Abstract
Both, the decreased L-type Ca2+ current (ICa,L) density and increased spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), have been associated with atrial fibrillation (AF). In this study, we tested the hypothesis that remodeling of 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the β-agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to β-adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation).
Highlights
Atrial fibrillation (AF) has been commonly associated with profound remodeling of calcium (Ca2+)-handling and regulatory proteins [1–4]
In atrial fibrillation (AF) the increase of basal Cyclic adenosine monophosphate (cAMP) levels was twice as big as in Ctl at the sarcolemma and nearby the RyR2 (Figure 4b). These results indicate that β-adrenergic signaling is compartmentalized by PDEs in human atrial myocytes and suggest that cAMP-dependent protein kinase A (PKA) signaling might have a prominent role regulating the phosphorylation of specific Ca2+-handling proteins, such as RyR2, in AF
Our study shows that AF is associated with (1) a cAMP/PKA-dependent reduction of the ICa,L density, (2) the upregulation of cAMP/PKA-dependent stimulation of sarcoplasmic reticulum (SR) Ca2+ release events large enough to produce in concurrent transient inward current (ITI), and 3) PDE-dependent specific cAMP signaling in each relevant compartment for Ca2+-handling regulation
Summary
Atrial fibrillation (AF) has been commonly associated with profound remodeling of calcium (Ca2+)-handling and regulatory proteins [1–4]. AF-myocytes displayed a reduced L-type Ca2+ current (ICa,L) density [2,5,6], as well as an increased spontaneous sarcoplasmic reticulum (SR) Ca2+ release [1] through the ryanodine receptor (RyR2) called Ca2+ sparks and Ca2+ waves. The first one, ser2809 (ser2808 in rodents) [3,5,6], has been shown to be phosphorylated by protein kinase A (PKA). The reduced ICa,L density has been shown to be in part linked to a phosphatase-dependent reduction in the phosphorylation state of the L-type Ca2+ channel [11]. The second one, ser2815 (ser2814 in rodents), has been shown to be phosphorylated by Ca2+/Calmodulin-dependent protein kinase II (CaMKII) [6,10]. Intracellular increase in cAMP levels can activate a guanine nucleotide exchange factor, the exchange protein activated by cAMP (Epac) [12], and phosphorylate RyR2 via CaMKII in a PKA-independent manner [13]
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