurrent episodic hypoxemia in OSA patients. For normoxic controls, aged-matched animals were kept in room air in the same housing and maintenance matching the hypoxic groups. The rats were treated for 28 days and were immediately used in the experiments. The animal room was controlled at a constant temperature (22 + 2oC), humidity and light:dark cycle (lights on 07:00-19:00).
Drug preparation Melatonin (Sigma, St. Louis, MO) solution was prepared fresh before injection by dissolving the indoleamine in absolute ethanol and further dilution with normal saline; the final concentration of ethanol was 2%. Melatonin in 10mg/kg body weight [30, 31] or vehicle (2% ethanol in normal saline) was administered intraperitoneally to rats in the melatonin-treated (MIH) and vehicle-treated (VIH) groups respectively each day 30 min before the hypoxic
This article is protected by copyright. All rights reserved.
Accepted Article
treatment.
Blood pressure measurement Systolic pressure was measured by a tail-cuff method. Briefly, the conscious animal was held inside a restraining cage and its tail was placed through the cuff with a photoelectric sensor. Changes in pressure applied to the cuff and arterial pulsation were amplified (IITC, Woodland Hills, CA, USA) and recorded on polygraph tracings. At least four measurements were taken for each animal and the average was calculated as the systolic pressure.
Histological analysis of Sirius Red staining Freshly isolated perfused rat hearts from normoxic and hypoxic groups were fixed in neutral buffered formalin for at least 72 h. Heart tissues were then processed routinely for dehydration with 70-100% graded alcohol and embedded in blocks with paraffin wax. Serial sections of 5 μm thickness were cut and mounted on silanized slides (DAKO, Denmark). Sections from all rat groups were kept in the oven overnight at 56oC. Quantitative analysis of collagen accumulation in heart tissues from all rat groups was visualized by Sirius Red staining and morphometric analysis. Briefly, tissue sections were stained with 0.1% picroSirius Red (Polysciences Inc., Washington, DC, USA) in saturated aqueous picric acid for 1 h at room temperature followed by differentiation in 0.01% hydrochloric acid. The Sirius Redstained collagen was then quantified by using LEICA Qwin Image Analyzer (Leica Microsystems Ltd., Milton Keynes, UK). All sections from normoxic and hypoxic rat groups were examined by the same person.
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Accepted Article
Isolated perfused heart preparation Rats from normoxic and hypoxic groups were decapitated and the hearts were quickly removed and placed in ice-cold Krebs-Henseleit (K-H) perfusion buffer before being mounted on the Langendorff apparatus for perfusion at 37 ºC with K-H buffer at constant pressure (100 cm of H2O) and equilibrated with 95% of O2/5% CO2. The buffer contained (in mM) 118.0 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3 and 11.0 glucose. All the hearts were subjected to regional ischemia described previously [16]. Briefly, a snare was formed by placing a silk suture around the left coronary artery of rat heart. The snare was pulled in order to occlude the coronary artery to produce ischemia. Reperfusion was done by releasing the snare. The isolated hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion which induced myocardial injury.
Measurement of the area of risk To determine the infarct size, the coronary artery was re-occluded at the end of reperfusion and the heart was perfused with 2.5% Evans blue to delineate the area of risk. The hearts were frozen at -70℃, cut into thin slices, which were perpendicular to the septum, from the apex to the base. Then incubated in sodium phosphate buffer containing 1% (w/v) 2,3,5triphenyl-tetrazolium chloride for 10 min in order to visualize the unstained infarct region. The infarct and the risk zone areas were determined by planimetry with software Image/J from the National Institutes of Health. (Bethesda, MD). The infarct size measured was expressed as a percentage of the risk zone.
Determination of myocardial injury by lactate dehydrogenase (LDH) efflux The effluent from each isolated perfused rat heart was collected at 10 min before regional ischemia, 5th min of reperfusion and the LDH was assayed spectrophotometrically by using a
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kit purchased from Sigma-Aldrich (St. Louis, MO), an intra-assay coefficient of variation of less than 10% and an inter-assay coefficient of variation of less than 12%. The LDH activity measured was expressed as units per liter. The limit of detection is about 1 ng/mL.
Measurement of malondialdehyde (MDA) The total lipid peroxides were measured as the amount of MDA using a BIOXYTECH® LPO-586™ kit (OxisResearch, Portland, OR), an intra-assay coefficient of variation of 5.8% and an inter-assay coefficient of variation of 7.2%. The reaction product was measured spectrophotometrically at 586nm. Standard curves were constructed with 1,1,3,3tetraethoxypropane as a standard. The limit of detection is about 1nM. The MDA level of in all rat hearts is expressed as μM, while the MDA concentration (μM) in heart was normalized to wet tissue weight (mg) and expressed as μmol/mg.
