Differential sensitivity of Ca 2+ wave and Ca 2+ spark events to ruthenium red in isolated permeabilised rabbit cardiomyocytes

Spontaneous Ca 2+ waves in cardiac muscle cells are thought to arise from the sequential firing of local Ca 2+ 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 Ca 2+ release in permeabilised rabbit ventricular cardiomyocytes. Perfusing with 600 n m Ca 2+ (50 μ m EGTA) caused regular spontaneous Ca 2+ waves that were imaged with the fluorescence from Fluo‐5F using a laser‐scanning confocal microscope. Addition of 4 μ m RuR caused complete inhibition of Ca 2+ waves in 50% of cardiomyocytes by 2 min and in 100% by 4 min. Separate experiments used 350 μ m EGTA (600 n m Ca 2+ ) to limit Ca 2+ 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 Ca 2+ sparks were altered, and after 4 min Ca 2+ 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 Ca 2+ during the initial 2–4 min of perfusion with RuR in both wave and spark conditions. Computational modelling suggests that the sensitivity of Ca 2+ waves to RuR block depends on the number of RyRs per cluster. Therefore inhibition of Ca 2+ waves without affecting Ca 2+ sparks may be explained by block of small, non‐spark producing clusters of RyRs that are important to the process of Ca 2+ wave propagation.