Difference in heart functions between the rat and the mouse under isoflurane anesthesia

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ABSTRACTS / Journal of Molecular and Cellular Cardiology 45 (2008) S1–S35

P-B-02 Angiotensinergic stimulation of vascular endothelium in mice causes hypotension, bradycardia and attenuated angiotensin response Takanobu Takezako1,2, Ramaswamy Ramchandran1, Yasser Saad1, Naoki Makino2, Sadashiva S. Karnik1 1 Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation 2 Division of Molecular and Clinical Gerontology, Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University Traditionally it is well known that Angiotensin II (Ang II) modulate contraction of vascular muscle cells (VSMCs) in the vasculature via AT1 receptor activation. However it is not clear whether AT1 receptor (AT1R) activation on endothelial cells (ECs) modulate contraction of VSMCs and whether impairment of this regulation contribute to hypertension. Delineating of this action of AT1R on ECs in the blood vessels has been challenging problem because of the predominance of the AT1R in VSMCs. We have obviated this limitation by generating the transgenic (TG) mice engineered to target expression of the constitutively active AT1R, N111G mutant specifically in ECs. Surprigingly, angiotensinergic stimulation of ECs resulted in hypotension and bradycardia in TG mice. The pressor response to acute infusion of Ang II was significantly reduced in TG mice. Production of hypotensive mediator, nitric oxide (NO) and aortic eNOS expression were increased in TG mice. AT1R blocker(ARB) rescued hypotension and bradycardia. In contrast, eNOS inhibitor rescued only hypotension. These results demonstrated that both hypotension and bradycardia are induced by AT1R activation on ECs but these are mediated different mechanism. Our results imply that the Ang II action by means of AT1R on ECs is antagonistic to vasoconstriction in general, and it may moderate the magnitude of functional response to Ang II in VSMCs. This control mechanism most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertension. Keywords: Angiotensin II; Endothelial cell; Hypotension doi:10.1016/j.yjmcc.2008.09.668

P-B-03 Suppressive effect of red wine polyphenolic compounds on platelet aggregation in dietary-induced hypercholesterolemic rats Yumi Katano1, Akira Ishihata2 1 Div.Theoretic. Nurs. & Pathophysiol, Yamagata Univ. Sch. Med. Yamagata, 990-9585 2 Div. Pathophysiol, Ohu Univ. Fukushima, Japan Platelet aggregation is enhanced in hyperlipidemia to become an important risk factor of coronary heart diseases. Previous studies suggested that platelet aggregation is inhibited by red wine polyphenolic compounds (RWPC) in vitro. We investigated the effect of chronic administration of RWPC on platelet aggregation and on plasma lipid levels in hypercholesterolemic rats. In addition, we investigated the effect of RWPC on plasma antioxidant levels in hypercholesterolemic rats. Male F344 rats (2 month-old) were fed standard diet (ST), 4% cholesterol-rich-diet (CH), or 4% cholesterolrich-diet plus 0.62% freeze-dried red wine extracts (CH+R) for 3 months. ADP-induced platelet aggregation was measured by a whole blood aggregometer. Plasma concentrations of total cholesterol, triglyceride and HDL-cholesterol were measured using a Spotchem EZ SP-4430 dry-chemistry blood analyzer. To evaluate antioxidant levels in each rat, reduced glutathione (GSH) content and superoxide dismutase (SOD) activity were measured. GSH content in CH was less than ST, while that of CH + R was not different from ST, although SOD activities were not affected by red wine consumption. Platelet

aggregation of CH was significantly enhanced compared with ST, while that of (CH + R) was not different from ST. Plasma concentration of lipids were significantly higher both in CH and (C + R) than ST, but were not different between CH and (CH+R). These results suggest that chronic administration of RWPC inhibits platelet aggregation in dietary-induced hypercholesterolemic rats. This effect was not due to the decrease of plasma lipid levels. Keywords: platelet aggregation; polyphenol; hypercholesterolemia doi:10.1016/j.yjmcc.2008.09.669

