Ivabradine causes abnormal intracellular calcium handlings and delayed afterdepolarizations to induce atrial fibrillation in rabbit hearts

Ivabradine causes abnormal intracellular calcium handlings and delayed afterdepolarizations to induce atrial fibrillation in rabbit hearts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Ivabradine causes abnormal intracellular calcium handlings and delayed afterdepolarizations to induce atrial fibrillation in rabbit hearts Chengyu Wang, Bingxun Li, Mingjie Lin, Qiaomei Yang, Gang Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7111461/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective The present paper is to determine the effects and underlying mechanisms of ivabradine (IVA) on atrial fibrillation (AF). Methods Electrophysiological changes were determined using Langendorff-perfused hearts and patch-clamp techniques. Parameters of Ca 2+ handling were evaluated by using calcium imaging and western blotting. Results IVA (0.1–10 µM) slowed HR in a concentration-dependent manner in isolated hearts of rabbit. IVA induced atrial arrhythmias in 26.1% and 76.9% of hearts paced at a basic cycle length of 350 and 570 ms, respectively. In hearts pretreated with either acetylcholine (ACh) or anemone toxin-II (ATX-II) which caused no inducible atrial arrhythmias, adding to IVA administration caused atrial arrhythmias in 61.9% (13/21) and 44.4% (8/18) of hearts, respectively. In atrial myocytes, IVA induced DADs by 41.7%, 62.5% and 50.0%, respectively, in the absence and presence of either ACh or ATX-II. IVA increased the frequency, amplitude and full width at half-maximum (FWHM) of Ca 2+ sparks and decreased Ca 2+ transport in association with increased protein expression of RyR2 and NCX1 and decreased SERCA2. Conclusion IVA increases atrial proarrhythmic risk in hearts with a slow HR, enhanced vagal tone and increased late sodium current by inducing DADs resulting from an enhanced intracellular Ca 2+ inhomeostasis. Ivabradine atrial arrhythmia action potential duration heart rate late sodium current vagal tone Ca2+ homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Drug-induced cardiac adverse effects are diverse and include both atrial and ventricular proarrhythmias[ 1 ]. Although not common, drug induced arrhythmias remain a major safety issue in drug development and in clinical practice and may increase under some pathological or pharmacological, even physiological conditions[ 2 , 3 ]. Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, can be triggered by multiple cardiovascular and non-cardiovascular conditions, which contribute to its pathogenesis through diverse mechanisms of action[ 4 , 5 ]. However, drug-induced AF received insufficient recognition and is often underestimated in the incidence, despite its potential significance in complicating clinical decision-making. Moreover, the lack of clinical data and inadequate understanding of the underlying mechanisms also limits identification and treatment for drug-induced AF[ 6 , 7 ]. Ivabradine (IVA), a class 0 antiarrhythmic agent according to the modernized classification of antiarrhythmic drugs[ 2 , 3 ], selectively inhibits hyperpolarization-activated cyclic nucleotide (HCN) channels to reduce the funny current ( I f ) and sinus heart rate (SHR) and has been used to treat inappropriate sinus tachycardia in addition to or as an alternative to digitalis, β-blockers or calcium channel inhibitors without affecting blood pressure and myocardial contractility[ 8 ]. However, the effect of IVA on AF remains uncertain[ 9 , 10 ]: on one hand, clinical research found that the unselective use of IVA was associated with increased risk of developing AF by about 13%[ 11 , 12 ]. However, experimental models showed that IVA reduced the risk of AF[ 10 , 13 , 14 ]. Moreover, researches also supported the application of IVA in controlling heart rate in AF patients[ 15 ]. Therefore, clinical implications of ivabradine in AF patients might remain controversial[ 16 ]. Mechanisms of AF, especially drug induced AF are complex and may be attributed to inflammation and endoplasmic reticulum stress to induce delayed afterdepolarization (DADs). Abnormalities of intracellular calcium