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Online supplements Activation of eNOS by a vanadium compound ameliorates pressure overload-induced cardiac injury in ovariectomized rats. 1Md. Shenuarin Bhuiyan, 1Norifumi Shioda, 2Masatoshi Shibuya, 2Yoshiharu Iwabuchi and 1,3Kohji Fukunaga 1 Department of Pharmacology and 2Department of Synthetic Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan and 3Tohoku University 21st Century COE Program “CRESCENDO,” Sendai, Japan Corresponding Author: Dr. Kohji Fukunaga Department of Pharmacology Graduate School of Pharmaceutical Sciences, Tohoku University Aramaki-Aoba, Aoba-ku, Sendai 980-8578, Japan Tel: 81-22-795-6837 Fax: 81-22-795-6835 E-mail: [email protected] 1 Supplemental materials and data: Detailed Materials and Methods: Materials Reagents and antibodies were obtained from the following sources: L-[2,3,4-3H]arginine , PerkinElmer Life Sciences, Boston, MA); anti-eNOS antibody (Sigma, St. Louis, MO); anti-phospho-eNOS antibody (Cell Signaling Technology, Beverly, MA); anti-phospho-Akt antibody (Ser-437) and total-Akt antibody (Cell Signaling Technology, Beverly, MA); anti-phospho-Akt antibody (Thr-308) (Upstate Biotechnology, Lake Placid, NY); anti-phospho-Myosin Light Chain (Ser-19) antibody and total Myosin Light Chain 2 antibody (Cell Signaling Technology, Beverly, MA); anti-Caveolin-3 antibody (BD Biosciences, San Jose, CA); anti-Heat shock protein-90 antibody (Upstate Biotechnology, Lake Placid, NY); anti-Calpastatin antibody and anti-dystrophin antibody (Chemicon International, Inc., Temecula, CA); anti-β tubulin antibody (Sigma, St. Louis, MO); anti-rabbit antibody (Amersham Biosciences Inc., Piscataway, NJ). Other reagents were of the highest quality available (Wako Pure Chemicals, Osaka, Japan). Animals All procedures for handling animals comply with the Guide for Care and Use of Laboratory Animals and were approved by the Animal Experimentation Committee of Tohoku University Graduate School of Pharmaceutical Sciences. Female Wistar rats (6 weeks old) weighing 150-200 gm were obtained from Japan SLC, Inc. (Hamamatsu, Japan). Rats were housed under climate-controlled conditions with a 12h light/dark cycle and provided with standard food and water ad libitum. An acclimation period of at least 1 week was provided before initiating the experimental protocol. 2 Experimental surgical procedure Female Wistar rats were randomly separated into four treatment groups: 1) ovariectomy (OVX)(n=8), 2) ovariectomy plus pressure overload plus vehicle treatment group (OVX-PO-vehicle) (n=8), 3) ovariectomy plus pressure overload plus VO(OPT) (1.25 mg/kg vanadium) treatment group (V 1.25) (n=8) and 4) ovariectomy plus pressure overload- plus VO(OPT) (2.5 mg/kg vanadium) treatment group (V 2.5)(n=8). Bilateral ovariectomy was produced in rats anesthetized with sodium pentobarbital (50 mg/kg, i.p) (Tokyo Kasei Kogyo, Tokyo, Japan)1,2. A sham operation was performed by exposing the ovaries without isolation. Two weeks after the sham operation or ovariectomy, pressure-overload was initiated in both groups by abdominal aortic banding as we described previously1,3. Briefly, rats were anesthetized, the abdominal aorta was exposed under sterile conditions through a midline abdominal incision, and a blunted 25-gauge needle (outside diameter, 0.5 mm) placed between the right and left renal arteries. A ligature (6-0 silk) was snugly tied around both the renal artery and the