The role of dye affinity in optical measurements of Cai2+ transients in cardiac muscle

摘要

Previous experiments in cultures of neonatal rat myocytes demonstrated that the shape of Cai²⁺ transients measured using high-affinity Ca²⁺-sensitive dyes may be misrepresented. The purpose of this study was to examine the role of dye affinity in Cai²⁺ 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 Ca²⁺-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 Cai²⁺ were made using optical fibers (diameter = 325 μm) glued with the microcapillaries. The durations of Cai²⁺ 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, Cai²⁺ 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 Cai²⁺ 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 Cai²⁺ upstrokes were comparable for low- and high-affinity dyes (P > 0.05). In conclusion, measurements of Cai²⁺ transient in ventricular myocardium are strongly affected by the affinity of Ca²⁺ dyes. The high-affinity dyes may overestimate the duration and alter the shape of Ca_i²⁺ transients.