Optical Mapping

Hearts (8 solutions × 2 mouse types × 5/7 replicates, N = 96) were perfused with the voltage-sensitive dye, di-4-ANEPPS at a concentration of 4 μM, and excess dye was washed out. Each heart was serially perfused with four solutions of either increasing or decreasing calcium concentration. Hearts were perfused with each solution for ∼8 min before being optically mapped. Motion was reduced by 2,3-butanedionemonoxime (10 mM), and the heart was stabilized against the front glass of the bath by applying slight pressure to the posterior surface. Hearts were paced using a unipolar silver wire placed on the anterior surface and a reference wire at the back of the bath. Stimuli of 1-V amplitude and 1-ms duration at a basic cycle length of 150 ms were applied. CV longitudinal (CV_L) and transverse (CV_T), anisotropic ratio (AR), and action potential duration (APD) were quantified as previously described (12). Briefly, the heart was excited by 510-nm light, and the excited light passed through a 610-nm filter and then was recorded using the MiCam Ultima CMOS L-camera at a sampling rate of 1,000 frames/s. The maximum rate of rise of the optical action potentials was assigned as activation times and CV vectors were determined by fitting the activation times at every pixel to a parabolic surface. Vectors up to 5 pixels away from a user-defined line indicating the direction of propagation, which fell within ∼3 or 5 mm (CV_T and CV_L, respectively) from the pacing site, not including the first 2 rows, and whose direction was not more than 7.5° from the direction of propagation, were included in the analysis. This region is roughly illustrated by the orange and green boxes in the isochrones maps (see Figs. 2 and ​and4).4). APD was calculated as the time interval between activation and 90% repolarization.

Extracellular calcium modulates conduction velocity during normonatremia. A: representative activation maps obtained by optically mapping a single heart during the serial perfusion of solutions with increasing [Ca²⁺]_o. Orange boxes to the top and bottom of the pacing site indicate the regions from which CV_T was averaged, and green boxes to the left and right indicate regions from which CV_L was averaged. B: average CV_T, CV_L, and AR (anisotropic ratio) from WT (black line) and HZ (gray line) hearts. C: percent change in CV_T between [Ca²⁺]_o = 1–1.8 mM and 1–3.4 mM in WT and HZ hearts demonstrates increased sensitivity of HZ hearts to W_P in the former and not the latter range. Values are means ± SD; n = 5. P < 0.05 relative to [Ca²⁺]_o = 1 mM in *WT and #HZ by paired Student's t-test.

Extracellular calcium modulates conduction velocity during hyponatremia. A: representative activation maps obtained by optically mapping a single heart during the serial perfusion of solutions with increasing [Ca²⁺]_o during hyponatremia. The orange box to the top of the pacing site indicates the region from which CV_T was averaged, and the green box to the left indicates the region from which CV_L was averaged in hearts with CV block over the left ventricle. B: average CV_T, CV_L, and AR (anisotropic ratio) from WT (black line) and HZ hearts (gray line). C: percent change in CV_T between [Ca²⁺]_o = 1 and 1.8 mM in WT and HZ hearts. Values are means ± SD; n = 5. P < 0.05 relative to [Ca²⁺]_o = 1 mM in *WT and #HZ by paired Student's t-test.