P-B-04 Metabolic Profile of Liver from Rats with Hypertension and Heart Failure Takao Kato1, Yasutaka Inuzuka1, Yodo Tamaki1, Tsuneaki Kawashima1, Shinichiro Niizuma1, Junji Okuda1, Yoshitaka Iwanaga1, Toru Kita1, Tomoyoshi Soga2, Tetsuo Shioi1 1 Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University 2 Institute for Advanced Bioscience, Keio University Metabolic abnormalities, such as insulin resistance, are frequently associated with cardiovascular diseases. Although the liver plays a critical role in metabolic regulation, little is known about hepatic metabolism in cardiovascular diseases. Using capillary electrophoresis time-of-flight mass spectrometry, we profiled liver metabolites of rats with hypertension and heart failure. Dahl salt-sensitive rats fed a high-salt diet developed hypertension (HT) at 11 weeks of age and congestive heart failure (CHF) at 17-19 weeks. Using metabolomic analyses of the livers, we quantified the amounts of 114 metabolites of glycolysis, TCA cycle, nucleic acid and amino acid syntheses. The amounts of 25 metabolites significantly differed between the control and HT, and the amounts of 26 metabolites differed significantly between HT and CHF. These findings indicate that alteration of hepatic metabolism is associated with the development of hypertension and heart failure. Since metabolic abnormality is known to influence the prognosis of patients with HT or CHF, the analysis of the metabolic response of the liver appears to be useful for identifying biomarkers and future therapeutic targets in hypertension and heart failure. Keywords: Metabolism; Hypertension; Heart Failure doi:10.1016/j.yjmcc.2008.09.670

P-B-05 Difference in heart functions between the rat and the mouse under isoflurane anesthesia M. Ito, Y. Sanzen, T. Imai, M. Tsuge, H. Sato, T. Kawada1, M. Nakazawa Niigata Univ, Niigata, Japan 1 Musashino Univ, Tokyo, Japan Volatile anesthetic is a useful tool for measurement of hemodynamic parameters of animal experiments. We have used 2% isoflurane for rat experiments, whereas we used 1.5% isoflurane which was optimal concentration for mouse experiment. Under these anesthetic conditions, the heart functions of mice measured with echocardiograph were significantly lower than those of rats. In the present study, we explored to find the cause of the difference. Six to 7 weeks old mail Wistar rats and ICR mice were used. Rats and mice were anesthetized with 2% and 1.5% isoflurane, respectively. Heart rate, echo-cardiograph, left ventricular pressure (LVP), dP/dt and left ventricular endodiastolic pressure were measured. Effects of atropine on these parameters were studied. Baroreceptor reflex were examined using phenylephrine and

ABSTRACTS / Journal of Molecular and Cellular Cardiology 45 (2008) S1–S35

nitroprusside injections. Moreover, plasma cathecholamines were measured. Heart rate, FS and EF of rats and of mice were 391 ± 17 and 353 ± 24 (beats/min), 48.1 ± 1.2 and 31.0 ± 2 (%), and 85.0 ± 1.0 and 65.6 ± 3.0 (%), respectively. Ten min after atropine, these values were 411 ± 18 and 413 ± 26 (beats/min), 48.1 ± 1.2 and 46.1 ± 3.8 (%), and 84.3 ± 2.3 and 82.8 ± 3.8 (%), respectively. LVP and dP/dt of the mice were lower than those of the rats. Baroreceptor reflex index of phenylephrine injection was significantly higher in the mice than in the rats, although that of nitroprusside was not different. Plasma noradrenaline, and dopamine concentration were higher in the mice than in the rats. These results suggest that isoflurane anesthesia activates parasympathetic nerve activity in the mice and we should pay attention to the interpretation of hemodynamic data of the mice obtained under isoflurane anesthesia. Keywords: Hemodynamics; Isoflurane; rat and mouse doi:10.1016/j.yjmcc.2008.09.671

P-B-06 Evolution of Ca2+ signalling and nano-domain development in rabbit inferior vena cava Jiazhen Minnie Dai, Kuo-Hsing Kuo, Cheng-Han Lee, Mark Nazer, Cornelis van Breemen Heart and Lung Institute, Vancouver, BC, Canada Activation of α1-adrenergic receptors by norepinephrine or its structural analogs typically results in the constriction of blood vessels. Such action in the venous vasculature represents a physiologically important mechanism in regulating venous return and ultimately the cardiac output of the heart. We have previously shown that phenylephrine(PE)-induced oscillatory Ca2+ signals underlie excitation-contraction coupling in adult rabbit inferior vena cava (IVC). These oscillations are caused by waves of regenerative Ca2+ release, which are maintained by sarcoplasmic reticulum (SR) reloading via junctions formed between plasma membrane and SR (PM-SR). Here we present evidence that these junctions together with oscillatory Ca2+ signaling develop post-natal. We examined the agonist-induced Ca2+ signal in in situ vascular smooth muscle cells (VSMC) of the neonatal rabbit IVC in relation to force generation. PE elicits a biphasic Ca2+ signal with an initial Ca2+ transient followed by a steady state plateau averaging 25 % of the peak value. In contrast to the adult, the plateau phase of PE-induced Ca2+ signal and tonic contraction in the VSMC of neonatal rabbit IVC was abolished by nifedipine. Employing electron microscopy, we found that VSMC in neonatal rabbit IVC posses less junctional SR, 6 ± 1%, as compared with the adult rabbit IVC, 18 ± 1%. Our results suggest that complex structures, defining a nano-domain (∼20 nm) and composition, including clusters of Ca2+ and Na+ transport proteins, of the PM-SR junctions develops during the neo-natal period and that this correlates with the appearance of oscillatory Ca2+ signaling in VSMC. Keywords: Ca2+ Oscillation; Excitation-contraction coupling; Cardiovascular doi:10.1016/j.yjmcc.2008.09.672

