Differential sensitivity of Ca2+wave and Ca2+spark events to ruthenium red in isolated permeabilised rabbit cardiomyocytes

Spontaneous Ca2+ waves in cardiac muscle cells are thought to arise from the sequential firing of local Ca2+ sparks via a fire-diffuse-fire mechanism. This study compares the ability of the ryanodine receptor (RyR) blocker ruthenium red (RuR) to inhibit these two types of Ca2+ release in permeabilised rabbit ventricular cardiomyocytes. Perfusing with 600 nm Ca2+ (50 mu m EGTA) caused regular spontaneous Ca2+ waves that were imaged with the fluorescence from Fluo-5F using a laser-scanning confocal microscope. Addition of 4 mu m RuR caused complete inhibition of Ca2+ waves in 50% of cardiomyocytes by 2 min and in 100% by 4 min. Separate experiments used 350 mu m EGTA (600 nm Ca2+) to limit Ca2+ diffusion but allow the underlying Ca2+ sparks to be imaged. The time course of RuR-induced inhibition did not match that of waves. After 2 min of RuR, none of the characteristics of the Ca2+ sparks were altered, and after 4 min Ca2+ spark frequency was reduced similar to 40%; no sparks could be detected after 10 min. Measurements of Ca2+ within the SR lumen using Fluo-5N showed an increase in intra-SR Ca2+ during the initial 2-4 min of perfusion with RuR in both wave and spark conditions. Computational modelling suggests that the sensitivity of Ca2+ waves to RuR block depends on the number of RyRs per cluster. Therefore inhibition of Ca2+ waves without affecting Ca2+ sparks may be explained by block of small, non-spark producing clusters of RyRs that are important to the process of Ca2+ wave propagation.status: publishe