Abstract 189: Simultaneous Imaging of the Single-Myocyte Optical Action Potentials and Intracellular Ca2+ Dynamics in Langendorff-Perfused Rat Heart by a Novel Dual-Imaging Confocal Microscopy
Rapid-scanning confocal imaging of intracellular Ca2+ ([Ca2+]i) dynamics in the heart has revealed various abnormal [Ca2+]i dynamics that indicate arrhythmogenic potential. For example, Ca2+ waves under [Ca2+]i overload and beat-to-beat alteration of Ca2+ transients may lead to triggered arrhythmia and inhomogeneous excitation and conduction, respectively. However, it is unknown whether such abnormal [Ca2+]i dynamics accompany altered electrical activities in the individual myocytes. To elucidate the link between [Ca2+]i dynamics and the membrane potentials (Vm) of the myocytes in the heart we developed a novel dual-imaging system for both the [Ca2+]i dynamics and optical Vm at cellular levels. Optical signals for [Ca2+]i and Vm were obtained using in situ confocal microscopy (x40 objective lens, 2.2 ms/frame, 96x64 pixels) after loading the rat heart with fluo3/AM and RH237, respectively. The subepicardial myocardium of the heart was excited by Argon/Krypton (488/568nm) laser. The dual, non-overlapping signals for fluo3 and RH237, obtained by splitting the emitted light with a dichroic filter (565nm) and the respective emission filters of 535/40 nm and 774/50nm, were detected by a high-speed CCD camera (14 bits). Following results were obtained from 17 hearts.
During sinus rhythm the myocytes exhibited spatially uniform Ca2+ transients with simultaneous action potentials.
Under perfusion with Ca2+-free solution Ca2+ transients were diminished while the action potentials remained.
The durations of these two parameters were stimulation-frequency dependent: the higher the frequency (up to 5Hz), the shorter the durations.
At a stimulation frequency of 4Hz or higher Ca2+ transients often showed beat-to-beat alternans while no discernible alternans was obtained in action potentials.
Under [Ca2+]i overload, cessation of high-frequency pacing at 5 Hz resulted in synchronous emergence of Ca2+ waves among individual cells which accompanied fluctuations of the optical Vm.
In summary, we succeed in acquiring [Ca2+]i dynamics Vm and optical of individual myocytes simultaneously in the whole heart. This novel approach would provide important information to understand the mechanism of Ca2+-mediated arrhythmias.