cells Article Abnormal Calcium Handling in Atrial Fibrillation Is Linked to Changes in Cyclic AMP Dependent Signaling Franziska Reinhardt 1,2,† , Kira Beneke 2,3,† , Nefeli Grammatica Pavlidou 2,3 , Lenard Conradi 1 , Hermann Reichenspurner 1,3 , Leif Hove-Madsen 4 and Cristina E. Molina 2,3, * 1 Department of Cardiovascular Surgery, University Heart & Vascular Center Hamburg UKE, 20251 Hamburg, Germany; fr.reinhardt@uke.de (F.R.); l.conradi@uke.de (L.C.); reichenspurner@uke.de (H.R.) 2 German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20251 Hamburg, Germany; k.beneke@uke.de (K.B.); nefeligramm@gmail.com (N.G.P.) 3 Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf (UKE), 20251 Hamburg, Germany 4 Biomedical Research Institute Barcelona, IIBB-CSIC and IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; leif.hove@iibb.csic.es * Correspondence: c.molina@uke.de; Tel.: +49-407-4105-7095 † These authors contributed equally to this work. 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 30 ,50 -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 Citation: Reinhardt, F.; Beneke, K.; atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly Pavlidou, N.G.; Conradi, L.; higher frequency of transient inward currents (ITI ) activated by spontaneous Ca2+ release was Reichenspurner, H.; Hove-Madsen, L.; confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger Molina, C.E. Abnormal Calcium effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. Handling in Atrial Fibrillation Is 2.5-fold reduction), while the β-agonist isoproterenol (ISO) caused a greater increase in Ctl patients Linked to Changes in Cyclic AMP (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). Dependent Signaling. Cells 2021, 10, 3042. https://doi.org/10.3390/ However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes cells10113042 (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 Academic Editors: Ursula Ravens and no effect on ITI . Fluorescence resonance energy transfer (FRET) experiments showed that although Rémi Peyronnet 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. Received: 18 August 2021 Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial Accepted: 2 November 2021 myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite Published: 5 November 2021 changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: atrial fibrillation (AF); cAMP-dependent regulation; protein kinase A (PKA); L-type published maps and institutional affil- calcium current (ICa,L ); transient inward current (ITI ); patch-clamp iations. 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Atrial fibrillation (AF) has been commonly associated with profound remodeling of This article is an open access article calcium (Ca2+ )-handling and regulatory proteins [1–4]. Thus, AF-myocytes displayed a distributed under the terms and reduced L-type Ca2+ current (ICa,L ) density [2,5,6], as well as an increased spontaneous conditions of the Creative Commons sarcoplasmic reticulum (SR) Ca2+ release [1] through the ryanodine receptor (RyR2) called Attribution (CC BY) license (https:// Ca2+ sparks and Ca2+ waves. The increased Ca2+ waves then resulted in a rise in concurrent creativecommons.org/licenses/by/ transient inward current (ITI ), due to Ca2+ extrusion by the Na+ -Ca2+ exchanger (NCX) [7], 4.0/). and the corresponding additional depolarization of the membrane. Cells 2021, 10, 3042. https://doi.org/10.3390/cells10113042 https://www.mdpi.com/journal/cells Cells 2021, 10, 3042 2 of 17 A common mechanism proposed to explain Ca2+ -handling remodeling is a change in the phosphorylation state of the related proteins [3,8–10]. Two distinct serine residues of RyR2 have displayed increased phosphorylation in AF. The first one, ser2809 (ser2808 in rodents) [3,5,6], has been shown to be phosphorylated by protein kinase A (PKA). 30 ,50 - Cyclic adenosine monophosphate (cAMP)-dependent activation of PKA is indeed a major pathway for modulation of protein phosphorylation. cAMP levels are in turn regulated by adenylyl cyclases (AC), which generate cAMP upon G protein-coupled receptor stimulation by catecholamines, and by phosphodiesterases (PDEs), which degrade cAMP and regulate its propagation, mediating compartmentalized cAMP signals [4]. Additionally, 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]. CaMKII activity is actively regulated by Ca2+ , calmodulin and phosphatases. Furthermore, intracellular increase in cAMP levels can also 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]. Therefore, remodeling of the phosphorylation state of Ca2+ regulatory proteins is emerging as an important mechanism modulating abnormal Ca2+ handling in AF. However, these findings suggest opposite modulatory phosphorylation states between the RyR2 [3,5] and the L-type Ca2+ channel [9], making it important to determine if this is due to alterations or compartmentalization of the regulating phosphorylation pathways. This study, therefore, tested the hypothesis that the remodeling of L-type Ca2+ current and SR Ca2+ channel activity in AF is due to changes in cAMP-dependent PKA signaling, the key pathway for β-adrenergic receptor-mediated regulation of the phosphorylation state of these channels. Hence, the effect of the pharmacological manipulation of cAMP- dependent signaling on ICa,L density, frequency of SR Ca2+ release events large enough to produce ITI and cAMP levels was investigated in atrial myocytes from patients in sinus rhythm (Ctl) and with AF. Specifically, PKA and CaMKII were inhibited to eliminate PKA and CaMKII-dependent Ca2+ channel phosphorylation, and β-adrenergic signaling was stimulated to maximize Ca2+ channel phosphorylation. Our data show that in AF, PKA-inhibition with H-89 had little effect on the ICa,L amplitude, but strongly reduced spontaneous Ca2+ release large enough to produce spon- taneous transient inward currents. These results suggest that, opposite to sinus rhythm, PKA-dependent phosphorylation is minimal for the L-type Ca2+ channel but eminent for the RyR2 at baseline in AF patients. However, β-adrenergic stimulation with isoprenaline increased ICa,L amplitude, ITI and cAMP levels in all compartments in both groups of patients. Therefore, changes in Ca2+ handling seen in patients with AF are likely due to modulations in cAMP-dependent regulation. 