concentration ([Ca 2+ ] i )-calmodulin kinase (CaMK)II-late sodium current (I Na )-intracellular sodium concentration ( [Na + ] i ) pathway under pathological conditions are reported to promote ventricular arrhythmias and AF[ 6 , 7 ]. An increase in late I Na has been shown to increase the proarrhythmic potential of low risk QT prolonging agents and to induce AF[ 17 , 18 ]. Because the low incidence of AF, in this study, effects of IVA by interacting with the [Ca 2+ ] i -CaMKII-late I Na -[Na + ] i pathway to increase [Ca 2+ ] i , and to induce DADs and AF under conditions of modulated late I Na and vagal activity were investigated in rabbit isolated hearts and atrial myocytes, respectively. Methods Electrophysiological study in isolated rabbit hearts New Zealand White rabbits (female, 2.5-3.0 kg) were anesthetized using xylazine (16 mg/kg by IM, Huamu; China) and ketamine (40 mg/kg by IM, CAHG; China). Isolated heart perfusion was completed according to the Langendorff system. The concentration-response relationships of IVA on the SHR were tested in spontaneously beating hearts (n = 24), while atrial monophasic APs (MAPs) and arrhythmic events were obtained from hearts paced at a fixed pacing cycle length (CL) after sinoatrial nodes were thermoablated. Hearts were treated with either increasing concentrations of IVA (n = 23) or in the absence and presence of low-concentration anemone toxin-II (ATX-II, n = 18) or acetylcholine (ACh, n = 21), respectively. ATX-II (2 nM) and ACh (0.3 µM) were confirmed not to cause atrial arrhythmias in hearts studied[ 19 ]. MAP, ECG and atrial arrhythmia recordings in isolated hearts The atrial MAPs and pseudo-12-lead ECGs were continuously monitored and digitized in real time. The atrial effective refractory period (ERP) and arrhythmic events were induced by the S1S2 programmed stimulations[ 17 ]. The postrepolarization refractory period (PRR) was calculated using the following equation: ERP-MAPD 90 . Atrial tachycardia (AT) was defined as a sequence of three or more consecutive, relatively regular spontaneous atrial beats occurring unexpectedly at a rate exceeding the spontaneous or pacing rate. An episode of AF was defined as a sequence of fast, irregular atrial signals in MAP and ECG recordings with irregular QRS complexes in a 12-channel ECG record. Atrial arrhythmias include AT and AF. Recordings of action potentials (APs) and triggered activity in single atrial myocytes Atrial myocytes were enzymatically isolated from New Zealand White rabbits as described in a previous study[ 20 ]. Quiescent and Ca 2+ -tolerant atrial myocytes (n = 28) were selected for recording APs. Atrial myocytes were bathed and perfused (2–3 mL/min) in a bath solution containing the following reagents (in mM; Sigma-Aldrich, MA, USA): 144 NaCl, 5.6 KCl, 1.2 MgCl 2 , 5 HEPES, 1.8 CaCl 2 and 11 Glucose at pH = 7.4 titrated with NaOH and maintained at 22–24°C. The patch pipette solution contained the following reagents (in mM; Sigma-Aldrich, USA): 110K-aspartate, 30 KCl, 5 NaCl, 10 HEPES, 0.1 EGTA, 5 Mg-ATP, 5 creatine phosphate, and 0.05 cAMP) at pH = 7.2 titrated with KOH. APs were induced in current-clamp mode at 1 s pacing cycle lengths. EADs were elicited by changing the stimulation frequency to 0.25 Hz, and DADs were determined following a baseline pacing CL of 9 s and 15 beats with a stimulation frequency of 2.5 Hz. DAD arises from the resting potential after full repolarization of an action potential and it may reach threshold for activation, while the EAD arises on the shoulder of a preceding action potential plateau and it is favored by slow preceding activation rate and prolonged action potentials[ 21 ]. Recording of Ca 2+ sparks in atrial myocytes using confocal imaging Atrial myocytes (n = 22) were bathed in an external solution composed of (in mM; Sigma-Aldrich, USA), 135 NaCl, 5.4 KCl, 1.0 MgCl 2 , 1.8 CaCl 2 , 10 glucose, 0.33 NaH 2 PO 4 , and 10 HEPES, pH = 7.4, with NaOH and incubated in 10 µM Fluo-4 AM (Thermo Fisher Scientific; USA) for 20 minutes. A laser-scanning confocal microscope system (TCS SP5; Leica; Germany) was applied to acquire the Ca 2+ sparks. Sparks were analyzed with SparkMaster[ 22 ], and the number, frequency, amplitude, full