Simultaneous Optical Mapping of Transmembrane Potential and Intracellular Calcium in Myocyte Cultures

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Abstract
Simultaneous Mapping of Vm and Cai2+. Introduction: Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Cai2+) are important for studying mechanisms of arrhythmias and defibrillation. The goals of this work were (1) to develop an optical technique for simultaneous multisite optical recordings of Vm and Cai2+, and (2) to determine the relationship between Vm and Cai2+ during normal impulse propagation in myocyte cultures. Methods and Results: Monolayers of neonatal rat myocytes were stained with fluorescent dye RH‐237 (Vm) and Fluo‐3AM (Cai2+). Both dyes were excited at the same wavelength range. The emitted fluorescent was optically separated into components corresponding to changes in Vm, and Cai2+ and measured using two 16 × 16 photodiode arrays at a spatial resolution of up to 27.5 μm per diode and sampling rate of 2.5 kHz. The optical setup was adjusted so that there was no optical cross‐talk between the two types of measurements, which was validated in experiments involving staining with either RH‐237 or Fluo‐3. The amplitude of Fluo‐3 signals rapidly decreased during experiments due to dye leakage. Dye leakage was substantially reduced by application of 1 mM probenecid, a blocker of organic anion transport, which had no effect on action potential duration and only minor effect on conduction velocity. In double‐stained preparations, during regular pacing Cai2+ transients had a rise time of 14.2 ± 2 msec, and they followed Vm upstrokes with a delay of 5.3 ± 1 msec (n = 9). Durations of Vm, and Cai2+ transients determined at 50% level of signal recovery were 54.6 ± 10 msec and 136 ± 8 msec, respectively. Application of 2 μM nifedipine reduced the amplitude and duration of Cai2+ transients without significantly affecting conduction velocity. Conclusion: The results demonstrate feasibility of simultaneous optical recordings of Vm and Cai2+ transients with high spatial and temporal resolution.