Effects of phosphodiesterase-5 inhibition with sildenafil on calcium waves in cardiac myocytes

Background

Diastolic Ca²⁺ waves in cardiac myocytes lead to arrhythmias by inducing delayed after-depolarisations. Waves occur when sarcoplasmic reticulum (SR) content reaches a threshold level. The phosphodiesterase-5 inhibitor, sildenafil, is antiarrhythmic in mammalian myocardial ischaemia models, and in rat myocytes it reduces Ca²⁺ transient amplitude and SR Ca²⁺ content. We sought to determine effects of sildenafil on propensity to Ca²⁺ waves in the large mammal.

Methods

Sheep ventricular myocytes were voltage clamped and Ca²⁺ fluorescence measured using fura-2. Cells were paced at 0·5 Hz with depolarisations from ?40mV to +10mV. When at steady state, waves were induced with 7·5–15 mM Ca²⁺. Upon regular waving, sildenafil (1 μM) was applied. To determine threshold SR Ca²⁺ content, caffeine (10 mM) was added immediately after a wave, and both wave and caffeine-induced Na⁺/Ca²⁺ exchanger current (I_NCX)were integrated.

Findings

Increasing external Ca²⁺ increased SR content and induced diastolic waves. Sildenafil abolished waves in seven of 11 cells. In cells where sildenafil terminated waves, SR content was reduced below threshold. In addition, sildenafil treatment was associated with reduced rate constant of SERCA (k_SERCA ?68·4% of control, p<0·0001), initial (first 4 s) increase in sarcolemmal efflux via I_NCX tail current (+190%, p=0·022), and reduced sarcolemmal influx via L-type Ca²⁺ current (I_Ca-L) (?29·8%, p=0·0015). In cells continuing to wave in sildenafil, SR threshold for waves was unchanged (123·8 μmol/L sildenafil vs 150·7, p=0·57). In unstimulated cells spontaneously waving in 10–15 mM Ca²⁺, sildenafil reduced wave frequency (6·3 waves per 20 s vs 2·7, p=0·0034). The effect of sildenafil on both wave models was abolished when cells were preincubated with the protein kinase G inhibitor, KT5823.

Interpretation

Sildenafil suppresses waves induced by elevated external Ca²⁺ via a protein kinase G-dependent mechanism. This suppression is mediated by reduced SR content, which itself is caused by reduced SERCA function and possible reduced I_Ca-L. These findings highlight novel antiarrhythmic properties of phosphodiesterase-5 inhibition.

Funding

British Heart Foundation.