2. Materials and Methods 2.1. Human Atrial Samples A total of 244 atrial human tissue samples were collected from patients undergoing cardiac surgery at the University Heart and Vascular Center Hamburg-Eppendorf (UKE). Atrial tissues were taken from patients in sinus rhythm (Ctl) or with atrial fibrillation (AF), subjected to atrial cannulation for extracorporeal circulation. All samples were taken with informed consent of the donors. The study was conducted in accordance with the Declaration of Helsinki principles and approved by the Ethical Committees of the Ärztekammer Hamburg (Ethical approval Number: WF-088/18, 26 February 2019). Patients treated with Ca2+ antagonists were excluded from the study. Overall patient characteristics, including diagnostics and treatment were summarized in Table 1. Cells 2021, 10, 3042 3 of 17 Table 1. Clinical and pharmacological details of patient included in this study. Continuous variables are presented as mean ± SD for normal-distributed data. Categorical data are given as number of patients (%). Characteristics Sinus Rhythm (Ctl) Atrial Fibrillation (AF) Patients (n) 148 96 Age (years) 60 ± 12 66 ± 14 Female (n (%)) 40 (27.0) 50 (52.1) Arterial hypertension (n (%)) 91 (61.5) 53 (55.2) Diabetes mellitus (n (%)) 35 (23.7) 30 (31.2) Dyslipemia (n (%)) 52 (35.1) 37 (38.5) Indication for surgery Coronary artery disease (n (%)) 48 (32.4) 11 (11.5) Valvular heart disease (n (%)) 70 (47.3) 80 (83.3) Both (n (%)) 30 (20.3) 5 (5.2) Echocardiography data Left atrial diameter (mm) 44 ± 9 51 ± 11 LVEF (%) 59 ± 8 58 ± 11 Medication ACE inhibitor (n (%)) 50 (33.8) 39 (40.6) AT1 receptor antagonist (n (%)) 44 (29.7) 46 (47.9) β-blocker (n (%)) 31 (20.9) 48 (50.0) Statins (n (%)) 56 (37.8) 29 (30.2) Amiodarone (n (%)) 0 (0) 29 (30.2) Diuretics (n (%)) 32 (21.6) 66 (68.7) Digitalis (n (%)) 54 (36.5) 29 (30.2) Nitrates (n (%)) 1 (0.7) 6 (6.2) 1 Abbreviations: LVEF: left ventricular ejection fraction, ACE inhibitor: angiotensin-converting enzyme inhibitor, AT1 receptor antagonist: angiotensin II receptor type 1 blockers. N-numbers represent number of patients whose samples were used for cell isolation and afterwards on patch-clamp recording and/or confocal and/or fluorescence resonance energy transfer experiments. 2.2. Isolation and Culture of Human Atrial Myocytes A total of 428 human atrial myocytes from 148 patients in sinus rhythm and 96 patients with AF were isolated as previously described [14,15] and used for experiments in this study. Right atrial appendage tissue samples, normally discarded during surgery, were cut into small pieces of tissue, and incubated at 35 ◦ C in a Ca2+ free solution containing 0.5 mg/mL collagenase (Worthington type 2, 323 U/mg; Lakewood, NJ, USA), 0.5 mg/mL proteinase (Sigma type XXIV, 11 U/mg solid; St. Louis, MO, USA) and 2% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA). After 30 min, the tissue was removed from the enzyme solution, and cells were disaggregated with a Pasteur pipette in Ca2+ -free solution containing 5% BSA and 2 µM blebbistatin (Sigma, St. Louis, MO, USA). The remaining tissue was digested for 3 × 15 min in a fresh Ca2+ free solution containing 0.4 mg/mL collagenase and 2% BSA. Freshly isolated cells were suspended in Ca2+ -free solution containing 5% BSA for patch-clamp experiments (duration ≈ 8 h) or in minimal essential medium (MEM: M 4780; Sigma, St. Louis, MO, USA) containing 1.2 mmol/L Ca2+ , 2.5% fetal bovine serum (FBS, Invitrogen, Cergy-Pontoise, France), 1% penicillin-streptomycin, 2% HEPES and 2 µM blebbistatin (pH 7.6) and plated on 35 mm, laminin-coated culture dishes (10 µg/mL laminin, 2 h; Sigma, St. Louis, MO, USA) for fluorescence resonance energy transfer (FRET) experiments. After 2 h of culture, the medium was replaced by 2 mL of FBS-free MEM with 2 µM blebbistatin. All experiments were performed at room temperature. Only elongated cells with clear cross striations and without granulation were used for experiments. 2.3. Patch-Clamp Technique Spontaneous ITI , caffeine-induced NCX currents and ICa,L were measured using a perforated patch-clamp technique in freshly isolated myocytes as previously described [1]. Cells 2021, 10, 3042 4 of 17 A HEKA EPC-10 amplifier (HEKA Elektronik GmbH, Lambrecht, Germany) was used to measure ICa,L elicited by a 200 ms depolarization from −80 to 0 mV and the tail currents activated upon repolarization to −80 mV at a pacing frequency of 2Hz. The ICa,L amplitude was determined as the difference between the peak inward current and the current level at the end of a 200 ms depolarization pulse. Na+ currents were eliminated by a 50 ms pre-pulse to −50 mV. ITI currents were measured at −80 mV and the spontaneous ITI frequency was used to measure Ca2+ waves induced by spontaneous SR Ca2+ release [7]. SR Ca2+ load was measured using the NCX current time integral elicited by a rapid application of 10 mmol/L caffeine [16], assuming a stoichiometry of 1Ca2+ :3Na+ for the NCX. Series resistance compensation was not performed. The extracellular medium (pH = 7.4) contained: NaCl 127 mM, TEA 5 mM, HEPES 10 mM, NaHCO3 4 mM, NaH2 PO4 0.33 mM, glucose 10 mM, pyruvic acid 5 mM, CaCl2 2 mM, and MgCl2 1.8 mM. The intracellular medium (pH = 7.2 with CsOH) contained: aspartic acid 109 mM, CsCl 47 mM, Mg2 ATP 3 mM, MgCl2 1 mM, Na2 phosphocreatine 5 mM, Li2 GTP 0.42 mM, HEPES 10 mM and 250 µg/mL amphotericin B. 2.4. Confocal Calcium Imaging Freshly isolated intact myocytes were loaded with a fluorescent Ca2+ dye (2.5 µM fluo-4 AM) for 20 min under control Tyrode perfusion (in mmol/L): 140 NaCl, 4 KCl, 1.1 MgCl2 , 10 HEPES, 10 glucose, 1.8 CaCl2 ; pH = 7.4, with NaOH). Ca2+ imaging was carried out with and a 63x glycerol-immersion objective and a resonance scanning Leica TCS SP8 X microscope (Leica Biosystems, Wetzlar, Germany) in the frame scanning mode at a frame rate of 90 Hz, with images being acquired every 30 s. Fluo-4 (Invitrogen, MA, USA) was excited at 488 nm and fluorescence emission measured between 500 and 650 nm. Image analysis was performed using IDL software (version 7.0.6, Research System Inc., Boulder, CO, USA). Images were corrected for the background fluorescence. The fluorescence values (F) were normalized by the basal fluorescence (F0 ) in order to obtain the fluorescence ratio (F/F0 ). Analysis to identify Ca2+ sparks was carried out using an automated detection system and a criterion that limited the detection of false events while detecting most Ca2+ sparks [17]. 