Preparation of isolated ventricular myocytes Single ventricular myocytes were isolated from the normoxic and hypoxic rats by using a collagenase method described previously [16]. After isolation, myocytes were allowed to stabilize for at least 30 min before any experiment.
Measurement of [Ca2+]i A spectrofluorometric method with Fura-2/AM as a Ca2+ indicator was used during the measurement of [Ca2+]i. Ventricular myocytes from either normoxic or hypoxic rats were incubated with 5 μM Fura-2/AM for 35 min. Fluorescent signals obtained at 340 nm (F340) and 380 nm (F380) excitation wave-lengths were recorded and stored in computer for data processing and analysis. The F340/F380 ratio was used to indicate cytosolic [Ca2+]i in the ventricular myocytes. During the measurement of electrically induced [Ca2+]i transients
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(E[Ca2+]i), myocytes were electrically stimulated at 0.2 Hz, whereas measurement of caffeine-induced [Ca2+]i transients (C[Ca2+]i) were done by applying drops of 10 mM caffeine directly to the ventricular myocytes. The amplitude of E[Ca2+]i and C[Ca2+]i were determined as the difference between the resting and the peak [Ca2+]i levels; the time for 50% decay of the transients (T50) was used to represent the decay of both transients. During the measurements the isolated fura-2-loaded cardiomyocytes from each group were incubated for 10 min with non-glucose K-H solution containing 10 mM 2-deoxy-D-glucose and 10 mM sodium dithionite to induce metabolic inhibition and anoxia (MI/A). Reperfusion (R) was followed by incubating the myocytes with normal K-H solution for further 10 min.
Isolation of SR and measurement of 45Ca2+ uptake SR vesicles from all rat groups were obtained by a method described previously [16] with some modifications. Briefly, freshly isolated cardiac myocytes from rats, destroyed by a low temperature (-75o C), were homogenized in extraction medium containing (in mM): 40 imidazole-HCl, 10 NaHCO3, 5 NaN3, 250 sucrose, 1 EDTA (t ~ 2o C; pH 7.0; 5 ml/g tissue), with a Polytron PT 35 homogenizer (Brinkmann, Wesbury, NY) at setting 9 for 10 s each. The homogenate was centrifuged for 5 min at 3000 × g to remove cellular debris. The supernatant was further centrifuged at 48 000 × g for 75 min in Sorvall SM-24 rotor and the supernatant was discarded. The pellet was suspended in 8 ml of a mixture 0.6 mM KCl and 20 mM imidazole-HCl (pH 7.0) and centrifuged at 48 000 × g for 60 min in Sorvall SM-24 rotor. The final pellet was re-homogenized in 1 ml 250 mM sucrose and 40 mM imidazoleHCl, using a Potter-Elvehjem homogenizer with Teflon pestle and stored at – 70o C. All solutions contained three protease inhibitors: soybean trypsin inhibitor (40 μg ml-1), 0.1% phenylmethylsulfonyl (PMSF) and leupeptin (0.5 μg ml-1). The ATP dependent transport Ca2+ to SR was measured at room temperature (22o C). 50 – 100 μg SR protein were added to 1 ml
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of a medium that contained 40 mM imidazole-HCl (pH 7.0), 100 mM KCl, 20 mM NaCl, 5 mM MgCl2, 4 mM ATP-Na2, 5 K-oxalate, 3 μCi 45CaCl2, 5 μM Ru360, an inhibitor of Ca2+ uptake in mitochondria and 5 μM calmidazolium, an inhibitor of Ca2+-ATPase of sarcolemma. A free concentration Ca2+ in this solution (5 μM) was determined by a Ca2+-EGTA buffer. After 10 min, aliquots of 0.9 ml were filtered through Millipore filters (0.45 μm, Bedford, MA, USA). Filters were washed three times with 4 ml cold (2– 4o C) solution containing 40 mM imidazole-HCl (pH 7.0), 100 mM KCl and 0.1 mM EGTA. The
45
Ca
2+
uptake by
SERCA, representing the activity of SERCA, was defined as the difference between the rate of
45
Ca
2+
uptake a K-oxalate containing solution in the presence and absence of 10 μM
cyclopiazonic acid (CPA), a specific inhibitor of SERCA. The difference in uptake in the presence and absence of 50 μM ryanodine, a specific blocker of RyR, was defined as the 45
Ca2+ release via the RyR receptor.