width at half-maximum (FWHM) and full duration at half-maximum (FDHM) of the detected sparks were obtained. A threshold criterion for spark detection of 3.8 was chosen for data analysis. Recording of Ca 2+ transport by SERCA in atrial myocytes Ca 2+ transport by SERCA was estimated from the rate constants (r) of single exponential curves fitted to the decaying part of the electrically and caffeine-evoked Ca 2+ transients, according to Choi and Eisner [ 23 ]. Cardiomyocytes (n = 11–18) were paced by an electrical field at 1.0Hz. The decay rate constant (r1) of electrically evoked Ca 2+ transients reflect Ca 2+ transport from cytoplasm to SERCA and outside the cell. The constant (r2) of decline of caffeine (20 mM)-evoked Ca 2+ transient reflects Ca 2+ transport outside to the myocytes. Finally, Ca 2+ transport by SERCA was estimated by subtracting r2 from r1 (r SERCA =r1-r2), and the relative contribution to relaxation of the Ca 2+ transporters was calculated according to the following formula: SERCA contribution= (r1-r2)/r1[ 23 ]. Western blotting Left atrial tissues (n = 3–6) were collected after isolated heart perfusion and homogenized using a tissue lyser. The levels of RyR2 (LS-C93425, LifeSpan BioSciences, USA), SERCA2 (4388s, Cell Signaling Technology, USA) and NCX1 (5507-I-AP, Proteintech, USA) were determined by Western blotting as described in our previous study[ 24 ]. Statistical analyses Data are reported as the Mean ± SEM. Statistical analyses were performed using IBM SPSS Statistics (version 20.0, IBM, New York, USA). The concentration-response relationships were analyzed using GraphPad Prism for Windows (version 6.02, GraphPad Software, Inc., San Diego, CA). When control/baseline and treatment values were obtained from the same heart/cell, the significance of the differences in the measures before and after interventions was determined by repeated measures one-way ANOVA followed by the Newman-Keul test. The χ 2 test and Fisher’s test were used to compare the incidences of atrial arrhythmias and trigger activities. Differences were considered significant at p < 0.05 . Results IVA decreased SHR in isolated rabbit hearts IVA (≥ 0.1 µM) reduced the HR in a concentration-dependent manner (10 µM IVA reduced the HR by 83.24 ± 7.30 bpm), and 10 µM IVA prolonged the PR interval, QRS width and QT interval ( p < 0.05 vs. baseline; Figure. 1 and Table 1 ) in heart treated with IVA alone (n = 8). ATX-II (n = 8) and ACh (n = 8) caused a small decrease in HR by 4.87 ± 1.59 and 10.34 ± 3.89 bpm, respectively. However, in the continued presence of either ATX-II or ACh, 10 µM IVA caused a smaller decrease in HR by 47.03 ± 9.07 bpm and 49.14 ± 4.34 bpm, respectively ( p < 0.05 vs. ATX-II or ACh alone, Fig. 1 ). Table 1 Effects of ivabradine on ECGs in isolated hearts at the sinus heart rate Ivabradine Concentration (µM) 0 0.1 0.3 1.0 3.0 6.0 10.0 Heart Rates (bpm) 161.90 ± 2.81 147.76 ± 5.77* 138.62 ± 6.65* 128.36 ± 5.63* 103.30 ± 6.04* 91.50 ± 6.08* 81.14 ± 5.15* PR interval (ms) 42.84 ± 3.02 39.88 ± 2.70 41.68 ± 2.80 44.12 ± 3.77* 48.80 ± 3.53* 54.68 ± 3.38* 61.96 ± 3.50* QRS width (ms) 62.00 ± 2.03 62.16 ± 2.65 60.13 ± 8.26 61.49 ± 6.16 62.16 ± 4.69 76.22 ± 15.19* 80.46 ± 7.33* QT interval (ms) 212.00 ± 3.56 211.02 ± 5.36 213.44 ± 6.49 210.56 ± 5.16 216.26 ± 3.69* 218.49 ± 2.87* 220.16 ± 6.94* *: p < 0.05 vs. baseline. IVA changed the atrial MAPD 90, EPR and PRR in paced hearts IVA, at high concentrations of 10 µM, prolonged the MAPD 90 , ERP and PRR when hearts were paced at a CL of 570 ms before and after IVA infusion from 50.59 ± 2.55, 92.50 ± 5.23 and 44.29 ± 3.03 ms to 58.93 ± 1.21, 126.25 ± 7.14 and 68.57 ± 5.52 ms, respectively (n = 8, p < 0.05 vs. baseline; Fig. 2 A). When the hearts were paced at a CL of 350 ms, IVA (10 µM) significantly prolonged the atrial MAPD 90 , ERP and PRR from 47.62 ± 1.72, 85.00 ± 3.45 and 40.75 ± 3.45 ms to 62.08 ± 3.01, 120.80 ± 6.09 and 61.75 ± 3.68 ms (n = 13, p < 0.05 vs. baseline; Figure. 