2.5. Fluorescence Resonance Energy Transfer (FRET)-Based Live Cell Imaging of Intracellular cAMP FRET measurements were performed on human atrial myocytes 48 h after transduction with the adenovirus encoding Epac1-camps [18], pm-Epac1 [19] and Epac1-JNC [20]. A K+ -Ringer solution containing: 144 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 1 mM CaCl2 , 10 mM HEPES, adjusted to 7.4 pH with NaOH at room temperature (RT) was used to maintain cells, and images were taken every 5 s. The FRET system used consisted of a standard inverted microscope (Leica DMI3000, Leica Biosystems, Wetzlar, Germany) with a 63×/1.4 oil immersion objective, an LED light source (pE-100, CoolLED), a beam splitter (DV2, Photometrics, Birmingham, UK), and a camera (CMOS camera optiMOS, Photometrics, Birmingham, UK). A single-wavelength light-emitting diode (LED, 440 nm) was used to excite the donor fluorophore (CFP), which was controlled by an Arduino digital-to-analog input-output board (Arduino). A dual view beam splitter (Cube 05-EM, 505 dcxr, D480/30m, D535/40) was used to split the emission light into donor (CFP) and acceptor (YFP) channels. The fluorescence emitted was then measured at 475/30 nm and 527/40 nm, for CFP and YFP respectively and expressed the percent of 475/527 nm. To acquire time-lapse images the software Micro-Manager 1.4.5 [21] was used. Data analysis was performed using Microsoft Excel (version 16.4, 20121301). 2.6. Data Analysis and Statistics Values were expressed as means ± SEM, and a two-tailed student’s t-test used to assess significant differences between data pairs with a significance value of p < 0.05. ANOVA was used for multiple effects comparison, whereas the Student–Newman–Keuls post-test was used to evaluate specific effects. Experimenters were blinded to clinical information. Cells 2021, 10, 3042 5 of 17 3. Results 3.1. cAMP-Dependent Regulation of L-Type Ca2+ Current and Spontaneous SR Ca2+ Release Show Opposite Changes in Atrial Fibrillation In agreement with previous studies [1,2,6], myocytes from patients with AF had a significantly smaller ICa,L density and a higher ITI frequency at baseline. Furthermore, SR Ca2+ loading was similar for both patient groups. Next, the effects of the PKA-inhibitor H-89 (10 µM) at baseline were tested to determine if the changes in ICa,L density or ITI frequency could be associated with changes in cAMP-dependent PKA signaling. In AF- myocytes ICa,L density was comparable before and after exposure to H-89, yet notably in Ctl myocytes ICa,L density was significantly reduced to levels seen in AF myocytes (Figure 1a). Inhibition with H-89 induced a small shift to the right on the current-voltage (I- V) relationship after H-89 treatment in AF myocytes (Figure 1b). The ITI current frequency was practically abolished in both patient groups by H-89 (Figure 1c), although no effect was observed on Ca2+ sparks frequency or density (Figure 1d). Indeed, the inhibition of ITI by H-89 was observed to be relatively stronger in patients with AF (7.6-fold), than Ctl ones (2.5-fold), indicating that the activation of RyR2 is more dependent on PKA at baseline in myocytes from AF patients. Jointly, these results suggest cAMP compartmentation which promoted a PKA-dependent ICa,L activation at baseline in Ctl patients, which is not seen in patients with AF, and a PKA-dependent RyR2 activation at baseline in AF patients, which could contribute, together with other factors, to the enhanced RyR2 activity in AF. 3.2. CaMKII Inhibition Reduces ICa,L and Ca2+ Sparks but Not ITI Frequency To assess whether the observed changes in intracellular Ca2+ -handling were also modulated by CaMKII-dependent regulation, the effects of the CaMKII inhibitor KN- 93 (1 µM) and its inactive analog KN-92 (1 µM) were investigated. ICa,L density was significantly reduced in response to KN-93 in myocytes from AF and Ctl patients (p = 0.05 and p < 0.005 respectively) (Figure 2a) without the I-V relationship being affected (results not shown). Neither KN-92 nor KN-93 had a significant effect on spontaneous ITI frequency in the same myocytes (Figure 2b). However, CaMKII inhibition with KN-93 significantly reduced Ca2+ spark density and frequency in AF (p < 0.0001) (Figure 2c), as well as the SR Ca2+ load (Figure S2). 3.3. β-Adrenergic Stimulates ICa,L Equally in Ctl and AF Patients but Has a Relatively Smaller Effect on ITI Frequency in AF To evaluate whether β-adrenergic signaling was affected in AF, myocytes from Ctl and AF patients were exposed to the β-adrenergic agonist isoproterenol (ISO) (100 nM). As shown in Figure 3a,b, ISO promoted a relative similar increase in ICa,L density in myocytes from both groups of patients (2.7-fold in Ctl vs. 3.6-fold in AF). However, superimposed current traces before and after ISO exposure shown in Figure 3a (left panel) highlight that ICa,L amplitude in patients with AF remained smaller in absolute terms after β-adrenergic stimulation with ISO compared to Ctl (right panel). Additionally, ISO caused a significant negative shift in the I-V relationship in both groups (p < 0.001 in both Ctl and AF) (Figure 3b). ISO eliminated the observed differences in ITI frequency (Figure 3c) and ITI amplitude (Figure S1) among Ctl and AF myocytes. This means that, in relative terms, the stimulatory effect of ISO on the spontaneous ITI frequency was smaller in myocytes from patients with AF (2.1-fold) than from Ctl patients (5.5-fold). To assess whether this effect was secondary to effects on SR Ca2+ loading or Na+ -Ca2+ exchanger (NCX) activity, a protocol for caffeine-induced Ca2+ release from the SR was used to elicit the Ca2+ load in basal and upon ISO stimulation. Thus, the SR Ca2+ content was cleared with a brief caffeine pulse before reloading the SR during 30 stimulation pulses. Representative traces of caffeine-induced