Simultaneous optical mapping of transmembrane potential and intracellular calcium in myocyte cultures.
BACKGROUND
Fast spatially resolved measurements of transmembrane potential (Vm) and intracellular calcium (Ca(i)2+) 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 Ca(i)2+, and (2) to determine the relationship between Vm and Ca(i)2+ during normal impulse propagation in myocyte cultures.


RESULTS
Monolayers of neonatal rat myocytes were stained with fluorescent dye RH-237 (Vm) and Fluo-3AM (Ca(i)2+). Both dyes were excited at the same wavelength range. The emitted fluorescence was optically separated into components corresponding to changes in Vm and Ca(i)2+ and measured using two 16 x 16 photodiode arrays at a spatial resolution of up to 27.5 microm 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 Ca(i)2+ 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 Ca(i)2+ transients determined at 50% level of signal recovery were 54.6 +/- 10 msec and 136 +/- 8 msec, respectively. Application of 2 microM nifedipine reduced the amplitude and duration of Ca(i)2+ transients without significantly affecting conduction velocity.


CONCLUSIONS
The results demonstrate feasibility of simultaneous optical recordings of Vm and Ca(i)2+ transients with high spatial and temporal resolution.
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Department of Biomedical Engineering, University of Alabama at Birmingham, 35294, USA. fasst@crml.uab.edu
Ideker
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D000814:Aniline Compounds; D000818:Animals; D000831:Animals, Newborn; D016477:Artifacts; D018780:Body Surface Potential Mapping / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D006321:Heart / Q000502:physiology*; D007424:Intracellular Fluid / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009200:Myocardial Contraction; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011339:Probenecid / Q000494:pharmacology; D011726:Pyridinium Compounds; D051381:Rats; D017208:Rats, Wistar; D014528:Uricosuric Agents / Q000494:pharmacology; D014741:Video Recording; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D014528:Uricosuric Agents; D014966:Xanthenes; C059715:Fluo-3; C048153:4-(6-(4-dibutylaminophenyl)-1,3,5-hexatrienyl)-1-(4'-sulfobutyl)pyridinium; D011339:Probenecid; D002118:Calcium