needle. The needle was then removed, leaving the internal diameter of the aorta approximately equal to that of the needle. Sham-operated animals had an untied ligature placed in the same location. After surgery, animals were housed under controlled environmental conditions with food (Purina Formulab Chow 5008) and water ad libitum. VO(OPT) administration VO(OPT) was dissolved in 5% glucose. Vehicle (5% glucose) or VO(OPT) (containing 1.25 and 2.5 mg of vanadium /kg) was administered orally for 14 days (once daily) in a volume of 0.1 ml/100g of body of rats, started from the 15th days after onset of aortic banding. During vehicle or VO(OPT) treatment the body weight and food intake of the 3 rats were observed every day. Haemodynamic measurements Haemodynamic measurements were done as we described previously 1,4,5,6. Briefly, rats were anesthetized with sodium pentobarbital (50mg/kg, i.p). The right carotid artery was cannulated with a polyethylene catheter (PE-50, length 25cm) filled with degassed saline containing heparin (300 UI/ml). Haemodynamic variables were measured with a pressure transducer (Nihon Kohden, Tokyo, Japan) connected to a polygraph (Nihon Kohden, Tokyo, Japan) and recorded using a thermal recorder (Nihon Kohden, Tokyo, Japan). Meticulous care was taken to ensure that the system remained free of air bubbles during the experimental period. After arterial blood pressure (AP-601G; Nihon Kohden, Tokyo, Japan) and heart rate (AT-601G; Nihon Kohden, Tokyo, Japan) measurements were obtained through the polygraph, the catheter was advanced to the left ventricular cavity. We then measured LV systolic and end-diastolic pressures (AP-601G; Nihon Kohden, Tokyo, Japan), the maximal rate of pressure development (+dp/dt), and the rate of relaxation (-dp/dt) of LV by cardiotachometer (EQ-601G; Nihon Kohden, Tokyo, Japan) using the polygraph. Western blot analysis Four weeks after aortic banding, rats were anesthetized, and hearts were excised and quickly perfused with phosphate-buffered saline to wash out blood from coronary vessels. Heart tissue was sliced at 2-mm thickness using a slicer (RBS-2; Zivic-Miller Laboratories, Zelienople, PA). LV tissue samples were then rapidly frozen in liquid nitrogen and stored at -80°C before use. For assays, each frozen sample was homogenized by methods we described previously1,7. An equal amount of protein for each sample (25 µg of total protein) was separated on 7.5-15% SDS-polyacrylamide gels and transferred onto polyvinylidene 4 difluoride membranes (Millipore Corporation, Billerica, MA). After being blocked with 5% low-fat milk in Tris-buffered saline plus Tween-20, membranes were incubated with specific primary antibodies overnight at 4°C. This was followed by a donkey anti-rabbit IgG coupled to horseradish peroxidase, and the blots were developed using the ECL immunoblotting detection system (Amersham Biosciences) and visualized on X-ray film (Fuji Film). Autoradiographic films were scanned by densitometry (Lasergraphics, Irvine, CA) and quantitated using Imagegause V3.41 (Fuji Film). The relative amounts of proteins were expressed as percent increase over sham. Isoproterenol administration Four weeks after aortic banding, a group of rats were treated with chronic isoproterenol as we described previously1. Vehicle (saline) or DL-isoproterenol (5mg/kg) (Sigma, St. Louis, MO) in saline was injected intraperitoneally (i.p.) in a volume of 0.1 ml/100 g of body weight once a day