Rho-kinase mediates reoxygenation-induced cardiomyocyte death and promotes mitochondrial transition pore opening

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

Abstract No. 189 Effects of hydroalcoholic extract of Cynodon dactylon (L.) pers. on ischemia/reperfusion-induced arrhythmias Moslem Najafi ⁎, Hossein Nazemiyeh, Alireza Garjani, Afshin Gharakhani, Hamed Ghavimi. School of Pharmacy, Tabriz University Med 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] Probable antiarrhythmic effects of Cynodon dactylon (L.) pers. (C. dactylon) against ischemia/reperfusion (I/R)-induced arrhythmias were investigated in isolated rat heart. The hearts were subjected to 30 min regional ischemia followed by 30 min reperfusion and perfused with rhizome hydroalcoholic extract of C. dactylon (50, 100 and 200 µg/ml). During ischemia, the extract (50 µg/ml) produced marked ( p b 0.01) reduction in the number, duration and incidence of ventricular tachycardia (VT). The total number of ischemic ventricular ectopic beats (VEBs) was lowered by 50– 100 µg/ml ( p b 0.001 and p b 0.05, respectively). At the reperfusion phase, C. dactylon (50 µg/ml) decreased incidence of VT from 100% (control) to 33% ( p b 0.05). The duration and number of VT and total VF incidence were reduced by the same concentration too ( p b 0.05 for all). Perfusion of the extract (50–100 µg/ml) was markedly lowered reversible VF duration from 218 ± 99 s to 0 s and 10 ± 5 s ( p b 0.01 and p b 0.05, respectively). Moreover, C. dactylon (50 µg/ml) decreased the number of total VEBs from 349 ± 73 to 66 ± 26 ( p b 0.01). The results showed protective effects of C. dactylon against I/R-induced arrhythmias in isolated rat hearts. Maybe, the improvement of hemodynamic factors such as heart contractility, left ventricular developed pressure (LVDP) and rate pressure product (RPP) and the presence of steroidal saponins and flavonoid glycosides in the extract have important roles in antiarrhythmic activity of C. dactylon. Keywords: Cynodon dactylon (L.) pers.; Ischemia/reperfusion; Arrhythmias doi:10.1016/j.yjmcc.2008.02.190

Abstract No. 190 p38(beta)-MAPK mediated cardioprotection by carbon monoxide (CO) in the isolated mouse heart James Clark ⁎, Negin Sarafraz, Michael Marber. Cardiovascular Division, Kings College London, United Kingdom ⁎ Corresponding author. The Rayne Institute, St Thomas' Hospital, SE1 7EH, London, United Kingdom. Tel.: +44 020 71881966; fax: +44 020 71881670. E-mail address: [email protected]

791

CO is regarded as a versatile signalling molecule, having essential regulatory roles in a variety of physiological conditions including inflammation, apoptosis and wound healing and it is accepted that CO can mediate cardioprotection. It is believed that different p38 isoforms (α, β, γ or Δ) play distinct roles in the heart and in the present study, we report that CO-mediated cardioprotection is dependent upon p38(beta) mitogen-activated protein kinase (MAPK). In the presence of a CO-releasing agent, CORM-3 (0– 100 µmol/l), perfused hearts and isolated adult mouse myocytes show pronounced concentration-dependent up-regulation of p38-MAPK activity and its downstream signalling (HSP27). p38-MAPK dual phosphorylation was not dependent upon the upstream kinase kinase MKK3, and was unaltered by coadministration of the p38 inhibitor SB203580 (1 µmol/l). Isolated wild type (p38(beta)+/+) hearts exposed to CORM-3 prior to 30 min global ischaemia showed a marked, and significant, reduction in infarct size after 120 min reperfusion. Equally, contractile dysfunction during reperfusion was prevented in hearts treated with CORM-3 prior to ischaemia; CORM-3 in the absence of ischaemia has no effect. The cardioprotective effects mediated by CORM-3 in this model were partially abolished by SB203580 (1 µmol/l) and completely abrogated in p38(beta)−/− mice. These novel findings suggest that CO can elicit its cardioprotective effects by selective activation of p38(beta)-MAPK through an as yet unidentified MKK3-independent mechanism. Keywords: p38 MAPK; Infarct; Cardioprotection doi:10.1016/j.yjmcc.2008.02.191

Abstract No. 191 Rho-kinase mediates reoxygenation-induced cardiomyocyte death and promotes mitochondrial transition pore opening Shabaz A. Hamid a, Sean M. Davidson b, Dwaine S. Burley a, Derek M. Yellon b, Gary F. Baxter a,⁎. a Cardiff University, United Kingdom. b Univerity College London, United Kingdom ⁎ Corresponding author. Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3NB, United Kingdom. Tel.: +44 29 2087 6309. E-mail address: [email protected]

We reported recently that Rho-kinase (ROCK) inhibition during reperfusion limits experimental infarct size [1]. The aims of the present study were to examine the role of the PI3K/Akt/ NO salvage pathway in the protective action of ROCK inhibition; and the effect of ROCK inhibition on mitochondrial permeability transition pore (mPTP) opening, a critical event in mediating lethal reperfusion injury. Adult rat cardiomyocytes were cultured and subjected to 60 min hypoxia (pO2 b 1 kPa) followed by 60 min reoxygenation. Cell death was assessed using propidium iodide (PI) staining. Under