NCX-currents recorded before and after ISO exposure can be seen in Figure 3d. Myocytes from both patient groups showed the same ability to load Ca2+ into the SR before ISO treatment (Figure 3d), and similar increased reloading capability after ISO. Noticeably, the relative effect of ISO was not significantly Cells 2021, 10, 3042 6 of 17 Cells 2021, 10, x FOR PEER REVIEW different in myocytes from Ctl and patients with AF. The time constant (tau) for the decay of the NCX-current upon caffeine was also comparable in myocytes from Ctl and AF patients at basal and upon ISO stimulation (Figure 3e). Figure 1. Effect of protein kinase A (PKA) inhibition on ICa,L , ITI and sarcoplasmic reticulum (SR) Figure 1. Effect of protein kinase A (PKA) inhibition on ICa,L, ITI and sarcoplasmic ret Ca2+ load. (a) (left): Representative ICa,L recordings in myocytes from a patient in sinus rhythm load. (a) Left: Representative ICa,L recordings in myocytes from a patient in sinus rhy (Ctl) and a patient with atrial fibrillation (AF) before (CON, black trace) and after exposure to the patientPKA selective withinhibitor atrial fibrillation (H-89, 10 µM,(AF) before(right): grey trace). (CON, blackeffect Average trace)of and H-89 after in Ctl exposure and AF to th inhibitor (H-89, 10 μM, grey trace). Right: Average effect of H-89 in Ctl and patients. (b) Mean effect of H-89 on the current-voltage relationship in Ctl and AF patients. (c) (left): AF pa effect of H-89 Representative onof the traces current-voltage spontaneous relationship ITI recorded in in Ctlbefore Ctl and AF myocytes and and AFafter patients. exposure(c) Left to traces of spontaneous ITI recorded in Ctl and AFTImyocytes before and patients. H-89. (right): Mean effects of H-89 on the spontaneous I frequency in Ctl and AF after exposure (d) Average effects of H-89 on Ca2+ sparks density (left) and frequency (right) before and after Mean effects of H-89 on the spontaneous ITI frequency in Ctl and AF patients. (d) A exposure to H-89. ROI: region of interest. Significant differences between treatments are indicated H-89 on Ca2+ sparks density (left) and frequency (right) before and after exposur with * and between groups with #. region of interest. Significant differences between treatments are indicated with groups with #. 3.2. CaMKII Inhibition Reduces ICa,L and Ca2+ Sparks but Not ITI Frequency Cells 2021, 10, x10,FOR Cells 2021, 3042 PEER REVIEW 7 of 17 Figure 2. Effects of Ca2+ /Calmodulin-dependent protein kinase II (CaMKII) inhibition on I , Figure2+2. Effects of Ca2+/Calmodulin-dependent protein kinase II (CaMKII)Ca,L inhibition ITI and Ca sparks. (a) (left): Representative I Ca,L recordings in myocytes from a patient in sinus Ca2+ sparks. (a) Left: Representative ICa,L recordings in myocytes from a patient in sin rhythm (Ctl) and a patient with atrial fibrillation (AF) before (CON, black trace) and after exposure to and a patient a selective CaMKII with atrial inhibitor fibrillation (KN-93, 1 µM, grey(AF) trace).before (right): (CON, Average black trace) effects of KN-92and after (1 µM, the exposu CaMKII inactive inhibitor CaMKII (KN-93, inhibitor) and KN-93 1 μM, in Ctl grey and AFtrace). Right: patients. Average (b) (left): effectsrecordings Representative of KN-92 (1 μ ofCaMKII spontaneous inhibitor) ITI before and KN-93 and after in Ctl exposure and AF to KN-92 andpatients. (b) Left:from KN-93 in myocytes Representative in Ctl and AF record patients. neous(right): Average ITI before and effects afterofexposure KN-92 and KN-93 to KN-92on theand spontaneous KN-93 Iin TI frequency myocytes in AF fromandin Ctl a CtlRight: myocytes. (c) Average effects of KN-92 and KN-93 on Ca 2+ sparks density (left) and frequency Average effects of KN-92 and KN-93 on the spontaneous ITI frequency in A (right) before and after exposure to KN-92 and KN-93. Significant differences between treatments are cytes. (c) Average effects of KN-92 and KN-93 on Ca2+ sparks density (left) and fre indicated with * and between groups with +. before and after exposure to KN-92 and KN-93. Significant differences between treat cated with * and between groups with +. 3.3. β-Adrenergic Stimulates ICa,L Equally in Ctl and AF Patients but Has a Relativ Effect on ITI Frequency in AF To evaluate whether β-adrenergic signaling was affected in AF, myoc and AF patients were exposed to the β-adrenergic agonist isoproterenol (I As shown in Figure 3a,b, ISO promoted a relative similar increase in ICa,L de same ability to load Ca2+ into the SR before ISO treatment (Figure 3d), creased reloading capability after ISO. Noticeably, the relative effect of IS nificantly different in myocytes from Ctl and patients with AF. The time co Cells 2021, 10, 3042 8 of 17 the decay of the NCX-current upon caffeine was also comparable in my and AF patients at basal and upon ISO stimulation (Figure 3e). Figure Figure 3. Effect 3. Effect of β-adrenergic of β-adrenergic stimulation on stimulation ICa,L , ITI and on ICa,L, ITI reticulum sarcoplasmic (SR) Ca2+ load. and sarcoplasmic reticulu (a)(a) (left): Representative Left: Representative ICa,L recordings in myocytes from a sinus rhythm (Ctl) patient ICa,L recordings in myocytes from a sinus rhythm (Ctl) pat and a patient with atrial fibrillation (AF), before (CON, black trace) and after exposure of the myocyte to 30 nM with atrial fibrillation (AF), before (CON, black trace) and after exposure of the m isoproterenol (ISO, grey trace). (right): Average effects of ISO in Ctl and AF patients. (b) Mean isoproterenol (ISO, grey trace). Right: Average effects of ISO in Ctl and AF patien current-voltage relationship for ICa,L in Ctl and AF myocytes before and after exposure to ISO. (c)rent-voltage relationship (left): Representative recordingsfor of ITIICa,L in and before Ctlafter andISO AFinmyocytes beforeAverage Ctl and AF. (right): and after effectsexposur ofRepresentative ISO on ITI in Ctl andrecordings AF myocytes. of TI before (d)I(right): and afterrecordings Representative ISO in Ctl and AF. Right: of caffeine-induced NCX- Average 2+ load in Ctl and AF. (e) Average ISO ITI in Ctl and AF myocytes. (d) Right: Representative recordings of caffeine-induc currents before and after ISO. (left): Average