title: The role of dye affinity in optical measurements of Cai(2+) transients in cardiac muscle.
abstract: Previous experiments in cultures of neonatal rat myocytes demonstrated that the shape of Cai(2+) transients measured using high-affinity Ca(2+)-sensitive dyes may be misrepresented. The purpose of this study was to examine the role of dye affinity in Cai(2+) measurements in intact adult cardiac tissue by comparing optical recordings obtained with high- and low-affinity dyes. Experiments were carried out in porcine left ventricular (LV) wedge preparations stained locally by intramural injection via microcapillaries (diameter = 150 μm) with a low-affinity Ca(2+)-sensitive dye Fluo-4FF or Fluo-2LA (nominal Kd, ~7-10 μmol/l), high-affinity dye Rhod-2 (Kd = 0.57 μmol/l), and Fluo-4 or Fluo-2MA (Kd, ~0.4 μmol/l); in addition, tissue was stained with transmembrane potential (Vm)-sensitive dye RH-237. Optical recordings of Vm and Cai(2+) were made using optical fibers (diameter = 325 μm) glued with the microcapillaries. The durations of Cai(2+) transients measured at 50% level of recovery (CaD50) using high-affinity Fluo-4/Fluo-2MA dyes were up to ~81% longer than those measured with low-affinity Fluo-4FF/Fluo-2LA at long pacing cycle lengths (CL). In Fluo-4/Fluo-2MA measurements at long CLs, Cai(2+) transients often (~50% of cases) exhibited slow upstroke rise and extended plateau. In Rhod-2 measurements, CaD50 was moderately longer (up to ~35%) than in Fluo-4FF recordings, but Cai(2+) transient shapes were similar. In all series of measurements, mean action potential duration values were not significantly different (P > 0.05). The delays between Vm and Cai(2+) upstrokes were comparable for low- and high-affinity dyes (P > 0.05). In conclusion, measurements of Cai(2+) transient in ventricular myocardium are strongly affected by the affinity of Ca(2+) dyes. The high-affinity dyes may overestimate the duration and alter the shape of Cai(2+) transients.
authors.affiliation: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama.
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.affiliation: Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama vfast@uab.edu.
authors.forename: Vladimir G
authors.initials: VG
authors.lastname: Fast
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006352:Heart Ventricles / Q000378:metabolism*; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000502:physiology*; D011726:Pyridinium Compounds / Q000493:pharmacokinetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; C048153:4-(6-(4-dibutylaminophenyl)-1,3,5-hexatrienyl)-1-(4'-sulfobutyl)pyridinium; D002118:Calcium
keywords: dye affinity; intracellular calcium transient; optical fiber; optical mapping