P-B-07 Molecular mechanisms of the antagonistic action between AT1 and AT2 receptors Yoshino Matsuo, Shin-ichiro Miura, Masahiro Fujino, Keijiro Saku Department of Cardiology, Fukuoka University School of Medicine, Fukuoka, Japan Although angiotensin II (Ang II) binds to Ang II type 1 (AT1) and type 2 (AT2) receptors, the functions of AT1 and AT2 receptors are

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antagonistic. The molecular mechanisms of this antagonism are not well known. Therefore, we examined AT1 and AT2 receptor-induced signal cross-talk in the cytoplasm and the importance of the hetero-dimerization of AT1 receptor with AT2 receptor on the cell surface. We used HepG2 cells and human coronary smooth muscle cells that endogenously expressed both receptors. The AT1 and AT2 receptors showed antagonistic effects toward inositol phosphate production, cell cycle regulatory protein p27kip1 expression and extracellular-signal-regulated kinase 1/2 activity. The AT1 receptors formed mainly homo-dimers but not hetero-dimers with AT2 receptor on the cell surface as determined by immunoprecipitation and induced cell signals. The ratio of the expression levels of homo-dimerized AT1 receptors, homo-dimerized AT2 receptors and hetero-dimerized AT1 and AT2 receptors on the cell surface was 25:20:1. In addition, the homo-dimerized AT1 receptor or AT2 receptor on the cell surface did not change after 24 hrs in the presence of telmisartan, an AT1 receptor blocker or PD123319, an AT2 receptor blocker. In conclusions, Ang II-induced AT1 receptor signals was blocked by AT2 receptor signals by their cross-talk in the cytoplasm but not by hetero-dimerization of both receptors on the cell surface. Keywords: angiotensin II receptors; cross-talk; dimerization doi:10.1016/j.yjmcc.2008.09.673

P-B-08 The Role of VEGFR1 Tyrosine Kinase Signaling in Enhancement of Angionegesis in Acute Hindlimb Ischemia in Mice Shintaro Kato1, H. Amano1, Y. Ito1, N. Aoyama1, M. Shibuya2, M. Majima1, Toru Izumi1 1 Kitasato University School of Medicine 2 Tokyo Medical and Dental University Purpose: Vascular endothelial growth factor (VEGF) has 3 receptors, VEGFR1 (Flt-1), VEGFR2 (Flk-1/KDR) and VEGFR3 (Flt-4). We have reported the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors, ‘hemangiocytes,’ constitute the major determinant of revascularization. We hypothesized that Flt-1 tyrosine kinase induces angiogenenic response by stimulating Stem Cell Factor (SCF) and MMP-9. Methods: Hindlimb ischemia was made by left femoral artery ligation of control mice (WT = C56Bl/6) and Flt-1 tyrosine kinase knockout mice (TKKO). Blood flow of the hindlimbs was assessed by scanning ischemic limbs and untreated control limbs with laser doppler. The plasma levels of concentration of VEGF, SCF and proMMP9 were assessed by using ELISA kits. In addition, white blood cells (WBC) count was measured by Celltact. Results: Blood flow recovery in TKKO was significantly delayed compared to WT (WT = 0.65 ± 0.09,TKKO = 0.39 ± 0.14 P < 0.05). WBC were significantly increased in WT at 1 day after ligation compare to TKKO (WT = 19000 ± 4305, TKKO = 10800 ± 4640, P < 0.05). Compared to WT, plasma concentration levels of SCF and pro-MMP9 were significantly reduced in TKKO (pro-MMP-9: WT = 48.59 ± 6.75, TKKO = 22.73 ± 4.55 P < 0.05. SCF: WT = 256 ± 25.35, TKKO = 142.4 ± 18.14 P < 0.05) . There was no difference in the concentration of the levels of VEGF in both mice. Conclusion: These results suggested that the signaling through the Flt-1 TK essential for recovering from acute ischemic condition. Administration of selective VEGFR1 agonist may open new therapeutic strategy in regenerative cardiovascular medicine. Keywords: Angiogenesis; VEGFR-1; Hindlimb ischemia doi:10.1016/j.yjmcc.2008.09.674