for 28 days in the OVX (n=8), OVX-PO-vehicle (n=8), V 1.25 (n=8) and V 2.5 (n=8) group rats. Statistical analysis Values are represented as means ± standard error of the mean (S.E.M.). Morphometric changes and SDS-PAGE electrophoresis results were evaluated for differences by one way analysis of variance (ANOVA) combined with Dunnett’s post hoc test. Survival was analyzed by Kaplan-Meier analysis. A value of P<0.05 was considered statistically significant. References: 1. Bhuiyan MS, Shioda N, Fukunaga K. Ovariectomy augments pressure overload-induced 5 hypertrophy associated with changes in Akt and nitric oxide synthase signaling pathways in female rats. Am J Physiol Endocrinol Metab. 2007;293:E1606-1614. 2. Kam KW, Kravtsov GM, Liu J, Wong TM. Increased PKA activity and its influence on isoprenaline-stimulated L-type Ca2+ channels in the heart from ovariectomized rats. Br J Pharmacol. 2005;144:972-981. 3. Jouannot P, Hatt P. Rat myocardial mechanics during pressure-induced hypertrophy development and reversal. Am J Physiol Cell Physiol. 1977;232:C355-C364. 4. Bhuiyan MS, Shibuya M, Shioda N, Moriguchi S, Kasahara J, Iwabuchi Y, Fukunaga K Cytoprotective effect of bis(1-oxy-2-pyridinethiolato)oxovanadiun(IV) on myocardial ischemia/reperfusion injury elicits inhibition of Fas ligand and Bim expression and elevation of FLIP expression. Eur J Pharmacol. 2007;571:180-188. 5. Bhuiyan MS, Takada Y, Shioda N, Moriguchi S, Kasahara J, Fukunaga K. Cardioprotective effect of vanadyl sulfate on ischemia/reperfusion-induced injury in rat heart in vivo is mediated by activation of protein kinase B and induction of FLICE-inhibitory protein. Cardiovasc Ther. 2008;26:1-14. 6. Bhuiyan MS, Fukunaga K. Inhibition of HtrA2/Omi ameliorates heart dysfunction following ischemia/reperfusion injury in rat heart in vivo. Eur J Pharmacol. 2007;557:168-177. 7. Takada Y, Hashimoto M, Kasahara J, Aihara K, Fukunaga K. Cytoprotective effect of sodium orthovanadate on ischemia/reperfusion-induced injury in the rat heart involves Akt activation and inhibition of fodrin breakdown and apoptosis. J Pharmacol Exp Ther. 2004;311:1249-1255. 6 Table S1: Effect of VO(OPT) on morphometric parameters Parameters No of animals (n) OVX OVX-PO V 1.25 V 2.5 8 7 8 8 Body weight (gm) 258.0±3.5 245.7±5.1 219.5±5.9‡|| 212.0±5.8‡|| Heart weight (gm) 0.95 ±0.03 1.15±0.03‡ 0.95±0.05|| 0.84±0.04¶ 0.76±0.02 0.92±0.03† 0.78±0.05§ 0.7±0.03|| 0.19±0.02 0.23±0.02 0.16±0.01§ 0.15±0.01|| 1.6±0.03 1.84±0.02‡ 1.59±0.06¶ 1.46±0.03*¶ Left ventricle weight (gm) Right ventricle weight (gm) Lung weight (gm) Data are expressed as means ± S.E.M. *, P<0.05, †, P<0.01 & ‡, P<0.001 versus the OVX group; §, P<0.05, ||, P<0.01 & ¶, P<0.001 versus the OVX-PO-vehicle treated group. OVX, ovariectomy; OVX-PO, ovariectomy plus pressure overload plus vehicle treatment group; V 1.25, ovariectomy plus pressure overload plus VO(OPT) (1.25 mg/kg vanadium) treatment group; V 2.5, ovariectomy plus pressure overload plus VO(OPT) (2.5 mg/kg vanadium) treatment group; 7 Table S2: Effect of VO(OPT) treatment on sham and OVX animals Parameters No of animals (n) sham sham-V 2.5 OVX OVX-V 2.5 8 5 8 5 Body weight (gm) 231.4±2.3 212.7±7.8 258.3±3.5 † 251.2±7.9 Heart weight (gm) 0.9±0.02 0.8±0.04 0.95±0.03 * 0.82±0.02 Left ventricle weight (gm) 0.71±0.01 0.61±0.03 0.76±0.02 0.67±0.02 Right ventricle weight (gm) 0.2±0.01 0.16±0.01 0.19±0.02 0.16±0.01 Lung weight (gm) 1.48±0.04 1.5±0.03 1.6±0.03 1.54±0.03 Data