Fibroblast growth factor-2 stimulates mitochondrial resistance to injury and phosphorylation of mitochondrial Connexin-43

ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 711–825

Pro-hypertrophic neurotransmitters, such as norepinephrine, are catabolized by monoamine oxidases (MAO) leading to hydrogen peroxide formation. Levels of both catecholamines and pro-oxidant species are increased in heart failure (HF). Yet, it is unknown whether and how MAO trigger maladaptive hypertrophy and contribute to HF. Here, we first demonstrated that stimulation of MAO-A activity can trigger pro-hypertrophic response in vitro. MAO-A substrate tyramine (10 μM, 24 h) led to a 2-fold increase in NFAT3 and 4, transcription factors involved in maladaptive hypertrophy. This was paralleled by excess ROS formation in mitochondria. These events were blunted or fully prevented by clorgyline (CLO, 1 μM), a specific MAO-A inhibitor. To test MAO-A role in in vivo HF, C57Bl6 mice were subjected to transverse aortic constriction (TAC). TAC mice received saline (control, n = 6) or clorgyline (1 mg/kg/day, n= 6). After 6 weeks, control mice displayed a 3.5-fold rise in MAO-A gene expression (pb 0.05) and poor inotropy. In contrast, CLO treated hearts showed well preserved pump function: fractional shortening was 61±0.8% vs 30±5.4 and ejection fraction 93.6±0.4% vs 60.2±8.2 (both pb 0.005). Furthermore, CLO mice showed reduced hypertrophy by echo: LV mass, LVEDD and LVESD were all markedly reduced (pb 0.005). Oxidative stress was also significantly reduced in CLO hearts. Fetal gene reprogramming (i.e. ANP expression) was 4-fold less in CLO mice. Cleaved caspase-3 and activated Akt levels were lower, indicating blunted pro-apoptotic/hypertrophic signaling. Thus, MAO-A activity triggers maladaptive pro-hypertrophy through increased ROS signaling. Its inhibition prevents cardiac decompensation in vivo. Keywords: Heart failure; Oxidative stress; LV function doi:10.1016/j.yjmcc.2008.02.185

Abstract No. 185 Overexpression of adiponectin receptors potentiates anti-inflammatory action of globular adiponectin in endothelial cells Peng Zhang, Ying Wang, Yanbo Fan, Zhihui Tang, Nanping Wang ⁎. Institute of Cardiovascular Science, Peking University, Beijing, China ⁎ Corresponding author. Institute of Cardiovascular Science, Peking University Health Science Centre, Beijing, 100083, China. Tel.: +86 1082801146; fax: +86 1082802769. E-mail address: [email protected] Objective: A decreased plasma level of adiponectin is associated with obesity and metabolic syndrome and correlated with endothelial dysfunction. This study aimed to investigate the regulated expression of the newly identified adiponectin receptors (AdipoR1 and 2) and their roles in the endothelial expression of intercellular adhesion molecule-1 (ICAM-1) in response to tumor necrosis factor-α (TNF-α). Methods and results: Immunohistochemical study and quantitative RT-PCR demonstrated that globular adiponectin

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suppressed the TNF-α-induced ICAM-1 expression in a dose-dependent manner in mouse aorta and human umbilical vein endothelial cells (HUVECs). Adenovirus-mediated overexpression of AdipoR1 and 2 in ECs signficantly enhanced the suppressive effect of a subeffective dose of adiponectin on TNF-α-induced ICAM-1 expression and NF-κB activation. Promoter reporter assays and small interfering RNA revealed that peroxisome proliferator-activated receptor-α may function as an important pathway downstream of adiponectin and its receptors. Furthermore, overexpression of AdipoRs in mouse carotid arteries markedly decreased the induction of ICAM-1 in vivo. Conclusions: We provide novel evidence that up-regulation of AdipoRs in ECs potentiates the anti-inflammatory effect of adiponectin; modulating adiponectin receptors may have potential therapeutic applications for cardiovascular complications associated with metabolic syndrome and diabetes. Keywords: Adiponectin receptors; Endothelium; Atherosclerosis doi:10.1016/j.yjmcc.2008.02.186

Abstract No. 186 Fibroblast growth factor-2 stimulates mitochondrial resistance to injury and phosphorylation of mitochondrial Connexin-43 Wattamon Srisakuldee, Zhanna Makazan, Barbara E. Nickel, James A.\ Thliveris, Elissavet Kardami⁎. Institute of Cardiovascular Sciences, St. Boniface Research Centre, 361 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6. Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada. Department of Physiol, University of Manitoba, Winnipeg, Canada ⁎ Corresponding author. Tel.: +1 204 235 3519; fax: +1 204 233 6723. E-mail address: [email protected] We have demonstrated that hearts treated with fibroblast growth factor-2 (FGF-2) display protein kinase C (PKC)-mediated and preconditioning-like resistance to injury, and that FGF-2-treated hearts exhibit hyperphosphorylation of the membrane channel protein Connexin-43 (Cx43) at intercalated disks. Since Cx43 is also present in mitochondrial inner membranes we examined the effect of FGF-2 administration on: the phosphorylation of cardiac mit-Cx43 at specific PKC target sites; mitochondrial resistance to mit. permeability transition pore (MPT) opening. Isolated rat hearts were perfused with oxygenated Krebs, supplemented or not with FGF-2. Cardiac sub-sarcolemmal mitochondria were isolated using differential centrifugation and Percoll gradient fractionation. Purity was ascertained by electron microscopy and Western-blot-based analysis for markers of several subcellular sites. Respiratory function was within normal range in mitochondria from FGF-2-treated and untreated hearts. Mitochondrial light scattering measurements were used as

