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

Abstract

BackgroundDiastolic 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. MethodsSheep 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 (INCX)were integrated. FindingsIncreasing 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 (kSERCA −68·4% of control, p<0·0001), initial (first 4 s) increase in sarcolemmal efflux via INCX tail current (+190%, p=0·022), and reduced sarcolemmal influx via L-type Ca2+ current (ICa-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. InterpretationSildenafil 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 ICa-L. These findings highlight novel antiarrhythmic properties of phosphodiesterase-5 inhibition. FundingBritish Heart Foundation.