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 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 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.Heart cells contract due to a rapid electrical event on the surface membrane that triggers a homogeneous release of Ca²⁺ from the sarcoplasmic reticulum (SR) into the cytoplasm. The SR can release Ca²⁺ without the electrical trigger in two ways: (i) a spatially limited Ca²⁺'spark' or (ii) a Ca²⁺'wave' that propagates throughout the cell. The latter event is thought to cause arrhythmias in the whole heart. This study provides experimental evidence to suggest that Ca²⁺ waves are not simply due to sequential activation of Ca²⁺ sparks. Instead SR Ca²⁺ release other than a spark appears to be essential for propagation of the Ca²⁺ wave. Pharmacological blocking of the 'non-spark' form of the release may be an effective strategy to prevent arrhythmias without compromising normal heart function.