ISO effect on SR Ca effect on time before andconstant after (Tau) ISO.inLeft: Ctl and AF. Significant Average differences ISO effect on between SR Ca2+treatments load in areCtlindicated and AF. (e) A with *, between groups with #. human isolated atrial myocytes from Ctl and AF patients with genetically encoded FRET- based biosensors for cytosolic cAMP (Epac1-camps), sarcolemmal cAMP (pm-Epac1) and cAMP in the vicinity of the RyR2 (Epac1-JNC). Application of ISO demonstrated that AF promotes desensitization on the β-adrenergic pathway. Figure 4a shows a significantly Cells 2021, 10, 3042 smaller increase in cytosolic cAMP levels upon ISO stimulation in AF myocytes compared 9 of 17 to Ctl myocytes (p < 0.0001). Interestingly, this deregulation on cAMP levels is differen- tially regulated in the different cellular compartments. Contrary to the reaction in the cy- tosol, the increase of cAMP levels upon ISO stimulation was similar between Ctl and AF 3.4. myocytes Stimulation Produces in the sarcolemma β-Adrenergic Different and the RyR2 Increase in cAMP compartments (FigureLevels 4a). in Ctl and AF in Different Cellular Next, Compartments we evaluated the role of PDEs in the control of basal cytosolic and local cAMP signaling FRET (using was firsttheused genetically encoded FRET-based to test whether the effects ofsensors Epac1-camps, ISO observed on ICa,L , Ca2+ load, pm-Epac1 and Epac1-JNC) sparks and ITI were by linked inhibiting all PDEs to cAMP expressed in the atria compartmentalized [22] using dynamics. IBMX Thus, we (100 μM) transduced human isolated atrial myocytes from Ctl and AF patients with genetically encodedand together with the PDE8 inhibitor PF-04957325 (100 nM), in atrial myocytes from Ctl FRET- AF patients. based Contrary biosensors to the effects for cytosolic cAMPelicited by ISO, application (Epac1-camps), sarcolemmal of IBMX+PF produced aand cAMP (pm-Epac1) similarly cAMP large in the increase vicinity of in thebasal RyR2 cytosolic cAMP levels (Epac1-JNC). by PDEof Application inhibition in Ctl and in ISO demonstrated AFAF that myocytes. However, IBMX+PF promoted differential increases of cAMP promotes desensitization on the β-adrenergic pathway. Figure 4a shows a significantly levels in Ctl ver- sus AF increase smaller depending in on the compartment. cytosolic cAMP levels Thus, upon in ISO AF the increase ofinbasal stimulation cAMP levels AF myocytes was compared twice as big as in Ctl at the sarcolemma and nearby the RyR2 (Figure 4b). to Ctl myocytes (p < 0.0001). Interestingly, this deregulation on cAMP levels is differentially These regulated in results indicate the different that β-adrenergic cellular compartments. signaling is compartmentalized Contrary by cytosol, to the reaction in the PDEs inthe human atrial myocytes and suggest that cAMP-dependent PKA signaling increase of cAMP levels upon ISO stimulation was similar between Ctl and AF myocytes might have a prominent role regulating the phosphorylation of specific Ca2+-handling proteins, such as in the sarcolemma and the RyR2 compartments (Figure 4a). RyR2, in AF. Figure4.4.Cytosolic Figure Cytosolic and and local local cAMP cAMP dynamics. dynamics. Left: (left):representative representativekinetics of fluorescence kinetics reso- of fluorescence reso- nance energy transfer (FRET) changes (expressed as CFP/YFP ratio) in the ryanodine receptor nance energy transfer (FRET) changes (expressed as CFP/YFP ratio) in the ryanodine receptor (RyR2) (RyR2) microdomain recorded in human atrial myocytes from patients in sinus rhythm (Ctl) and microdomain recorded in human atrial myocytes from patients in sinus rhythm (Ctl) and with atrial with atrial fibrillation (AF), transduced with Epac1-camps-JNC sensor and treated with (a) isopro- fibrillation (AF), terenol (ISO, 100transduced nM) and (b)with Epac1-camps-JNC the non-selective sensor and inhibitor phosphodiesterase treated with (a) 100 (IBMX, isoproterenol μM) and the(ISO, 100 nM) and (b) the type phosphodiesterase non-selective phosphodiesterase 8 selective inhibitor inhibitor PF-04957325 (100 (IBMX, 100 µM) nM). Right: and the average phosphodi- increases in cAMP levels esterase type 8inselective the cytosol and inPF-04957325 inhibitor different compartments (sarcolemma (100 nM). (right): averageand RyR2) ofinliving increases cAMP human levels in the cytosol and in different compartments (sarcolemma and RyR2) of living human atrial myocytes, measured as total cAMP produced after exposure (a) to ISO and (b) to the inhibitor IBMX. Significant differences between treatments are indicated with *. Next, we evaluated the role of PDEs in the control of basal cytosolic and local cAMP signaling (using the genetically encoded FRET-based sensors Epac1-camps, pm-Epac1 and Epac1-JNC) by inhibiting all PDEs expressed in the atria [22] using IBMX (100 µM) together with the PDE8 inhibitor PF-04957325 (100 nM), in atrial myocytes from Ctl and AF patients. Contrary to the effects elicited by ISO, application of IBMX+PF produced a similarly large increase in basal cytosolic cAMP levels by PDE inhibition in Ctl and in AF myocytes. However, IBMX+PF promoted differential increases of cAMP levels in Ctl versus AF depending on the compartment. Thus, in AF the increase of basal 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 PKA signaling might have a prominent role regulating the phosphorylation of specific Ca2+ -handling proteins, such as RyR2, in AF. Cells 2021, 10, x FOR PEER REVIEW 10 of 18 Cells 2021, 10, 3042 atrial myocytes, measured as total cAMP produced after exposure (a) to ISO and (b) to the inhibitor 10 of 17 IBMX. Significant differences between treatments are indicated with *. 