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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Abstract

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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Materials

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Figures

  •  FigureFigure 12.17.1 A dual optical mapping instrument based on two Hamamatsu photodiode arrays is designed with sealed optics to reduce noise from thermal convection currents in front of the arrays. The photodiode arrays (PDA 1 and PDA 2) and the first-stage amplifiers are housed in the beige boxes where PDA 1 serves to map Cai signals and PDA 2 maps Vm signals. The first dichroic box is located immediately behind the camera lens and splits the excitation beam coming from the lamp housing. The second dichroic box is located behind the first dichroic box and plots the fluorescence from the heart with the low-wavelength image focused on PDA 1 and the long-wavelength image focused on PDA 2.
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  •  FigureFigure 12.17.2 Schematics of optical apparatus with direct illumination. An alternative design for dual optical mapping was used for large fields of view where the light beam from tungsten-halogen lamps can be directly focused on the heart and fluorescence images from the heart are split with a dichroic element and refocused on the surface of two Hamamatsu PDAs. The dichroic element can be rotated and replaced with a mirror to focus an image of the heart on a graticule placed on a parafocal plane and with the exact dimensions of the surface of the PDA. The image of the heart that is focused on the graticule will be automatically focused on the surfaces of the two PDAs.
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  •  FigureFigure 12.17.3 Dual optical mapping apparatus based on two CMOS from SciMedia. A photograph of a dual mapping system based on two CMOS cameras has general features similar to those shown in Figure 12.17.1, except for the dimensions of the CMOS compared to the PDAs, which require changes in the lenses used to focus the excitation beam and to focus images of the heart on the smaller CMOS sensors (1 × 1 cm2) compared to the larger PDA sensing surface (1.8 × 1.8 cm2).
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  •  FigureFigure 12.17.4 Simultaneous recordings of Vm and Cai from CMOS cameras. With the apparatus shown in Figure 12.17.3, maps of Vm and Cai can be recorded at high spatial and temporal resolution. The left panel shows an image of the heart taken by one of the CMOS cameras with white light illumination. The heart is placed in a chamber designed to abate motion artifacts, and a silhouette of the CMOS sensing surface (square outline) is drawn on the heart to identify the region of the heart that is being mapped by the two CMOS cameras. Each CMOS camera recorded AP or Cai from 100 × 100 pixels at a frame rate of 2 kHz. Examples of simultaneous recordings of APs and Cai transients are shown from three locations on the heart (right traces).
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  •  FigureFigure 12.17.5 APs recorded with CMOS cameras from SciMedia and RedShirt. A guinea pig heart was labeled with PGH1-PEG750 (200 µl of 1 mM dye) and APs were mapped with the Ultima-L camera then the CardioCMOS with areas of 600 and 770 µm, respectively. Frame rate: 1000 frames/sec.
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  •  FigureFigure 12.17.6 Emission spectra of Rhod-2 and RH 237 in a guinea pig heart. A guinea pig heart was labeled with RH237 or Rhod-2 as previously described (Choi and Salama, 2000), and the fluorescence emission spectra of the dyes were recorded with a high-speed spectrograph. The considerable overlap between the two emission spectra required the careful selection of filters to avoid cross-talk between the two signals.
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  •  FigureFigure 12.17.7 Simultaneous Vm and Cai recordings from the heart of different species. Simultaneous recordings of Vm and Cai were obtained with PGH1 and Rhod-2 from guinea pig (A), rabbit (B), and mouse (C) hearts. The heart was excited at 540 nm, the emission for Rhod-2 was measured at 585 ± 30 nm, and the PGH I emission was measured above 700 nm. In rabbit hearts, Vm and Cai are shown at slow (a) and fast (b) sweep speeds, after the addition of an IKr blocker (E4031) (c), and PGH1 was measured at long excitation wavelengths (ex = 690 nm and >750 nm).
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  •  FigureFigure 12.17.8 Mouse Vm and Cai recordings from RH 237 and GCaMP2. A heart from a mouse that was genetically encoded with GCaMP2 was isolated, perfused in a Langendorff apparatus, and stained with RH 237. APs were recorded from RH 237 (ex = 540 nm and em > 630 nm) and Cai from GCaMP2 (ex = 480 ± 15 nm and em = 520 ± 150 nm).
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  •  FigureFigure 12.17.9 Kinetics of GCaMP2 versus Rhod-2. The kinetics of GCaMP2 Ca2+c transients (left traces) were slower than those recorded with Rhod-2 (right traces). The effect was negligible at long cycle lengths (top traces) and became increasingly more severe at short cycle lengths (middle and bottom traces) where the GCaMP2 signal fails to recover back to baseline.
