Simultaneous Optical Mapping of Intracellular Free Calcium and Action Potentials from Langendorff Perfused Hearts

Guy Salama1, Seong‐min Hwang1

1 University of Pittsburgh, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.17
DOI:  10.1002/0471142956.cy1217s49
Online Posting Date:  July, 2009
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The cardiac action potential (AP) controls the rise and fall of intracellular free Ca2+ (Cai), and thus the amplitude and kinetics of force generation. Besides excitation‐contraction coupling, the reverse process where Cai influences the AP through Cai‐dependent ionic currents has been implicated as the mechanism underlying QT alternans and cardiac arrhythmias in heart failure, ischemia/reperfusion, cardiac myopathy, myocardial infarction, congenital and drug‐induced long QT syndrome, and ventricular fibrillation. The development of dual optical mapping at high spatial and temporal resolution provides a powerful tool to investigate the role of Cai anomalies in eliciting cardiac arrhythmias. This unit describes experimental protocols to map APs and Cai transients from perfused hearts by labeling the heart with two fluorescent dyes, one to measure transmembrane potential (Vm), the other Cai transients. High spatial and temporal resolution is achieved by selecting Vm and Cai probes with the same excitation but different emission wavelengths, to avoid cross‐talk and mechanical components. Curr. Protoc. Cytom. 49:12.17.1‐12.17.32. © 2009 by John Wiley & Sons, Inc.

Keywords: action potential (AP); intracellular free Ca2+ (Cai); photodiode array (PDA); complementary metal oxide silicon (CMOS) camera; Voltage Sensitive Dye (VSD); Cai indicator; Rhod‐2AM; Pittsburgh I (PGH I); RH237

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Table of Contents

  • Introduction
  • Protocols
  • Selection of Vm and Cai Probes
  • Properties of RHOD‐2AM in Perfused Hearts
  • Dealing with Motion Artifacts
  • Kinetics of AP and Ca2+c Transients in Guinea Pig Hearts
  • Findings and Significance
  • Literature Cited
  • Figures
  • Tables
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