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 μm EGTA) caused regular spontaneous Ca²(+) waves that were imaged with the fluorescence from Fluo-5F using a laser-scanning confocal microscope. Addition of 4 μm RuR caused complete inhibition of Ca²(+) waves in 50% of cardiomyocytes by 2 min and in 100% by 4 min. Separate experiments used 350 μm EGTA (600 nm Ca²(+)) to limit Ca²(+) diffusion but allow the underlying Ca(2+) 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 ∼40%; no sparks could be detected after 10 min. Measurements of Ca(2+) 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.