3.5. Carvedilol, but Not β1-Blocker Treatment, Attenuates PKA Dependent Increase of ITI in AF 3.5. Carvedilol, The abovebut Not βsuggest results 1 -Blocker Treatment, that Attenuates remodeling PKA Dependent of β-adrenergic Increase signaling of ITI in AF in AF patients mightThe above results contribute to the suggest observedthat remodeling elevation of theofspontaneous β-adrenergic ITIsignaling frequencyininAF patients myocytes might from contribute these to To patients. thefurther observed elevation evaluate this,ofthetheeffect spontaneous of patientsITItreated frequencywithinβ-blockers myocytes from on (BBs) these ICa,Lpatients. density,ToITI further evaluate frequency and SR this, Cathe effect 2+ load was ofexplored. patients treated Comparingwith β-blockers atrial my- (BBs) on I density, I frequency and SR Ca 2+ load was explored. Comparing atrial ocytes fromCa,L Ctl or AF patients TI who were treated with BBs and from patients who did not myocytes receive thisfrom Ctl or (no treatment AF patients who 5a–c), BBs) (Figure were treated with BBs no significant and fromwas difference patients foundwhoon Idid Ca,L not receive this treatment (no BBs) (Figure 5a–c), no significant difference density (Figure 5a), SR Ca2+ load (Figure 5b), or ITI frequency (Figure 5c). Statistical analy- was found on ICa,L density 2+ sis taking into(Figure account5a), SR Ca load confounding (Figure effects 5b), or ITI frequency of concurrent (Figure disease, risk 5c). and factors Statistical other analysis taking into account confounding effects of concurrent disease, treatments different than BBs (including ACE inhibitors, angiotensin receptor blockers risk factors and other treatments different than BBs (including ACE inhibitors, angiotensin and amiodarone) revealed no differences in none of the tested parameters. However, sep- receptor blockers and amiodarone) revealed no differences in none of the tested parameters. aration of BBs treatment in β1-blockers and Carvedilol (Figure 5d–f) revealed that myo- However, separation of BBs cytes treatment from in β1 -blockers AF patients who received and Carvedilol Carvedilol (Figure treatment 5d–f) hadrevealed reducedthat myocytes (Fig- ITI frequency from AF patients ure 5). who received Carvedilol treatment had reduced ITI frequency (Figure 5). Figure Figure5.5.Effect Effectofofβ-blocker β-blocker treatment treatment of of patients on on patients Ca2+Ca 2+ -handling. -handling. Left:(left): average effectseffects average of the of treat- the ment of patients with β-blockers (BBs) or without BBs (no BBs) on (a) I , treatment of patients with β-blockers (BBs) or without BBs (no BBs) on (a) ICa,L , (b) sarcoplasmic Ca,L (b) sarcoplasmic reticu- lum (SR) Ca(SR) reticulum 2+ load 2+ Ca and (c) and load ITI, in(c)patients in sinus in ITI , in patients rhythm (Ctl) and(Ctl) sinus rhythm withand atrial withfibrillation (AF). Right: atrial fibrillation (AF). Carvediol effects on (d) I Ca,L, (e) SR Ca2+ load and 2+ (f) I TI, , in Ctl and AF. Statistical significance was (right): Carvediol effects on (d) ICa,L , (e) SR Ca load and (f) ITI , in Ctl and AF. Statistical significance evaluated using a multivariate linear regression model adjusted for the confounding effects of com- was evaluated using a multivariate linear regression model adjusted for the confounding effects of mon clinical factors showing a bias between BBs treatment as well as factors suspected to affect Ca2+- common clinical factors showing a bias between BBs treatment as well as factors suspected to affect handling. Significant differences between treatments are indicated with * and between groups with 2+ -handling. Significant differences between treatments are indicated with * and between groups +.Ca Each point represents a patient mean value recorded in myocytes from a total of 53 patients with with +. Each point represents a patient mean value recorded in myocytes from a total of 53 patients with AF and 104 patients without AF. 23 of the AF patients and 37 of the no AF patients were treated with BBs. Cells 2021, 10, 3042 11 of 17 4. Discussion 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 SR Ca2+ release events large enough to produce ITI , and 3) PDE-dependent specific cAMP signaling in each relevant compartment for Ca2+ -handling regulation. While inhibition of CaMKII reduced Ca2+ sparks frequency, it did not reduce ITI frequency in myocytes from patients with AF. Furthermore, Carvedilol, but not β1 -blockers, strongly reduced the ITI frequency. Together our findings suggest that cAMP/PKA-dependent compartmentalized signaling has a potential to prevent large proarrhythmic SR Ca2+ release events and/or increase ICa,L density in AF and that Carvedilol, but not β1 -blockers, may be a new therapeutic strategy to reduce Ca2+ release-induced atrial arrhythmias. 4.1. PKA-Dependent Modulation of Ca2+ Homeostasis in AF In line with previous studies [2,6,23] atrial myocytes from patients with AF showed a marked reduction in ICa,L density. Although different mechanisms have been proposed to explain this reduction, i.e., reduced channel expression [24,25], redox modulation [26], or phosphatase 2-mediated dephosphorylation [11], none of them fully clarify this phe- nomenon. Here we show that PKA-inhibition had almost no effect on ICa,L density in AF myocytes while significantly reducing ICa,L density in Ctl myocytes (Figure 1a), suggesting that L-type Ca2+ channels were already dephosphorylated in AF. However, given that ISO had similar relative effects on the ICa,L density in myocytes from patients with and without AF, a down-regulation of L-type Ca2+ -channel expression and/or changes in the redox state of the Ca2+ channel are also likely in AF. PKA- [3,5,6] as well as CaMKII- [6,10] mediated RyR2 hyperphosphorylation has been associated with the increased RyR2 activity in AF patients. Voigt et al. [6] also found significantly higher cAMP levels in right atrial samples from patients with AF than those from patients in sinus rhythm. In accordance with these findings, our study found a stronger effect of the PKA-inhibitor H-89 on the ITI frequency in AF than in Ctl myocytes (Figure 1c), as well as a smaller relative effect of β-adrenergic stimulation with ISO in AF (Figure 3c). Together, these results highlight the role of the cAMP-dependent PKA mediated stimulation of RyR2 activity and abnormal depolarization in AF. Hyperphosphorylation of the RyR2 has been associated with heart failure [27,28] and treatment with BBs reduces RyR2 phosphorylation [28] and improves Ca2+ -handling in these patients. However, in our study the ITI frequency did not differ in AF myocytes from patients with or without BBs treatment (Figure 5c), suggesting that BBs treatment had no protective