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ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 711–825

an estimate of swelling (MPT opening) in response to increased calcium: mitochondria from FGF-2-treated hearts had increased resistance to swelling compared to those from untreated hearts. This was associated with a potent increase in Cx43 phosphorylation at PKC sites serines 262 and 368 in mitochondria from FGF2-treated hearts. We conclude that increased mitochondrial resistance to MPT opening contributes to FGF-2 cardioprotection, and that mit.Cx43, through its hyper-phosphorylation at PKC sites, acts as a sensor of protective signalling, and thus is a possible contributor to an injury-resistant state. Keywords: Mitochondrial swelling; Cardioprotection; Connexin 43 phosphorylation doi:10.1016/j.yjmcc.2008.02.187

Abstract No. 187 Overexpression of FGF-2 increases cardiac viability after injury assessed by Tissue Doppler imaging Sarah K. Jimenez ⁎, Davinder S. Jassal, Tielan Fang, Elissavet Kardami, Peter A. Cattini. University of Manitoba, Winnipeg, MB, Canada. St. Boniface Hospital Research Centre, Winnipeg, MB, Canada ⁎ Corresponding author. Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, MB, Canada R3E 3J7. Tel.: +1 204 789 3503; fax: +1 204 789 3934. E-mail address: [email protected] Fibroblast growth factor-2 (FGF-2) is implicated in normal growth and development as well as response to injury in many tissues, including the heart. We showed previously that FGF-2 is cardioprotective when provided acutely to cardiomyocytes, to ex vivo perfused hearts, and in vivo. However, FGF-2 is normally produced and released in the heart, suggesting that a chronic increase in endogenous FGF-2 levels will result in injury-resistant hearts. In agreement, isolated hearts from FGF-2-overexpressing transgenic (TG) mice displayed increased cardiomyocyte survival after global ischemia and reperfusion. Echocardiography has now been used to assess cardioprotection in an isoproterenol model of injury in FGF-2 TG versus non-TG mice in vivo. Mice were assessed at 24 h as well as 2 and 4 weeks after isoproterenol administration. A significant decrease in Tissue Doppler parameters was observed in both TG and non-TG mice when compared to baseline, but non-TG mouse hearts had significantly reduced left ventricular endocardial velocity when compared to the TG mice, predicting an improved outcome for the FGF-2-overexpressing mice. An assessment of cellular infiltration in heart sections stained with haematoxylin and eosin revealed a significant ∼ 6-fold increase in the size of these regions in TG mice when compared to their non-TG counterparts at 24 h, which persisted to 6 weeks post-injury. The presence of cells positive for c-kit, a marker of certain progenitor cells, was confirmed in the infiltrate. Thus, in spite of the increased region of infiltration, it is possible that these cells may

contribute to the beneficial effect observed in hearts up to 4 weeks post-injury by Tissue Doppler imaging in vivo. Keywords: Cardioprotection; Growth factors; Echocardiography doi:10.1016/j.yjmcc.2008.02.188

Abstract No. 188 Cardioprotective effects of pharmacologic preconditioning by natural honey against ischemia/reperfusion injury Moslem Najafi ⁎, Tahereh Eteraf Oskouei, Farzaneh Rafie, Elnaz Mahdizadeh-Aghdam. School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Islamic Republic of Iran ⁎ Corresponding author. Department of Pharmacology, School of Pharmacy, Tabriz University of Medical Sciences, 51666, Tabriz, Islamic Republic of Iran. Tel.: +98 411 3341315; fax: +98 411 3344798. E-mail address: [email protected] Effects of natural honey as a pharmacologic preconditioning agent on ischemia/reperfusion (I/R) induced arrhythmias and infarct size were investigated in isolated rat heart. The hearts were subjected to 30 min regional ischemia followed by 120 min reperfusion. In control group, the hearts were perfused by a modified Krebs–Henseleit solution, however, in the test groups they were perfused by enriched Krebs solution with honey (0.25, 0.5 and 1%) 10 min before to 10 min after ischemia. The arrhythmias were analyzed based on the Lambeth conventions. The infarct size was determined by TTZ and planimetry methods. At the ischemia, the number of ventricular ectopic beats (VEBs) in the control group was 667 ± 116 while perfusion of honey (0.25, 0.5 and 1%) reduced it to 128 ± 35 ( p b 0.01), 161 ± 35 ( p b 0.01) and 303 ± 94 ( p b 0.05), respectively. The time spent in ischemic ventricular tachycardia (VT) was markedly lowered by the same concentrations. In addition, honey (0.25 and 0.5%) decreased the incidence of VT from 100% (control) to 13% ( p b 0.001) and 25% ( p b 0.01), respectively. During reperfusion, the number of VEBs and VT were significantly reduced by all used concentrations. Honey (0.25%) significantly decreased the incidence and time spent for reversible ventricular fibrillation. Moreover, perfusion of honey (0.25, 0.5 and 1%) reduced infarct size from 46.3 ± 2.9% (control value) to 3.3 ± 1.3, 9.2 ± 1.9 and 11.7 ± 2.2%, respectively ( p b 0.001 for all). The results showed antiarrhythmic and anti-infarct properties of natural honey as a preconditioning agent. Antioxidant activity of honey, scavenging of free radicals and presence of energy sources such as glucose might be involved in these protective effects. Keywords: Natural honey; Ischemia/reperfusion; Arrhythmias doi:10.1016/j.yjmcc.2008.02.189