2A). ATX-II and ACh caused either prolongation or shortening of the MAPD 90 , ERP and PRR in heats were paced at a CL of 350 ms, respectively (n = 8 and 8, respectively, p < 0.05 vs. baseline, Fig. 2 B-C). IVA (1–10 µM) prolonged the MAPD 90 in 0.3 µM ACh-treated hearts ( p < 0.05 vs. ACh alone; Fig. 2 B-a) but shortened the MAPD 90 in 2 nM ATX-II-treated hearts ( p < 0.05 vs. ATX-II alone; Fig. 2 C-a). For the ERP and PRR, IVA (1–10 µM) showed prolonging effects in all hearts ( p < 0.05 vs. ATX-II or ACh alone; Fig. 2 B-b-c and 2 C-b-c). These results suggest that the effect of IVA on atrial electric parameters depends on the hearts’ substrate in rabbits. Effects of IVA on the APs of atrial myocytes IVA (0.3 µM) reduced the AP amplitude (APA) and maximum upstroke velocity of the AP (V max ) ( p < 0.05 vs. baseline, Table 2 ), and shortened the APD 30 (n = 12, p < 0.05 vs. baseline) without changing the resting membrane potential (RMP). A high concentration of IVA (10 µM) reduced the RMP and shortened the APD 50 and APD 90 ( p < 0.05 vs. baseline). Table 2 Effects of ivabradine on action potentials in control atrial myocytes Ivabradine Concentration (µM) 0 0.1 0.3 1 3 RMP (mV) -78.72 ± 2.40 -78.37 ± 2.53 -77.22 ± 2.67 -76.96 ± 2.38 -72.45 ± 3.81* APA (mV) 139.02 ± 5.43 136.58 ± 6.04 128.03 ± 5.37* 125.62 ± 5.24* 107.64 ± 6.27* V max (V/s) 187.47 ± 14.85 165.89 ± 16.80 175.12 ± 14.73* 163.59 ± 12.16* 147.53 ± 12.00* APD 30 (ms) 131.06 ± 4.17 122.91 ± 5.94* 111.00 ± 9.92* 89.60 ± 8.02* 88.91 ± 13.22* APD 50 (ms) 154.20 ± 14.16 150.98 ± 13.17 150.06 ± 10.43 130.28 ± 11.44 100.29 ± 17.94* APD 90 (ms) 222.36 ± 12.31 227.55 ± 13.36 222.56 ± 13.88 203.72 ± 17.46 178.93 ± 16.76* *: p < 0.05 vs. baseline. RMP: resting membrane potential; APA: action potential amplitude; V max : maximum upstroke velocity of the action potential; APD 30 : action potential duration at which repolarization was 30% completed; APD 50 : action potential duration at which repolarization was 50% completed; APD 90 : action potential duration at which repolarization was 90% completed. ATX-II (1 nM) slowed the V max and prolonged the APD 30 , APD 50 and APD 90 ( p < 0.05 vs. baseline, n = 8, Table 3 and Fig. 3 A) without changing the RMP and APA. In the presence of ATX-II, the administration of IVA (0.3 and 3 µM) shortened the APD 30 , APD 50 and APD 90 (p < 0.05 vs. ATX-II alone). However, the administration of ACh alone increased the RMP, decreased the APA, and shortened the APD 30 , APD 50 and APD 90 values of atrial myocytes (n = 8, p < 0.05 vs. baseline, Table 3 and Fig. 3 B), respectively. In the continued presence of ACh, IVA (0.3 and 3 µM) maximally prolonged the ACh-induced shortening of the APD 30 , APD 50 and APD 90 ( p < 0.05 vs. ACh alone). The present results indicate that IVA affects the depolarization of APs in atrial cells while influencing the whole AP process in ATX-II- and ACh-pretreated cells. Table 3 Effects of ivabradine on action potentials in Ach or ATX-II pretreated rabbit atrial myocytes ATX-II (1 nM) + ivabradine ACh (0.3 µM) + ivabradine 0 µM ivabradine 0.3 µM ivabradine 3 µM ivabradine 0 µM ivabradine 0.3 µM ivabradine 3 µM ivabradine RMP (mV) -78.03 ± 3.03 -79.34 ± 3.60 -77.27 ± 2.94 -65.72 ± 3.61*# -66.16 ± 4.15# -74.87 ± 4.42 APA (mV) 138.56 ± 6.65 137.62 ± 6.24 128.83 ± 6.12# 123.78 ± 4.13*# 124.55 ± 8.91 107.87 ± 11.09 V max (V/s) 167.63 ± 21.58* 162.89 ± 23.59 160.24 ± 23.64 149.55 ± 15.41 143.51 ± 14.35 134.32 ± 13.64 APD 30 (ms) 159.67 ± 13.07* 126.35 ± 22.08* 91.13 ± 26.66* 91.99 ± 17.60* 97.43 ± 15.73* 101.49 ± 17.61* APD 50 (ms) 179.97 ± 20.69* 152.70 ± 23.14* 122.71 ± 25.47* 121.31 ± 11.11* 142.04 ± 17.85* 131.85 ± 13.47* APD 90 (ms) 260.94 ± 19.26* 227.76 ± 25.16* 190.71 ± 29.52* 126.41 ± 10.48*# 146.47 ± 8.89*# 140.18 ± 5.15* *: p < 0. 05 vs. baseline or ATX-II or ACh alone, #: p < 0.05 vs. ivabradine alone group. RMP: resting membrane potential; APA: action potential amplitude; V max : maximum upstroke velocity of the action potential; APD 30 : action potential duration at which repolarization was 30% completed; APD 50 : action potential duration at which repolarization was 50% completed; APD 90 : action potential duration at which repolarization was 90% completed. IVA caused atrial arrhythmias in hearts and DADs in atrial myocytes Atrial arrhythmias, were not observed before IVA administration (Fig. 4 A), but these were induced by programmed stimulations in the presence of IVA in 6 of 23 