Plasma membrane purification and NCX assay. For purification of plasma membrane vesicles from all rat groups the procedure described previously [16] was used with some modifications. Briefly, freshly isolated cardiac myocytes from rats, destroyed by low temperature (-75o C), was homogenized in ice-cold buffer containing 0.6 M sucrose and 10 mM imidazole-HCl (pH 7.0). The homogenization consisted of 2 bursts of 7 s each at maximum speed with Polytron PT 35. The homogenate was centrifuged at 1000 × g for 5 min. The supernatant was centrifuged at 12 000 × g for 10 min in Sorvall SM-24 rotor. The 12,000 × g supernatant was diluted in 1.5 volumes of 160 mM NaCl and 20 mM HEPES-Tris (pH 7.4). This vesicle suspension was completed to 30 ml with the same solution supplemented with 0.25 M sucrose. The fraction was then centrifuged 160,000 × g for 70 min (Beckman, L8-M; rotor Ti 50.4). The pellet representing the sarcolemma enriched fraction was dissolved in 0.5 ml of solution (A): 100 mM NaCl, 50 mM
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LiCl, 6 mM KCl, 20 mM HEPES-Tris (pH 7.4) and assayed for Na+/Ca2+ exchange activity. All solutions contained all three protease inhibitors, soybean trypsin inhibitor (40 mg/ml), PMSF (0.1%) and leupeptin (0.5 μg/ml). Na+/Ca2+ exchange was estimated as a specific Na+dependent Ca2+ uptake following the protocol described previously [32] with some modifications. Briefly, 4 μl of the vesicle suspensions were incubated for 50 min at 22o C to load by Na+ via passive diffusion with their suspension medium, i.e. solution A. Afterwards, 15 μl of the vesicle suspension were placed on the side of polystyrene. Eppendorf tube containing 85 μl K-reaction medium: 160 mM KCl, 0.1 mM CaCl2, 100 μCi 45CaCl2 0.2mM EGTA, 2 μM valinomycin, 2 μM Ru360 to prevent the contribution of Na+/Ca2+ exchange of mitochondria and 20 mM HEPES-Tris (pH 7.4). The Ca2+ influx was stopped by diluting the reaction mixture after 2, 5 or 10 seconds with 5 ml of ice-cold termination medium: 160 mM KCl and 2 mM LaCl3. Na+-dependent specific Ca2+ uptake was defined as the total Ca2+ uptake minus unspecific Ca2+ uptake in medium A containing 0.2mM EGTA, 0.1 mM CaCl2, 100 μCi 45CaCl2, 2μM valinomycin, i.e. in solution where no Na+ gradient existed across the membrane. All samples were filtered under through Millipore filters (0.45 µM, Bedford, MA, USA) washed twice with 6 ml of 140 mM KCl and 0.1 mM LaCl2.
Western blot analysis for SR calcium handling proteins, eNOS and CAMKII Total proteins (20 μg per lane) from the homogenate of isolated ventricular myocytes were separated by SDS-PAGE (10% for SERCA, NCX, eNOS and CAMKII; 6% for RyR) and transferred to PDVF membrane. The PVDF membrane was blocked with 5% milk in Trisbuffered saline-Tween and incubated with primary antibody [goat anti-SERCA2 polyclonal antibody, goat anti-eNOS and rabbit anti-p-eNOS (ser-1177) polyclonal antibody (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-RyR2 monoclonal antibody (1:2000 dilution; Affinity BioReagent, Golden, CO), mouse anti-NCX1 monoclonal antibody
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(1:1000 dilution; Abcam, Cambridge, UK) and rabbit anti-CAMKII polyclonal antibody (1:1000 dilution, Stressgen Biotechnologies, Victoria, BC, Canada) at 4℃ overnight. The nitrocellulose membranes were then washed with Tris-buffered saline-Tween (0.05%) solution and incubated with corresponding horseradish peroxidase-conjugated second antibody (1:10000) and mouse anti-β-actin (1:10000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. The reaction was visualized by chemiluminescence with ECL Plus Western Blotting Detection System (Amersham, Buckinghamsire, UK).
RT-PCR Total RNAs from the isolated ventricular myocytes of normoxic and CIH rats were extracted by using the Illustra RNAspin mini RNA Isolation Kit (GE Health Care, Piscataway, NJ, USA). 1μg of RNA was reverse transcribed into cDNA with SuperscriptTM first strand synthesis system (Invitrogen Life Technologies, Carlsbad, CA, USA) for RT-PCR. PCR was done in RoboCycler (Stratagene, La Jolla, CA, USA) with Taq polymerase, AmpliTaq GoldTM (Roche Moelcular Systems, Pleasanton, CA, USA). Target genes for amplification, primer sets and thermal conditions were listed in Table 1.
Statistical analysis Values were expressed as means+/-SEM. One-way ANOVA (Newman-Keuls Multiple comparison test) and unpaired t-test were used to determine the differences among the multiple groups. The significance level was set at P