effect on those patients. Importantly, Carvedilol treatment had reduced ITI frequency, indicating that BBs negative results were not due to a wash-out effect of the myocytes isolation. Although the selective effect of Carvedilol on the ITI frequency could be due to its ability to inhibit β-adrenergic receptors [28], to modify RyR2 gating [29,30] or to prevent RyR2 oxidation [31], it is the only BB capable of reducing Ca2+ waves [27]. By contrast, myocytes from patients treated with B1 Bs showed no changes in ICa,L density, SR Ca2+ load or ITI frequency. These observations provide a mechanistic explanation for clinical studies showing that the treatment with Carvedilol, but not B1 Bs, reduces the incidence of post-operative AF [32–35] and ischemia-induced arrhythmia [34]. In line with this, we here show that PKA inhibition effectively reduces spontaneous ITI , suggesting that this may be a key to preventing atrial arrhythmias where abnormal Ca2+ release is an underlying mechanism. 4.2. CaMKII-Dependent Regulation of Ca2+ Sparks in AF Hyperphosphorylation of the RyR2 mediated by CaMKII has also been linked to the increased RyR2 activity seen in AF [3,6,36–41] and has been proposed as a key mechanism underlying the elevation of spontaneous Ca2+ release in AF. In agreement with these studies, we found that CaMKII inhibition significantly reduced the Ca2+ sparks frequency, although we found no significant effects on ITI frequency in AF nor Ctl myocytes. Of Cells 2021, 10, 3042 12 of 17 note, none of the previous studies evaluated the effect of CaMKII inhibition in human atrial myocytes on Ca2+ waves frequency, only on Ca2+ sparks frequency, Ca2+ leak or RyR2 open probability. Furthermore, although CaMKII was previously highlighted as responsible for the observed effects on Ca2+ -handling, many of those studies also found a decrease in RyR2 phosphorylation (total and at Ser2808), in incidence of spontaneous Ca2+ events and in the corresponding delayed afterdepolarizations when PKA was inhibited. Our findings demonstrate that a reduction of Ca2+ sparks or passive SR Ca2+ leak that had no effect on the resting membrane potential does not necessarily imply a reduction of large Ca2+ waves which induce a transient inward current and membrane depolarizations (Figure 2b,c). In line with this observation, it has been shown that VK-II-86, a minimally-β- blocking Carvedilol analog, reduces arrhythmias by suppressing Ca2+ waves and RyR2 open duration but increases Ca2+ sparks frequency [29]. Furthermore, knocking out phospholamban in heterozygous R4496C mice strongly increases Ca2+ sparks but reduces cell-wide Ca2+ waves preventing ventricular arrhythmia in these mice [42]. 4.3. Key Ca2+ -Handling Compartments Are Regulated by Specific cAMP Microdomains The observed concurrent decrease in L-type Ca2+ channel phosphorylation and hy- perphosphorylation of most of RyR2 in AF suggests compartmentation of cAMP/PKA signals in restricted microdomains separately regulating the phosphorylation of each Ca2+ -handling compartment. Up to date, the majority of studies on cAMP compartmen- tation and the effects of PDE inhibition have been performed on ventricular cells and mostly in animal models [18,43–48], and only a few studies evaluated the contribution of cAMP/PKA compartmentation on AF pathophysiology. Increased cAMP activity [6], as well as decreased total cAMP hydrolytic activity and PDE-dependent modulation of cytosolic cAMP levels [14,15] were described in AF. Stimulation of both 5-HT [15,22,49] and adenosine A2A receptors [5,50] has been linked to arrhythmias by increasing cAMP and its RyR2-dependent activation [4]. Decreased PDE activity could explain the loss in persistent AF of PDE3/PDE4 control on the propensity of 5-HT-evoked arrhythmias observed in human atrial trabeculae from patients in sinus rhythm [22]. PDE1a, PDE1c, PDE2a, PDE3a, PDE3b, PDE4a, PDE4b, PDE4d and PDE8a were found to be expressed in human atria [51]. Pharmacological inhibition of PDE4 in Ctl patients increased Ca2+ sparks and waves frequency, ICa,L and arrhythmias [14]. However, PDEs redistribution could increase their influence on cAMP signals in discrete Ca2+ -handling compartments in AF. Thus, although PDE4 activity is reduced in AF, this PDE was suggested to be, at least in part, responsible for the enhanced frequency of spontaneous SR Ca2+ release in AF [14,52]. Moreover, PDE4 inhibition increased the incidence of arrhythmias in human atrial strips during β-adrenergic stimulation [14]. Our FRET experiments to directly measure cAMP levels in living atrial myocytes from Ctl and AF patients demonstrated that although cytosolic cAMP levels increased less in AF compared to Ctl upon ISO stimulation, they are differentially regulated in the different Ca2+ -handling compartments (Figure 4a). Indeed, cAMP/PKA signals can reach L-type Ca2+ channels to the same extent in AF as in Ctl myocytes upon β-adrenergic stimulation, providing an explanation to the same relative effect on the ICa,L density upon ISO stimulation in myocytes from the two groups of patients. Furthermore, inhibition of all PDEs increased basal levels of cAMP nearby L-type Ca2+ channels twice as much in AF compared to Ctl (Figure 4b), suggesting that PDEs may be responsible for the dephosphorylation of the L-type Ca2+ channel, reducing ICa,L in AF. At the same time, a larger increase on basal cAMP after PDE inhibition nearby RyR2 in AF (Figure 4b) would suggest an increase of PDEs in this compartment and thus, a reduction of cAMP-dependent PKA phosphorylation of these receptors. Within the hyper- phosphorylated context of AF, the increase in CaMKII phosphorylation of RyR2, would maintain SR Ca2+ load by increasing Ca2+ sparks and SR Ca2+ leak avoiding proarrhythmic effects of Ca2+ waves and ITI , which at least at a certain point of the AF progression would Cells 2021, 10, 3042 13 of 17 need to be accompanied by an attempted restriction of the strong PKA phosphorylation by PDEs in this compartment. 