hearts (26.1%, p < 0.05 vs. baseline) and 10 of 13 hearts (76.9%, p < 0.05 vs. baseline) in hearts paced at CL of 350 and 570 ms, respectively (Fig. 4 B). In contrast, in hearts paced at 350 ms and pretreated with either ATX-II or ACh (no atrial arrhythmia), the incidence of IVA induced atrial arrhythmias significantly increased in 8 of 18 (44.4%, p < 0.05 vs. ATX-II alone) and 13 of 21 (61.9%, p < 0.05 vs. ACh alone) hearts, respectively (Fig. 4 C). The atrial single-cell patch-clamp tests indicated that IVA induced DADs but not EADs, with incidences of 41.7% (5/12), 62.5% (5/8) and 50.0% (4/8) in control, ATX-II-treated and ACh-treated cells, respectively ( p < 0.05 vs. baseline, Fig. 5 ). IVA altered the properties of Ca 2+ sparks and Ca 2+ transport function with regulating related protein expression Compared to baseline (no IVA, Fig. 6 A), IVA (0.3 µM) increased the spark frequency, amplitude and FWHM of the Ca 2+ spark by 1.9-, 4.2- and 2.7-fold ( p < 0.05 , Fig. 6 B), respectively, and the FDHM remained unchanged (Fig. 6 B). These results suggest that IVA mainly affects the spatial characteristics but not the temporal properties of Ca 2+ sparks. As shown in Fig. 7 A, SERCA transporting function (r SERCA ) was depressed by 0.3 µM and 3 µM but not 0.03 µM IVA from 77.53 ± 1.27% to 62.86 ± 4.93% and 57.59%±2.56% (n = 11–18, p < 0.05 vs. baseline) in atrial myocytes, along with decreasing SERCA2 expression by ≥ 0.1 µM IVA in atrium in a concentration-dependent manner ( p < 0.05 vs. baseline, Fig. 7 B). The expression of RyR2 and NCX1 was increased in hearts treated with 1–10 µM and 10 µM IVA (n = 3–6, p < 0.05 vs. baseline, Fig. 7 C-D). Discussion The main findings of this study include the following: (1) the intrinsic heart rate reduction by IVA was attenuated in hearts with either increased vagal activity or late I Na ; (2) IVA changed the MAPD 90 , and prolonged ERP and PRR in the atria and decreased the APA and V max in myocytes at relatively low therapeutic concentrations (≥ 0.1 µM) and lengthened the QRS and QT intervals in the high concentration range (> 3 µM) in isolated hearts; (3) the modulation by IVA of the atrial MAPD 90 and APD was condition dependent, i.e., it prolonged the MAPD 90 /APD in ACh-treated hearts but shortened the MAPD 90 /APD in ATX-II-treated hearts or cells; (4) IVA (0.03-10 µM) induced a greater incidence of atrial arrhythmias either at slow HR or in the presence of ATX-II or ACh as well as DADs in atrial myocytes; and (5) IVA increased the frequency, amplitude, and FWHM of calcium sparks, depressed Ca 2+ transport by SERCA, upregulated RyR2 and NCX1 protein expression, and downregulated SERCA2 protein expression, leading to intracellular Ca 2+ inhomeostasis. The intrinsic SHR was reduced by IVA at relevant clinical concentrations (about 0.02–0.05 µM [ 25 ]), which is conformed to that IVA inhibited I f and slowed HR [ 26 ]. Interestingly, in isolated hearts treated with ATX-II or ACh to increase late I Na or vagal activity, respectively, the amplitude of HR blunted by IVA was reduced, which might result from the slower basal HR by drugs or cardiac pathological conditions. The MAPD 90 , ERP and PRR prolongation evoked by high concentrations of IVA alone were in consistent with previous research in rabbit hearts [ 27 ], which could be attributed to the I Kr blockade (IC 50 = 2.8 µM) at concentrations greater than the therapeutic range [ 28 ], suggesting a potential of QT prolongation and the tendency to develop TdP if it was overdosed [ 29 ]. IVA caused atrial arrhythmias with low incidence in control heart but increased incidence in hearts with either a slow HR, or in heart with augment late I Na or increased vagal activity. IVA induced a higher incidence of atrial arrhythmias in hearts paced at a CL of 570 ms than those paced at a CL of 350 ms (76.9% vs. 26.1%, p < 0.05). In hearts with increased late I Na by ATX-II or simulate vagal activity by using ACh, IVA changed MAPD 90 , lengthened the ERP and PRR at high concentrations range, and induced atrial arrhythmias in 44.4% and 61.9% respectively, when hearts were paced at a fixed CL of 350 ms, suggesting that the risk of atrial arrhythmia induced by IVA was increased under these