Programme Contents

The investigation of dantrolene sodium analogues on SR calcium loading and release, and calsequestrin Ca²⁺ binding properties in cardiac muscle

Recently the skeletal muscle relaxant dantrolene sodium (DaNa) has been investigated for its ability to modulate SR calcium release in cardiac muscle, with the potential of it being used as a treatment of heart failure. How this modulation occurs is still not well understood. The use of dantrolene analogues may provide some useful insight into the structure activity relationship (SAR) as well as provide future lines of investigation for potential therapeutics of heart failure. Here we investigated the effect of DaNa, azumolene, and two synthesised DaNa analogues on Ca²⁺ loading and release in skinned cardiomyocytes and on calsequestrin (CSQ₂) Ca²⁺ binding properties.

DaNa and azumolene were bought commercially (Sigma) while analogues GIF-0185 (1) and GIF-0166 (2) were synthesised using the procedure described by Hosoya et al., (2003). The structure of dantrolene and the analogues can be seen in the figure below with the differences highlighted in red and will be referred to as analogue 1 and 2. Fresh sheep hearts were obtained from Hardwick’s abattoirs Kyneton, Victoria and the right ventricle was enzymatically digested to produce single cardiac cells. The cells were then allowed to adhere to a 96-well plate (black, clear bottom), by incubating overnight (5% CO₂, 37°C). The cells were subsequently skinned (sarcolemma made leaky) for 10 to 45 min in a sodium-based solution containing 0.1% w/v saponin, 8mM ATP and 1mM EGTATotal. After skinning the cells were washed in a solution containing 49.9mM HDTATotal + 0.1mM EGTATotal and resuspended in the same solution containing the Ca²⁺-sensitive fluorescent dye Fluo4 (5μM). Ca²⁺ dependence on SR loading and release was investigated using Ca²⁺ loading solutions (pCa = −log10[Ca²⁺] 6.54, 6.26, 6.00 and 5.83 0.1mM EGTATotal, 8mM ATP, 1mM Mg²⁺ for 3.0 min). SR Ca²⁺ release was induced by the addition of caffeine (24mM final concentration). The fluorescent signal from Fluo4 (Ex 494, Em 516 nm) was continually monitored using the Flexstation 3 (read rate 1.15s for up to 5 min). This was compared to the maximal Ca²⁺ signal recorded after destroying all the membranous compartments by exposure to 1% v/v Triton X-100 for up to 5 minutes. The effect of dantrolene sodium and the analogues (1μM) on SR Ca²⁺ loading and release were investigated. Purified recombinant CSQ₂ was used to study the effect of all compounds on calcium binding. A plasmid containing the human calsequestrin 2 gene (GeneCopoeia) was inserted into single step KRX competent cells (Promega) and expressed as per the protocol provided (Promega). CSQ₂ was purified using a GST affinity column (Sigma) and GST tag cleaved using TEV protease (Sigma). CSQ₂ native fluorescence (Ex 280nm, Em 340nm), turbidity (Abs 350nm) and free Ca²⁺ in solution (measure with Rhod5N; Ex 551nm, Em 576nm) were monitored in the presence of increasing micromolar and millimolar concentrations of Ca²⁺ in the absence and presence of the drugs (10μM).

DaNa enhanced both SR Ca²⁺ loading and subsequent release in the presence of lower pCa solutions (pCa 5.83 and 6.26) compared with control, and increased both CSQ₂ polymerization and Ca²⁺ binding. Azumolene slightly increased SR loading and release but had no effect on CSQ₂ Ca²⁺ binding properties. Analogues 1 and 2 had different effects; with analogue 1 enhancing SR Ca²⁺ release when present in the release solution only, but reduced CSQ₂ polymerization; while analogue 2 consistently enhanced SR Ca²⁺ loading and subsequent release for all pCa used. Analogue 2 also significantly enhanced CSQ₂ polymerization at both micromolar and millimolar Ca²⁺ concentrations. It appears that substitution in the para position of the benzene ring had more effect on the gating properties of the RyR2, while the nitro group was associated with changes in CSQ₂ polymerization and Ca²⁺ binding. The specificity of the analogues may allow SR Ca²⁺ capacity or RyR2 leak to be targeted independently in heart failure.

Hosoya T, Aoyama H, Ikemoto T, Kihara Y, Hiramatsu T, Endo M, Suzuki M. (2003) Bioorg Med Chem 11, 663-673.