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  •  FigureFigure 12.17.10 Chemical structure of Rhod-2.
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  •  FigureFigure 12.17.11 Subcellular distribution of Rhod-2 in guinea pig myocytes. Panels (A) and (B): confocal fluorescence images of a guinea pig ventricular myocytes bathed with TMRE (10 nM) in the absence (A) and presence (B) of 10 µM FCCP) (FCCP is an H+ ionophore which collapses —membrane potential—across the mitochondria). TMRE was rapidly accumulated in the mitochondria (10 to 15 min), and exhibited the typical punctate pattern associated with mitochondria aligned in myocytes below the surface membrane (Chacon et al., 1996). Panels (C) and (D) illustrate confocal fluorescence images of myocytes loaded with Rhod-2AM (5 µM) at 37°C, resulting in very different dye distribution compared to the punctate pattern of mitochondria shown in panel A. The subsequent addition of FCCP collapsed the mitochondrial potential but had no effect on Rhod-2 fluorescence (panel D). To test the possibility that Rhod-2 remained trapped in mitochondria, myocytes were loaded with Rhod-2AM (2 µM), then a low concentration of detergent was added to the bathing medium to allow cytosolic Rhod-2 to diffuse out of the cells without changing the mitochondrial membrane permeability. In panel (E), Rhod-2 loading produced what could be mistaken for the punctate appearance of mitochondrial stains, but when digitonin (20 µM) was then added to permeabilize the surface membrane (but not the mitochondrial membrane), Rhod-2 diffused out of the myocyte and there was no detectable level of Rhod-2 in the cell (panel F). Note that the cytosol and the mitochondria contained high levels of Ca2+ (2 µM), such that Rhod-2 trapped in the mitochondria and nondiffusible Rhod-2 in the cytosol would fluoresce strongly and would be readily detected in controls. The color-coded bar represents the relative fluorescence intensity.
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  •  FigureFigure 12.17.12 A cardiac chamber abates motion artifacts with no effect on left ventricular pressure. (A) A guinea pig heart placed in a chamber designed with front, rear and side branches to minimize motion. (B) Simultaneous left ventricular (LV) pressure and fluorescence measurements of motion artifacts (MA) obtained by loading the heart with fluorescein used as a control dye. In plot a, MA are clearly noticeable from the fluorescein control before tightening the chamber around the heart. Tightening the chamber produced a marked reduction of MA with no detectable change in LV pressure.
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  •  FigureFigure 12.17.13 Rise time of Ca2+c transients. In a Rhod-2AM-loaded guinea pig heart, the rise time of Ca2+c transients was measured by superimposing the second derivative of the Ca2+c upstroke and the trace; rise time was the time between the maximum and minimum of the second derivative.
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  •  FigureFigure 12.17.14 Kinetics of guinea pig Ca2+c transients. Ca2+c transients were measured from a guinea pig heart loaded with Rhod-2AM, placed in the chamber described in Figure 12.17.12, and perfused with cytochalasin-D to abate motion artifacts. The downstroke of Ca2+c transients exhibited complex biphasic recoveries to baseline and decreases in durations that were dependent on Ca2+ in the perfusate: (A), 1; (B), 0.75; (C), 0.5; and (D), 0.25 mM free Ca2+.
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  •  FigureFigure 12.17.15 Simultaneous Vm and Cai recordings from a Langendorff perfused rabbit heart treated with E4031 (0.5 µM) to induce LQT2. Vm and Cai measured during a normal cardiac beat (A) and during EADs (B) were normalized between 0 and 1 to plot Cai versus Vm and thereby generate phase maps. Phase trajectories were counterclockwise during a normal beat (control A) and clockwise during EADs (EADs B), indicating that during EADs, Cai elevation preceded the voltage depolarization.
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  •  FigureFigure 12.17.16 Vm (white) and Cai (red) were simultaneously recorded from a rabbit heart before (A) and after perfusion with the IKr blocker dofetilide (0.2 µM) for 2 min (B), 5 min (C), 7 min (D), and 10 min (E). The marked Cai instabilities in B and C clearly precede Vm instabilities and demonstrate a loss of voltage control.
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 Internet Resources
    http://www.dalsa.com/markets/ccd_vs_cmos.asp

Discussion of CCD versus CMOS on Dalsa Corporation Web site.

    http://www.olympusmicro.com/primer/techniques/fluorescence/fluorosources.html

Abramowitz, M. and Davidson, M.W. Microscopy Primer: Light Sources.

    http://repairfaq.ece.drexel.edu/sam/laserdio.htm

Goldwasser, S.M. 2008. Sam's Laser FAQ: Diode lasers.

    http://www.lumileds.com/pdfs/DS34.pdf

Philips/Lumileds. 2004. LUXEON V LED Emitter Datasheet.

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