Abstract
A major concern of using phosphodiesterase (PDE) inhibitors in heart failure is their potential to increase mortality by inducing arrhythmias. By diminishing cyclic adenosine monophosphate (cAMP) hydrolysis, they promote protein kinase A (PKA) activity under β-adrenergic receptor (β-AR) stimulation, hence enhancing Ca(2+) cycling and contraction. Yet, cAMP also activates CaMKII via PKA or the exchange protein Epac, but it remains unknown whether these pathways are involved in the pro-arrhythmic effect of PDE inhibitors. Excitation-contraction coupling was investigated in isolated adult rat ventricular myocytes loaded with Fura-2 and paced at 1 Hz allowing coincident measurement of intracellular Ca(2+) and sarcomere shortening. The PDE4 inhibitor Ro 20-1724 (Ro) promoted the inotropic effects of the non-selective β-AR agonist isoprenaline (Iso) and also spontaneous diastolic Ca(2+) waves (SCWs). PDE4 inhibition potentiated RyR2 and PLB phosphorylation at specific PKA and CaMKII sites increasing sarcoplasmic reticulum (SR) Ca(2+) load and SR Ca(2+) leak measured in a 0Na(+)/0Ca(2+) solution ± tetracaine. PKA inhibition suppressed all the effects of Iso ± Ro, whereas CaMKII inhibition prevented SR Ca(2+) leak and diminished SCW incidence without affecting the inotropic effects of Ro. Inhibition of Epac2 but not Epac1 diminished the occurrence of SCWs. PDE3 inhibition with cilostamide induced an SR Ca(2+) leak, which was also blocked by CaMKII inhibition. Our results show that PDE inhibitors exert inotropic effects via PKA but lead to SCWs via both PKA and CaMKII activation partly via Epac2, suggesting the potential use of CaMKII inhibitors as adjuncts to PDE inhibition to limit their pro-arrhythmic effects.
Highlights
Upon stress or during exercise, norepinephrine released by the sympathetic nerve terminals activates the -adrenergic receptors (β-ARs) to elicit positive inotropic, chronotropic and lusitropic effects. β-ARs couple primarily to Gαs proteins, leading to stimulation of adenylyl cyclases (AC) and cyclic adenosine monophosphate production
L-type Ca2+ channels (LTCC), RyR2 and PLB are substrates for Calmodulin-dependent kinase II (CaMKII) which contributes to the inotropic and lusitropic effects of β-AR agonists. This is especially true upon excessive β-AR activation as occurring under pathological conditions, notably in heart failure (HF), where chronic β-AR stimulation is accompanied by perturbations of the cyclic adenosine monophosphate (cAMP) signaling pathway4 and increased CaMKII activity
To investigate the role of PDE4 in controlling the excitation-contraction coupling (ECC) and the pro-arrhythmic effects of its inhibition, Ca2+ transients (CaT) and sarcomere shortening (SS) were simultaneously recorded in ARVMs loaded with 1 μM Fura-2 and paced at 1 Hz (Fig. 1A)
Summary
Upon stress or during exercise, norepinephrine released by the sympathetic nerve terminals activates the -adrenergic receptors (β-ARs) to elicit positive inotropic, chronotropic and lusitropic effects. β-ARs couple primarily to Gαs proteins, leading to stimulation of adenylyl cyclases (AC) and cyclic adenosine monophosphate (cAMP) production. CAMP activates the cAMP-dependent protein kinase (PKA) which phosphorylates key proteins of the cardiac excitation-contraction coupling (ECC) process, including the sarcolemmal L-type Ca2+ channels (LTCC), the ryanodine receptors (RyR2) of the sarcoplasmic reticulum (SR) and phospholamban (PLB), a constitutive inhibitor of the SR Ca2+ pump, SERCA2. LTCC, RyR2 and PLB are substrates for CaMKII which contributes to the inotropic and lusitropic effects of β-AR agonists. This is especially true upon excessive β-AR activation as occurring under pathological conditions, notably in heart failure (HF), where chronic β-AR stimulation is accompanied by perturbations of the cAMP signaling pathway and increased CaMKII activity.. This is especially true upon excessive β-AR activation as occurring under pathological conditions, notably in heart failure (HF), where chronic β-AR stimulation is accompanied by perturbations of the cAMP signaling pathway and increased CaMKII activity. Interestingly, CaMKII has been identified as the main suspect to provoke the Ca2+ handling disturbances observed upon excessive β-AR stimulation in physiological conditions and in HF. Thereafter, Epac (exchange protein directly activated by cAMP), a direct target of cAMP, has emerged as a link between β-AR/cAMP signaling and CaMKII activation to promote, independently of PKA, a pro-arrhythmogenic SR Ca2+ leak.
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