conditions. The risk of AF increased by 24% in patients treated with IVA in clinical studies [ 12 ]. Wu et al. reported that an increase in late I Na by ATX-II potentiated the proarrhythmic activity of low-risk arrhythmic drugs [ 30 ]. The prolongation of the MAPD caused by drugs that purely inhibit I Kr is synergistically increased in hearts treated with late I Na enhancers [ 30 ]. However, drugs that potentially inhibit late I Na cause an increase (such as pentobarbital) or sometimes a shortening (such as ranolazine) of the MAPD [ 17 ]. I f is a kind of Na + /K + mixed current mainly involved in the automatic depolarization of sinoatrial node cells in phase 4 [ 31 ]. IVA decreased the amplitude and V max of APs without affecting the AP duration at relatively low concentrations, which might be attributed to the inhibitory effect on the I Na in atrial myocytes. IVA mainly affected the APD and triggered activities, i.e., DADs, of atrial cells pretreated with either ACh or ATX-II. When IVA was applied to ATX-II-/ACh-treated cells, the APD 30 , APD 50 and APD 90 were either shortened or prolonged, indicating that IVA could also affect I K1 and I KACh under certain conditions without affecting the RMP, APA and V max in the low therapeutic concentration range [ 32 ]. Finally, IVA increased DADs but not EADs in both the absence and presence of either ACh or ATX-II in atrial myocytes. DADs are related to intracellular calcium overload and abnormal Ca 2+ handling associated with an increase in Na + /Ca 2+ exchange [ 33 , 34 ]. Ca 2+ instability occurs in AF and contributes to atrial arrhythmias and the maintenance of AF[ 35 ]. These mechanisms may be attributed to changes in the Ca 2+ release flux as the Ca 2+ gradient crosses the SR membrane or to luminal Ca 2+ -dependent RyR regulation. Diastolic Ca 2+ sparks are spontaneous bouts of localized inter-RyR Ca 2+ -induced Ca 2+ release (CICR) that are likely triggered by a rare stochastic opening of a single RyR channel. A spark occurs if the RyR Ca 2+ flux amplitude mediated by that rare channel opening is sufficient to drive inter-RyR CICR. IVA mainly enhanced the frequency, amplitude and FWHM (spatial characteristics) with little effect on the FDHM (temporal properties) of Ca 2+ sparks. The results of this study indicated that IVA increased Ca 2+ release and that Ca 2+ -based arrhythmogenic substrates may contribute to the initiation of AF caused by IVA. Collectively, IVA-induced AF might be related to atrial DADs and activation of Ca 2+ sparks. In addition, we found that SERCA function was decreased by 0.3 µM IVA in rabbit atrial myocytes. Decreased SERCA expression was also observed in our study (Fig. 7 ). The predominant resource of increased intracellular Ca 2+ is yet to be fully determined in this study and is worth further investigation. When [Ca 2+ ] i increase due to spontaneous Ca 2+ release events, a Ca 2+ -based membrane current is activated during diastole. This arrhythmogenic transient inward current (I ti ), which is carried by the sarcolemma NCX, is responsible for DAD generation. Enhanced late I Na is one of the causes of increased [Ca 2+ ] i because it increases [Na + ] i and then [Ca 2+ ] i through NCX to facilitate DAD formation; therefore, it was used in this project to augment the proarrhythmic risk[ 30 ] of IVA. The results of this study indicated that ivabradine-induced AF is more, feasible in slow rate and further verifications are warranted. Drug-induced AF may have diverse mechanisms. Adequate understanding of the mechanisms underlying the increased risk of drug-induced AF is critical for the prevention and management of this kind of AF. The results of this study indicated that intracellular Ca 2+ inhomeostasis-associated triggered activities and the reduction of HR by IVA might have synergistic effects to increase the risk of IVA-induced AF. Further study will be needed to determine how IVA induces Ca 2+ handling abnormalities and the characteristics of IVA-induced AF under different pathophysiological conditions. Conclusions IVA reduced sinus rate, evoked Ca 2+ sparks and caused DAD-driven trigger activities to induce or increase the risk of AF in a condition-dependent manner. Slower HR, enhancement