Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C39
Blebbistatin - the excitation-contraction uncoupler significantly affects cardiac electrophysiological and calcium homeostasis
R. K. Narang1, K. E. Brack1, G. Ng1,2
1. Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom. 2. Biomedical Research Unit, Glenfield General Hospital, Leicester, United Kingdom.
Purpose: Movement artefacts pose challenge in optical mapping studies. Blebbistatin (BS) is a recently discovered inhibitor of myosin II isoform and has been adopted as the mechanical uncoupler of choice for optical mapping. Previous studies in cellular and whole heart preparations suggested that BS has no direct electrophysiological effects. Our aims were to 1) measure the effects of BS on monophasic action potential duration (MAPD), maximum slope of MAPD restitution (RT), ventricular effective refractory period (ERP), ventricular fibrillation threshold (VFT), Ca2+ transient duration (CaTD) and optically recorded action potentials and 2) to determine if BS would alter the effect of sympathetic nerve stimulation (SNS) on ERP and VFT. Methods: Aim 1: Langendorff perfused hearts (NZW rabbits, n=8) were obtained after pentobarbitone euthanasia (160mg/kg, i.v.). Left ventricular (LV) MAPD (90% decay) was measured during constant ventricular pacing (300ms CL). RT was obtained using a single extra-stimulus protocol with maximum slope measured using contact electrodes. VFT was measured as the minimum current required inducing sustained VF with burst pacing (30x30ms). LV epicardial Ca2+ transients were measured using Fura-2 AM fluorescence and CaTD measured at 90% decay. Optical action potentials (APs) were recorded using 5µM Di-4-ANEPPS. Aim 2: Measurements were taken in the absence and presence of SNS (4.8Hz, 4.9V, n=3) in the isolated innervated rabbit heart. Following pre-sedation (ketamine (10mg/kg), medetomidine hydrochloride (0.2mg/kg) and butorphanol (0.05mg/kg) i.m.), general anaesthesia was established with propofol (1%w/v ad libitum, i.v.) during which vessels were ligated and cervico-thoracic tissues isolated to give the innervated heart preparation (1). Animals were euthanised with pentobarbitone overdose (160mg/kg, i.v.). The resulting ex vivo preparation was perfused via the descending aorta with constant flow. All measurements were taken at baseline (BL) and after 60min perfusion of BS-5µM. Data are mean±SEM, *P<0.05 using t-test. Results: Aim 1: BS significantly prolonged MAPD (113.2±2.2 to 163.0±3.1ms, [Fig 1A]) and ERP (135.0±4.1 to 166.7±2.5ms) whilst increasing RT slope (0.60±0.08 to 1.45±0.27) and VFT (3.5±0.5 to 12.9±3.8mA). CaTD (Fig 1B) and optical APs (Fig 1C-D) were similarly prolonged. Aim 2: Although there was a trend for BS to alter the percentage change in SNS induced shortening of ERP or decrease in VFT (Figure 2A-B), this failed to reach significance. Conclusion: BS had significant effects on ventricular electrophysiology and Ca2+ homeostasis in the isolated heart. BS caused a non-significant reduction on the ventricular electrophysiological response to SNS. The use of BS should be treated with caution in optical mapping studies.
Where applicable, experiments conform with Society ethical requirements