792

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

control conditions 34 ±2% of cells were PI +ve and this index was reduced to 15± 3% in cells reoxygenated in the presence of the selective ROCK inhibitor Y27632 5 μM (P b 0.01). The addition of the NO synthase inhibitor L-NAME (100 μM) or the PI3K inhibitor wortmannin (10 nM) at reoxygenation abolished the protective effect of Y27632 (PI +ve cells 29± 1% and 29 ± 2% respectively, P is not significantly different to control). In separate experiments, the effect of Y27632 on mPTP opening was studied in rhodamine ester-loaded cardiomyocytes. Y27632 5 μM increased the time to depolarisation (an index of mPTP opening) from 232 ± 30 s to 383 ±52 s (P b 0.01). This effect on delayed mPTP opening was abolished by wortmannin or L-NAME. Together, these data provide further evidence for a specific protective action of ROCK inhibition against reoxygenationinduced cell death. This beneficial effect of ROCK inhibition is mediated by promotion of the PI3K/Akt/NO pathway and subsequent inhibition of MPTP opening.

also a nucleolar localization signal. Inhibition of nuclear translocation prevented increased protein synthesis in response to phenylephrine treatment and decreased total MLP expression. These data suggest that MLP can both regulate its own expression and also myocyte hypertrophy through nuclear translocation. Furthermore, when myocytes were cyclically stretched for 48 h in the absence of nuclear MLP, there was a diminution of the usual enhancement in BNP expression and the sarcomeres lost their integrity. Collectively, these findings suggest that MLP is required for transcriptional regulation and hypertrophic remodelling of the myocytes in response to stretch or other stimulatory agonists. Supported by the following grants: HL62426, HL077995 and AHA 0630307N. Keywords: Mechanosensing; Nucleus; Hypertrophy doi:10.1016/j.yjmcc.2008.02.193

Keywords: Rho kinase; Reperfusion injury; Mitochondrial permeability transition pore

Reference Abstract No. 193 [1] Hamid SA, et al. Am J Physiol 2007;292:H2598–606. doi:10.1016/j.yjmcc.2008.02.192

Abstract No. 192 Nucleocytoplasmic shuttling of muscle LIM protein is required for myocyte remodelling and adaptation to hypertrophic stimuli Samuel Y. Boateng b,⁎, Samuel E. Senyo a , Lixin Qi a, Brenda Russell a. a University of Illinois at Chicago, USA. b University of Reading, United Kingdom ⁎ Corresponding author. School of Biological Sciences, University of Reading, Whiteknights, Reading, RG6 6AH, United Kingdom. Tel.: +44 118 3787041. E-mail address: [email protected] Muscle LIM protein (MLP) is a mechanosensor in the z-disk and a nucleocytoplasmic shuttling protein in cardiac myocytes. In this study, we examined MLP function and subcellular distribution in cultured rat neonatal myocytes. In myocytes treated with verapamil, nuclear MLP decreased while phenylephrine increased it, suggesting that calcium and/or contractility play a role in the subcellular localization of the protein. When RNA polymerase II was inhibited with α-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerases I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative NLS of MLP, we inhibited the nuclear import of the protein in cultured rat neonatal myocytes. We show that the NLS of MLP is

Activation of STAT3 within the embryonic heart in response to anoxia–reoxygenation Sarah Pedretti ⁎, Stéphany Gardier, Eric Raddatz. Department of Physiology, Faculty of Biology and Medicine, University of Lausanne, 7 rue du Bugnon, CH-1005, Lausanne, Switzerland ⁎ Corresponding author. Tel.: +41 21692 5582; fax: +41 21692 5595. E-mail address: [email protected] The Signal Transducer and Activator of Transcription 3 (STAT3) is required for early cardiogenesis, involved in protection of adult cardiomyocytes against ischemia–reperfusion and activated by oxidative stress. Whether STAT3 is involved in the response of the embryonic/fetal heart to transient oxygen deprivation remains unknown. We assessed the level of STAT3 phosphorylation in atria (A), ventricle (V) and outflow tract (OT) during anoxia–reoxygenation and also after an exogenous oxidant stress. Total and phosphorylated forms of STAT3 were determined by Western immunoblotting in A, Vand OT of hearts isolated from 4-day-old chick embryos under basal conditions, during anoxia (30 min)–reoxygenation (60 min) and after exposure to H2O2 (1 mM for 1 h). Level of STA3 phosphorylation was normalized by the total form. Beating rate of A, V and OT was checked through experiments. Under basal conditions, the content of total STAT3 corrected for myocardial protein was 25% higher in OT than in A and V, whereas the level of STAT3 phosphorylation was the highest in A (twofold relative to V and OT). In V, STAT3 phosphorylation was unchanged during anoxia but increased threefold relative to preanoxic level upon reoxygenation, peaking fourfold after 60 min. Exposure to H2O2 increased STAT3 phosphorylation in V (twofold) but had no significant effect in A and OT. After exposure to H2O2, atrial activity persisted in 50% of the hearts (n = 93) whereas atrioventricular propagation was preserved in 25% only. These findings indicate that in the developing heart, STAT3 is