Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC252
Mechanical and electrical effects of superoxide donor menadione in rat myocardium are associated with increased diastolic intracellular Ca2+
A. Lake1, L. Rogers1, K. White1, M. Hardy1, Z. Yang1, E. White1
1. IMSB, University of Leeds, Leeds, United Kingdom.
Reactive oxygen species such as superoxide are involved in many cardiac diseases. Menadione is a superoxide donor that has varied effects upon the mechanical and electrical activity of cardiac muscle(Anderson & Dutta 1991; Choi et al, 2005). It has been suggested that its effects are associated with changes in intracellular Ca2+ but this has not been tested nor have simultaneous mechanical and electrical responses been recorded. The aim of this study was to test the hypothesis that the mechanical and electrical effects of menadione occur in the presence of dysfunctional intracellular Ca2+ regulation. Male Wistar rats 200-250g were killed, hearts removed and Langendorff perfused with a Krebs-Heinsleit solution at 37°C. Left ventricular pressure was monitored by an indwelling balloon connected to a pressure transducer and monophasic action potentials (MAPs) were recorded from the epicardial surface of the left ventricle. Hearts were alternately paced at 5Hz or allowed to intrinsically pace for 5 min periods before and during a 30 min exposure to 50 µM menadione (dissolved in 0.01% vol/vol methanol). Single left ventricular myocytes were loaded with the fluorescent Ca2+ indicator fura-4 AM and intracellular Ca2+ transients and cell length simultaneously measured in cells stimulated at 1Hz before and during exposure to menadione. Data are presented as means ± SEM. In paced preparations exposure to menadione caused a significant increase in diastolic pressure of 50 ± 9.9 mm Hg (P< 0.001); decrease in developed pressure of 59 ± 14.4 mm Hg (P < 0.001) and rate of relaxation by 886 ± 241 mm Hg. s-1 (P < 0.001). There was also a significant reduction in MAP duration at 80% repolarisation by 48 ± 7.8 % from an initial mean duration of 26.4 ± 2.3 ms (P < 0.001) (1-way RM ANOVA). When hearts were not paced, intrinsic heart rate fell significantly following exposure to menadione by 82 ± 21 beats. min-1 (P < 0.05, Student's paired t-test) and heart rate became less regular, as indexed by its S.D. (P < 0.05, Wilcoxon signed rank test) (N = 7 hearts). These effects did not reverse upon wash and were not observed in hearts exposed to vehicle alone (P > 0.05, N= 4 hearts). In single myocytes, menadione caused a significant increase in diastolic Ca2+ (P < 0.05) and a decrease in resting cell length (P < 0.001)(n = 5 cells, N = 3 animals, Student's paired t-test). Our observations are consistent with menadione causing dysregulation of Ca2+ handling possibly via effects on the sarcoplasmic reticulum Ca2+-ATPase(SERCA2) and intracellular Ca2+ overload which leads to an increase in diastolic Ca2+ and diastolic contracture together with a shortening of the action potential, possibly via Ca2+-induced inactivation of L-type Ca2+ channels.
Where applicable, experiments conform with Society ethical requirements