Effects of superoxide donor menadione in adult Rat myocardium are associated with increased diastolic intracellular calcium

Luke J. Rogers 0 1 Andrew John Lake 0 1 Katherine White 0 1 Matthew Hardy 0 1 Ed White 0 1 0 Supervisor: Ed White, School of Biomedical Sciences, University of Leeds , Leeds LS2 9JT, UK 1 School of Biomedical Sciences, University of Leeds , Leeds LS2 9JT, UK Superoxide anions have been associated with many aspects of cardiovascular disease. Menadione is a superoxide anion donor that alters the heart's electrical and mechanical functions. The aim of this study was to demonstrate simultaneous changes in intracellular Ca2+ ([Ca2+]i) and mechanical activity in intact adult cardiac myocytes, and mechanical activity and electrical activity in isolated whole hearts in order to provide greater insight into the mechanisms associated with the detrimental effects of menadione on the myocardium. Isolated hearts from adult male Wistar rats (n = 11, 200-250 g) were Langendorff perfused at 38C with a Krebs-Henseleit solution. A saline-filled balloon was placed in the left ventricle (LV) in order to measure diastolic and developed pressure. Monophasic action potentials were simultaneously recorded from the epicardial surface. External stimulation at 5 Hz and intrinsic pacing were used throughout a 10 min control period and 30 min exposure to 50 M menadione. Single LV myocytes (n = 7 from n = 4 animals) were loaded with the Ca2+-indicator Fura4-AM, stimulated at 1 Hz and exposed to 50 M menadione. Myocyte length was simultaneously measured with [Ca 2+]i using a video edge detection system. In isolated hearts, exposure to menadione significantly decreased contractility and action potential duration (with a similar time course); intrinsic heart rate and rhythmicity. Diastolic pressure was significantly increased. In single adult myocytes, menadione caused a significant increase in diastolic [Ca2+]i and a decrease in resting cell length and led to spontaneous release of [Ca2+]i. We conclude that the effects of menadione upon electrical and mechanical activity of the heart are at least in part a consequence of dysregulation of [Ca2+]i handling and the subsequent increase in diastolic [Ca2+] alterations in [Ca2+]i are consistent with the generation of delayed after depolarization arrhythmias. Introduction Menadione is a potent superoxide donor (Choi et al., 2005) that has a negative inotropic effect on isolated hearts, which, it has been suggested, is linked to intracellular Ca2+ ([Ca2+]i) regulation (Anderson and Dutta, 1991). In addition, it has been demonstrated that menadione alters the electrical response of cardiac tissue preparations (Choi et al., 2005; Ha et al., 2007). However, the simultaneous measurement of the mechanical and electrical effects of menadione has not previously been reported, nor has its effects on [Ca2+] i transients in intact adult myocytes been measured. The objective was to test the hypothesis that the mechanical and electrical effects of the superoxide anion donor menadione can be explained by the presence of dysfunctional [Ca2+]i regulation. Materials and Methods Isolated whole hearts Adult male Wistar rats (n = 13, 200250 g) were killed by stunning and cervical dislocation in accordance with the UK Home Office regulations (Animal [Scientific Procedures] Act 1986). Hearts were isolated, weighed and Langendorff perfused (Stones et al., 2009) with a KrebsHenseleit (KH) solution at 38C at a constant flow rate of 7 ml min1 g1 heart weight. A saline-filled balloon, connected to a pressure transducer, was placed in the left ventricle (LV) via the dissected left atrium to measure diastolic and developed pressure (DP). The balloon was inflated (typically to a volume of 0.1 ml) until diastolic pressure began to register and transient systolic pressures were visible. Monophasic action potentials (MAPs) were simultaneously recorded from the epicardial surface of the LV (Benoist et al., 2011). Alternating, 5 min periods of external stimulation, delivered via platinum contact electrodes at a frequency of 5 Hz and intrinsic pacing (no external stimulation), were used throughout a 10 min control and 30 min exposure to 50-M menadione (Sigma, Aldrich) followed by a return to menadione-free solution. Alternatively, after 10 min control solution, hearts (n = 2) were exposed to glibenclamide (a blocker of ATP-modulated potassium current, IKATP) for 10 min followed by glibenclamide plus menadione for 30 min. Pressure values and rates of pressure development and MAP durations were measured with Lab Chart 7 software (ADInstruments, Australia). Single LV myocytes LV myocytes were isolated from n = 4 adult Wistar rat hearts as described previously by McCrossan, Billeter and White (2004). Myocytes selected for study were quiescent when not stimulated and had clear and regular striations. Myocytes were loaded with the Ca2+-indicator Fura4-AM (2 M for 20 min) stimulated at 1 Hz and exposed to 50 M menadione (n = 7). Cells were alternately excited by light at 340 and 380 nm (optoscan monochromator, Cairn Research, UK) and the ratio of emitted light at 510 nm was our index of [Ca2+]i. Myocyte length was simultaneously measured using a video edge detection system (Crescent Electronics, Sandy, UT, USA). [Ca2+]i transients and cell shortening were analysed with pClamp 9 (Axon Instruments). The KH solution contained (in mM) NaCl 118.5; NaHCO3 25.0; KCl 4.2; KH2PO4 1.2 mM; MgSO47H2O 1.2; glucose 11.1 and CaCl2 2.0. Menadione was dissolved in methanol to make a 50 mM stock solution which was added to KH solution to give a final concentration of 50 M menadione and 0.1% methanol. Glibenclamide, at a final concentration of 50 M, was dissolved in 0.1% methanol. Exposure to 0.1% methanol in KH solution had no statistically significant effects on isolated whole hearts or single myocytes. The effects of menadione were not reversible on removal of the agent. Statistical analysis Statistical significance was tested using one-way repeated measures ANOVA (RMANOVA) unless stated otherwise, P values < 0.05 were regarded as significant. Data are expressed as mean SEM. Isolated whole hearts Menadione caused a significant increase in diastolic pressure approximately 10 min after exposure compared to vehicle alone (Fig. 1). The end-diastolic pressure (EDP) rose from 19.7 12.84 mmHg, prior to exposure, to 50.7 9.9 mmHg, (P < 0.001) after 25 min. As a consequence of this, DP fell from 76.5 15.72 to 17.5 3.0 mmHg over the same time period (P < 0.001). The mean changes for EDP and DP are shown in Fig. 2A and B, respectively. Exposure to menadione for 25 min also significantly reduced the rate of peak pressure development (max dP/dT) (from 4037.0 446.2 to 2254.2 98.0 mmHg s1, P < 0.001) and the peak rate of relaxation (min dP/dT) (from 3126.6 259.0 to 2240.0 36.5 mmHg s1, P < 0.001). The mean changes of max and min dP/dT are shown in Fig. 2C and D, respectively. MAP durations at 20, 50 and 80% repolarization (APD20, APD50, APD80, respectively) were simultaneously recorded with LV pressure (Fig. 3 (...truncated)