5. Conclusions Many conditions, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), heart failure (HF), and AF, have been linked to abnormal SR Ca2+ release and increased phosphorylation of the RyR2 [3,5,27,28]. Additionally, BBs have been reported to reduce RyR2 phosphorylation in CPVT and HF [28,53]. Here we show that selective PKA inhibition effectively reduces spontaneous SR Ca2+ release in myocytes from patients with AF without affecting Ca2+ entry via L-type Ca2+ channels, therefore highlighting modulation of PKA activity as a potential key target to prevent atrial arrhythmia where abnormal Ca2+ -release is an underlying mechanism. In line with this finding, spontaneous SR Ca2+ release was significantly reduced in patients treated with Carvedilol, the only BB capable of reducing SR Ca2+ waves [29]. In conclusion, our results show that cAMP-dependent PKA signaling is highly com- partmentalized and regulated by PDEs, that PKA inhibition effectively reduces spontaneous SR Ca2+ release in AF, and that Carvedilol treatment reduces the incidence of spontaneous Ca2+ release events and ITI in myocytes from patients with AF. Furthermore, increasing evidence indicates that β-adrenergic signaling can activate different pathways other than PKA. Thus, β-adrenergic stimulation can also activate CaMKII via cAMP in a PKA-independent (activating Epac, nitric oxide synthase 1 and protein kinase G) [54–56] pathway, increasing Ca2+ sparks frequency and diminishing Ca2+ sparks amplitude, while decreasing SR Ca2+ load [54]. In line with this observation, it has been shown that Carvedilol reduces arrhythmias by suppressing Ca2+ waves and RyR2 open duration but increasing Ca2+ sparks frequency [29]. Interestingly, we found increased expression of Epac2 in AF [51], the Epac isoform which is highly concentrated at Z lines and thus implicated in SR Ca2+ -handling [57]. In this study, we propose a model where PKA and CaMKII phosphorylation complement each other in order to adapt Ca2+ - handling to AF remodeling progression. CaMKII would enhance RyR2 phosphorylation at the dyadic clefts during the first episodes of AF or at the beginning of the progression of the arrhythmia increasing SR Ca2+ leak as an attempt to reduce SR Ca2+ load. A similar mechanism was proposed by Zhou et al. [29], and Díaz et al. [58], as well as in response to sudden increases in heart rate, adrenergic stress or stimulation in normal hearts [59–63]. However, and in line with previous CaMKII studies regarding its affinity and RyR2 Ca2+ sensitivity [63–65], during diseases such as persistent AF with chronic adrenergic stimulation, enhanced cytosolic and dedicated CaMKII activity would worsen the effects of PKA-hyperphosphorylation by contributing to abnormal diastolic Ca2+ release from the SR, and triggering arrhythmias. Although in atrial myocytes, L-type Ca2+ channels (LTCC) are equally distributed in and out of the tubular system [66], AF-associated cell hypertrophy and structural changes in the sarcolemma and RyR2 microdomains [67,68] may affect LTCC function, Ca2+ -induced Ca2+ -release events, RyR2 coupling to LTCC, PDE microdomains, RyR2 Ca2+ sensitivity and CaMKII activity. Further investigation regarding how structural changes in AF remodeling affect PKA, CaMKII and PDE interactions in basal and during β-adrenergic stimulation would provide valuable insights into AF pathophysiology. 6. Study Limitations Due to the isolation process potentially washing out acute effects of BB treatment, this study could only confirm long-term effects of BBs on the remodeling of Ca2+ handling in atrial myocytes. However, the exclusion of patients who received concomitant treatment that theoretically could mask any BB effects did not alter our results. Several reasons, such as differences in patient cohort or in experimental conditions, could account for the discrepancies between this and previous studies. Usage of the perforated patch-clamp technique in the present study, preserving cAMP-dependent sig- Cells 2021, 10, 3042 14 of 17 naling, compared to using the ruptured patch-clamp technique which has been reported to attenuate cAMP-dependent signaling and removes the effects observed on ICa,L during perforated-patch recordings [40], is likely an important factor, as any interference in cAMP- dependent signaling could bias towards CaMKII-dependent signaling. The present study used a physiological Ca2+ concentration (2 mM) rather than a high Ca2+ concentration (5 mM), commonly used to increase SR Ca2+ release events, and which would produce an overestimation of CaMKII-dependent signaling. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cells10113042/s1, Figure S1: Effects of H-89, isoproterenol (ISO) and Ca2+ /Calmodulin- dependent protein kinase II (CaMKII) inhibition with KN-92 and KN-93 on ITI amplitude. Average effects of protein kinase A (PKA) inhibition with H-89 (top left panel), PKA stimulation with ISO (top right panel), and CaMKII inhibition (bottom panel) with KN-92 (the inactive CaMKII inhibitor) and KN-93 in sinus rhythm (Ctl) and atrial fibrillation (AF) patients. Significant differences between treatments are indicated with * and between groups with +, Figure S2: Effects of Ca2+ /Calmodulin- dependent protein kinase II (CaMKII) inhibition on sarcoplasmic reticulum (SR) Ca2+ load. Average effect of CaMKII inhibition with KN-92 (the inactive CaMKII inhibitor) and KN-93 on SR Ca2+ load in sinus rhythm (Ctl) and atrial fibrillation (AF). Significant differences between treatments are indicated with *. Author Contributions: Conceptualization, C.E.M. and L.H.-M.; investigation, F.R., K.B. and N.G.P.; resources, L.C. and H.R.; writing—original draft preparation, K.B. and C.E.M.; supervision, C.E.M.; funding acquisition, C.E.M. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (ES 569/2-1 to CM) and the German Centre for Cardiovascular Research (DZHK), Spanish Ministry of Science and Innovation (PID2020-116927RB-C21 to L.H-M). Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki principles and approved by the Ethical Committees of the Universitätsklinikum Hamburg- Eppendorf, Germany (Ethical approval Number: WF-088/18). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We thank the UKE Microscopy Imaging Facility (UMIF, University Medical Center Hamburg-Eppendorf) for technical support and providing the Leica TCS SP8 X microscope. 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