of vagal activity and late I Na facilitated the proarrhythmic effects of IVA. Abbreviations IVA, ivabradine; HCN, hyperpolarization-activated cyclic nucleotide; AF, atrial fibrillation; AP, action potential; ATX-II, anemone toxin-II; ACh, acetylcholine; SHR, sinus heart rate; MAP, monophasic AP; MAPD, MAP duration; MPAD 90 , MAP duration at which repolarization was 90% completed; ERP, atrial effective refractory period; PRR, post-repolarization refractoriness; RMP, resting membrane potential; APA, AP amplitude; Vmax, maximum upstroke velocity of the AP; DAD, delayed afterdepolarization; EAD, early afterdepolarization; FWHM, full width at half-maximum; FDHM, full duration at half-maximum; RyR, ryanodine receptor; NCX, Na + /Ca 2+ exchanger; SERCA, sarcoplasmic reticulum calcium pump/sarcoplasmic reticulum calcium-ATPases. Declarations Ethics approval and consent to participate Animal used in this study were purchased from Beijing Fangyuanyuan Breeding Farm and conformed to the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies [36] and was approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (201879). Consent for publication All authors have read this manuscript and agreed publication in its current version. Availability of data and materials N/A Competing Interests The authors declare that there are no conflicts of interest. Funding This study was funded by the National Natural Science Foundation of China (Grant Nos. 82370312). Authors' contributions C.W. and L.W. conceived and designed the study. C.W., B.L., M.L., Q.Y., G.L., X.L., Q.Z., and S.Y. performed the experiments and collected the data. C.W., B.L., M.L., and L.W. analyzed and interpreted the data. C.W. and L.W. drafted the manuscript. All authors (C.W., B.L., M.L., Q.Y., G.L., X.L., Q.Z., S.Y., L.W.) critically reviewed, edited, and approved the final manuscript. Acknowledgements N/A References Tisdale JE, Chung MK, Campbell KB, Hammadah M, Joglar JA, Leclerc J, Rajagopalan B: Drug-Induced Arrhythmias: A Scientific Statement From the American Heart Association . Circulation 2020, 142 (15):e214-e233. Lei M, Wu L, Terrar DA, Huang CL: Modernized Classification of Cardiac Antiarrhythmic Drugs . Circulation 2018, 138 (17):1879-1896. Lei M, Wu L, Terrar DA, Huang CL: The modernized classification of cardiac antiarrhythmic drugs: Its application to clinical practice . Heart Rhythm 2025. Nattel S, Heijman J, Zhou L, Dobrev D: Molecular Basis of Atrial Fibrillation Pathophysiology and Therapy: A Translational Perspective . Circ Res 2020, 127 (1):51-72. Dobrev D, Heijman J, Hiram R, Li N, Nattel S: Inflammatory signalling in atrial cardiomyocytes: a novel unifying principle in atrial fibrillation pathophysiology . 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Martin RI, Pogoryelova O, Koref MS, Bourke JP, Teare MD, Keavney BD: Atrial fibrillation associated with ivabradine treatment: meta-analysis of randomised controlled trials . Heart (British Cardiac Society) 2014, 100 (19):1506-1510. Tanboğa İ H, Topçu S, Aksakal E, Gulcu O, Aksakal E, Aksu U, Oduncu V, Ulusoy FR, Sevimli S, Kaymaz C: The Risk of Atrial Fibrillation With Ivabradine Treatment: A Meta-analysis With Trial Sequential Analysis of More Than 40000 Patients . Clinical cardiology 2016, 39 (10):615-620. Wang J, Yang YM, Li Y, Zhu J, Lian H, Shao XH, Zhang H, Fu YC, Zhang LF: Long-term treatment with ivabradine in transgenic atrial fibrillation mice counteracts hyperpolarization-activated cyclic nucleotide gated channel overexpression . Journal of cardiovascular electrophysiology 2019, 30 (2):242-252. Frommeyer G, Sterneberg M, Dechering DG, Ellermann C, Bögeholz N, Kochhäuser S, Pott C, Fehr M, Eckardt L: Effective suppression of atrial fibrillation by ivabradine: Novel target for an established drug? International journal of cardiology 2017, 236 :237-243. Fontenla A, Tamargo J, Salgado R, López-Gil M, Mejía E, Matía R, Toquero J, Montilla I, Rajjoub EA, García-Fernandez FJ et al : Ivabradine for controlling heart rate in permanent atrial fibrillation: A translational clinical trial . Heart Rhythm 2023, 20 (6):822-830. Katsi V, Skalis G, Kallistratos MS, Tsioufis K, Makris T, Manolis AJ, Tousoulis D: Ivabradine and metoprolol in fixed