Dantrolene sodium increases calcium binding by human recombinant cardiac calsequestrin and calcium loading by sheep cardiac sarcoplasmic reticulum.

Abstract

Dantrolene interacts with ryanodine receptors in skeletal and cardiac muscle affecting sarcoplasmic reticulum calcium release. Since dantrolene is lipophilic it could also affect intra-sarcoplasmic reticulum calcium handling proteins such as calsequestrin. This study investigated whether dantrolene (1-30µmol/L) alters the polymerization state and the calcium binding capacity of recombinant cardiac calsequestrin and cardiac sarcoplasmic reticulum calcium handling. Human recombinant cardiac calsequestrin was used to make simultaneous measurements of turbidity (to indicate calsequestrin polymerization) and calcium binding to calsequestrin in the presence and absence of dantrolene. Caffeine-induced Ca2+ transients were used to investigate the effects of dantrolene on sarcoplasmic reticulum calcium loading and release in saponin-permeabilized cardiomyocytes laid down in monolayers in 96-well array plates. Dantrolene (1-30µmol/L) increased the polymerization state of calsequestrin and its calcium binding capacity. In the presence of dantrolene, calsequestrin-dependent turbidity increased 2.5-11.5-fold at 1.0mmol/L calcium added to unbuffered Ca2+ solutions and 3-10-fold when calcium was raised from 0.06 to 30µmol/L. The dantrolene-dependent increase in turbidity at 30µmol/L dantrolene was associated with a 3-fold increase in the number of calcium ions bound per calsequestrin molecule at 30 and 100µmol/L calcium. The caffeine-induced releasable calcium loaded by the sarcoplasmic reticulum at 0.63µmol/L free calcium in permeabilized cardiomyocytes was also increased in the presence of 30µmol/L dantrolene. Dantrolene alters the polymerization state and the calcium binding properties of cardiac calsequestrin and increases sarcoplasmic reticulum calcium loading in permeabilized cardiomyocytes.