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

Authors


Corresponding author G. L. Smith: Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK.  Email: g.smith@bio.gla.ac.uk

Abstract

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.

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