are expressed as means ± S.E.M. *, P<0.05, and †, P<0.01 versus the sham group; sham-V 2.5, sham plus VO(OPT) containing 2.5 mg/kg vanadium treatment group; OVX, ovariectomy; OVX-V 2.5, ovariectomy plus VO(OPT) containing 2.5 mg/kg vanadium treatment group; 8 5 HW/BW (mg/gm) A 4 3 2 1 B LW/BW (mg/gm) 0 8 7 6 5 4 3 2 1 0 sham sham-V2.5 OVX OVX-V2.5 sham sham-V2.5 OVX OVX-V2.5 Figure S1: Effect of VO(OPT) (2.5mg/kg vanadium) on HW/BW(A) and LW/BW(B) ratio in the sham and OVX rats. Each bar represents the mean ± S.E.M. 9 400 HR (beats/min) A 300 200 100 0 sham sham-V2.5 OVX OVX-V2.5 160 MABP (mmHg) B †† 120 80 40 0 sham sham-V2.5 OVX OVX-V2.5 Figure S2: Effect of VO(OPT) (2.5mg/kg vanadium) on heart rate and mean arterial blood pressure in the sham and OVX rats. Both sham and OVX rats were orally treated with VO(OPT) (containing 2.5 mg/kg vanadium) once day for 14 days and heart rate (HR (A) and mean arterial blood pressure (MABP)(B) were measured after the treatment period. Each bar represents the mean ± S.E.M. *, P<0.05, versus the sham group; †, P<0.01 versus the OVX group. 10 sham OVX V 1.25 Body weights (gm) A sham-V2.5 OVX-V2.5 V 2.5 300 200 100 0 0 B 1 2 3 Sham OVX V 1.25 30 Food intake (gm) 5 Days 8 11 14 sham-V2.5 OVX-V2.5 V 2.5 20 10 0 0 3 6 9 12 15 Days Figure S3: Effect of VO(OPT) on body weight (A) and food intake (B). Rats were treated with VO(OPT) (containing 2.5mg/kg vanadiaum) for 14 days and body weight and food intake were observed each day. Values expressed as mean ± S.E.M. for body weights and mean food intake per rats. 11 4 HW/BW (mg/gm) A 3 2 OVX 1W 2W 3W 4W 7 LW/BW (mg/gm) B ** ** 6 5 4 OVX 1W 2W 3W 4W Figure S4. Time course of cardiac hypertrophy. Heart-to-body weight ratio (HW/BW) (A) and lung-to-body weight ratio (LW/BW) (B) increased time-dependently. Number of rats in OVX, TAC 1 week (1W), TAC 2 week (2W), TAC 3 week (3W) and TAC 4 week (4W) are 4,4,4,5and 5 respectively. **, P<0.01 versus the OVX group. 12 A pAkt (Ser 473) Total Akt OVX pAkt (% of sham) 250 1W 2W 3W 4W * 200 150 100 50 ** 0 OVX 1W 2W 3W 4W B peNOS (Ser 1179) eNOS OVX 1W 2W peNOS 120 NOS (% of sham) 3W 4W eNOS 100 * 80 * 60 40 20 0 OVX 1W 2W 3W 4W Figure S5. Time course of LV Akt and eNOS activity. A, left ventricular Akt phosphorylation (Ser 473) increased 1 week after pressure overload and there after decreased time-dependently with no change in total Akt level. B, left ventricular eNOS and Akt mediated eNOS phosphorylation (Ser 1179) decreased time-dependently. Number of rats in OVX, TAC 1 week (1W), TAC 2 week (2W), TAC 3 week (3W) and TAC 4 week (4W) are 4,4,4,5and 5 respectively. *, P<0.05 and **, P<0.01 versus the OVX group. 13 Figure S6: Putative mechanism of VO(OPT) mediated cardioprotection. In normal female heart, eNOS is localized to the caveolae through its interaction with caveolin 3 and compartmentalized with L-type Ca2+ channel and β-aderenergic receptor. Activation of Akt signaling ultimately leads to eNOS phosphorylation, eNOS activation and thereby activates 14 nitric oxide signaling pathways. The resulting combination of these effects subsequently confers ventricular dilation and cardioprotection. Pressure overload-induced hypertrophy severely impairs eNOS and Akt signaling pathways, thereby imbalances the NO mediated cardioprotective action. Treatment with VO(OPT) activate the Akt activity and enhances Akt mediated eNOS activity and subsequently confers cardioprotection against myocardial hypertrophy. 15