Rhodamine 6G

An expanded Methods section is available in the Online Data Supplement. All procedures involving animals were approved by the Animal Care and Use Committee of the University of California, Davis and adhered to the NIH Guide for the Care and Use of Laboratory Animals. Male New Zealand White rabbits (n=29) were anesthetized with an intravenous injection of pentobarbital sodium (50mg/kg). Hearts were excised, Langendorff-perfused at 37°C and loaded with RH237 and Rhod-2AM (Molecular Probes, Eugene, OR) for simultaneous fluorescent imaging of V_m and intracellular Ca²⁺. An electrocardiogram (ECG) was continuously recorded and pacing was from the basal left ventricle (LV). Blebbistatin (Tocris Bioscience, Ellisville, MO; 10–20µM) was used to eliminate motion artifact during optical recordings.^{20 (link)} Fluorescent signals were recorded using two CMOS cameras (MiCam Ultima-L, SciMedia) with a sampling rate of 1kHz and 100×100 pixels with a 35x35mm field of view.
Subepicardial injections were delivered via 30G needles with a 90° bend 1.5mm from the tip. Injections of normal Tyrodes (NT, 50µL: control) and NE (50µL, low-dose [30–60µM] or high-dose [125–250µM]) were delivered at different anatomical locations (LV base/LV apex/RV base/RV apex) in 15 hearts. In a subset of hearts (n=8), co-injection of NE and the fluorophore rhodamine-6G (R6G, 50µM, 528/547nm ex/em, Sigma, St. Louis, MO) was performed to visualize the epicardial area and transmural depth of tissue exposed to NE. In another subset (n=8 hearts), 25µM carbenoxolone (CBX) was added to produce partial gap junction (GJ) uncoupling. A further 14 hearts were used to study the effect of the β-AR antagonist propranolol (5–10µM, n=3), low perfusate [Ca²⁺] (0.33mM, n=3), the SR Ca²⁺-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA, 30µM, n=2) and ablation of the RV endocardium with Lugol’s solution (n=3) on the occurrence of NE-induced PVCs. Local caffeine injections (10–40mM, 50µL, n=4) and global perfusion of isoproterenol (1µM, n=4) were also studied.
Data analysis was performed using two analysis programs (BV_Analyze, Brainvision, Japan; and Optiq, Cairn, UK). Activation time was determined as 50% rise time. For APs, repolarization time at 80% return to baseline was used to calculate action potential duration (APD₈₀). For CaTs, duration was measured at 50% (CaTD₅₀) and the time course of decay was quantified using the time constant (τ) of a single exponential fit of the decline (30–100%).^{21 (link)} V_m activation time was subtracted from Ca²⁺ activation time to produce maps of V_m-Ca²⁺ delay. Phase plots of the V_m/Ca²⁺ relationship were generated by plotting the normalized V_m values (x-axis) against the normalized Ca²⁺ values (y-axis) for the time course of a single AP, where counterclockwise chirality indicates normal V_m-Ca²⁺ coupling, and clockwise chirality indicates abnormal V_m-Ca²⁺ coupling.^{22 (link)} Conduction velocity (CV) was calculated as in Bayly et al.²³ Continuous variables are presented as mean±SD. Comparisons between two groups of continuous data were made using a Student’s t-test, paired where appropriate, and categorical data using a Fisher’s exact test. Multiple comparisons were made using one- or two-way analysis of variance (ANOVA) with Bonferroni’s post-testing. P<0.05 was considered statistically significant.