Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC147
Concurrent Optical Mapping of Voltage and Calcium in Rat Isolated Hearts using One Camera
C. Bollensdorff1,2, P. Lee2,3, L. Loew4, A. Quinn1,2, J. P. Wuskell4, P. Kohl1,2
1. DPAG, Oxford University, Oxford, United Kingdom. 2. Cardiac Biophysics and Systems Biology Group, National Heart and Lung Institute, Imperial College London, Harefield, United Kingdom. 3. Life Sciences Interface Doctoral Training Centre, University of Oxford, Oxford, United Kingdom. 4. Richard D. Berlin Center for Cell Analysis and Modeling, University of Connecticut, Farmington, Connecticut, United States.
Concurrent measurement of membrane voltage (Vm) and intracellular calcium ([Ca2+]i), key parameters underlying excitation-contraction coupling, is necessary for exploring the cross-talk between these two processes. The development of voltage- and ion-sensitive fluorescent dyes has enabled non-contact measurements, with increasing spatial and temporal resolution. The use of dual camera systems is the most commonly used approach to visualise Vm and [Ca2+]i. However, this approach is technically challenging and expensive. We have developed a single-camera system utilising frame-accurate light-emitting-diode (LED) activation to measure Vm and [Ca2+]i ratiometrically, using Di-4-ANBDQPQ (for Vm) and Fura-2 (for [Ca2+]i. High-speed coordination between the LEDs (ranging from UV to red) and a 128x128 EMCCD camera system (Cascade; Photometrics) is achieved via custom, micro-processor based control, which also allows high flexibility in LED excitation sequence. Emission fluorescence is passed through a multiband filter. As a proof-of-principle application, ratiometric Vm and [Ca2+]i signals were measured during electrical and mechanical stimulation at the same location on the left ventricle in rat hearts. Hearts (n=8) were excised after cervical dislocation (female SD, 10-12wk), according to Sch1 of the Home Office Animals (Scientific Procedures) Act of 1986, and Langendorff perfused with Krebs solution (in mM: NaCl 123, CaCl2 1.8, KCl 4, MgCl2 1.2, NaH2PO4 1.4, NaHCO3 24, Glucose 10). Fura-2 (10μM) was reperfused for 40 min after initial bolus injection, and Di-4-ANBDQPQ was applied by bolus injection (20µl of 27.3mM stock in EtOH, over 5 min to 25 ml of perfusate). The speed of action potential propagation and the time delay between the peaks of Vm and [Ca2+]i were compared for the two modes of stimulation. No significant difference were found in propagation speed of electrically and mechanically triggered excitations (0.65±0.04mm/ms vs 0.65±0.07mm/ms, respectively). Furthermore, there was no significant difference in the time-delay between peak Vm and [Ca2+]i (22.0±1.4ms vs 22.5±1.3ms for electrical and mechanical stimulation, respectively. We present a simple, versatile and scalable technique for concurrent ratiometric imaging of Vm and [Ca2+]i in the isolated rat heart. For proof-of-principle, we integrated this technology with ECG-timed local electrical or mechanical stimulation to study mechano-electric coupling at the organ level, and show that activation patterns, down-stream of the ectopic excitation, are not dissimilar regardless of the trigger.
Where applicable, experiments conform with Society ethical requirements