dose combination: When, why and how to use it . Pharmacol Res 2019, 146 :104279. Chu Y, Yang Q, Ren L, Yu S, Liu Z, Chen Y, Wei X, Huang S, Song L, Zhang P et al : Late Sodium Current in Atrial Cardiomyocytes Contributes to the Induced and Spontaneous Atrial Fibrillation in Rabbit Hearts . J Cardiovasc Pharmacol 2020, 76 (4):437-444. Yu S, Li G, Huang CL, Lei M, Wu L: Late sodium current associated cardiac electrophysiological and mechanical dysfunction . Pflugers Arch 2018, 470 (3):461-469. Wu L, Rajamani S, Shryock JC, Li H, Ruskin J, Antzelevitch C, Belardinelli L: Augmentation of late sodium current unmasks the proarrhythmic effects of amiodarone . Cardiovascular research 2008, 77 (3):481-488. Liu X, Ren L, Yu S, Li G, He P, Yang Q, Wei X, Thai PN, Wu L, Huo Y: Late sodium current in synergism with Ca(2+)/calmodulin-dependent protein kinase II contributes to β-adrenergic activation-induced atrial fibrillation . Philos Trans R Soc Lond B Biol Sci 2023, 378 (1879):20220163. Wit AL: Afterdepolarizations and triggered activity as a mechanism for clinical arrhythmias . Pacing and clinical electrophysiology : PACE 2018. Picht E, Zima AV, Blatter LA, Bers DM: SparkMaster: automated calcium spark analysis with ImageJ . American journal of physiology Cell physiology 2007, 293 (3):C1073-1081. Choi HS, Eisner DA: The role of sarcolemmal Ca2+-ATPase in the regulation of resting calcium concentration in rat ventricular myocytes . The Journal of physiology 1999, 515 ( Pt 1) (Pt 1):109-118. Zhang Q, Ma JH, Li H, Wei XH, Zheng J, Li G, Wang CY, Wu Y, He QH, Wu L: Increase in CO(2) levels by upregulating late sodium current is proarrhythmic in the heart . Heart rhythm 2019, 16 (7):1098-1106. Sheldon RS, Grubb BP, 2nd, Olshansky B, Shen WK, Calkins H, Brignole M, Raj SR, Krahn AD, Morillo CA, Stewart JM et al : 2015 heart rhythm society expert consensus statement on the diagnosis and treatment of postural tachycardia syndrome, inappropriate sinus tachycardia, and vasovagal syncope . Heart rhythm 2015, 12 (6):e41-63. Ceconi C, Cargnoni A, Francolini G, Parinello G, Ferrari R: Heart rate reduction with ivabradine improves energy metabolism and mechanical function of isolated ischaemic rabbit heart . Cardiovascular research 2009, 84 (1):72-82. Suenari K, Cheng CC, Chen YC, Lin YK, Nakano Y, Kihara Y, Chen SA, Chen YJ: Effects of ivabradine on the pulmonary vein electrical activity and modulation of pacemaker currents and calcium homeostasis . Journal of cardiovascular electrophysiology 2012, 23 (2):200-206. DiFrancesco D, Camm JA: Heart rate lowering by specific and selective I(f) current inhibition with ivabradine: a new therapeutic perspective in cardiovascular disease . Drugs 2004, 64 (16):1757-1765. Wu L, Ma J, Li H, Wang C, Grandi E, Zhang P, Luo A, Bers DM, Shryock JC, Belardinelli L: Late sodium current contributes to the reverse rate-dependent effect of IKr inhibition on ventricular repolarization . Circulation 2011, 123 (16):1713-1720. Wu L, Rajamani S, Li H, January CT, Shryock JC, Belardinelli L: Reduction of repolarization reserve unmasks the proarrhythmic role of endogenous late Na(+) current in the heart . American journal of physiology Heart and circulatory physiology 2009, 297 (3):H1048-1057. Kleinbongard P, Gedik N, Witting P, Freedman B, Klöcker N, Heusch G: Pleiotropic, heart rate-independent cardioprotection by ivabradine . British journal of pharmacology 2015, 172 (17):4380-4390. van der Heyden MA, Jespersen T: Pharmacological exploration of the resting membrane potential reserve: Impact on atrial fibrillation . European journal of pharmacology 2016, 771 :56-64. Voigt N, Heijman J, Wang Q, Chiang DY, Li N, Karck M, Wehrens XHT, Nattel S, Dobrev D: Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation . Circulation 2014, 129 (2):145-156. Voigt N, Li N, Wang Q, Wang W, Trafford AW, Abu-Taha I, Sun Q, Wieland T, Ravens U, Nattel S et al : Enhanced sarcoplasmic reticulum Ca2+ leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation . Circulation 2012, 125 (17):2059-2070. Dridi H, Kushnir A, Zalk R, Yuan Q, Melville Z, Marks AR: Intracellular calcium leak in heart failure and atrial fibrillation: a unifying mechanism and therapeutic target . Nature reviews Cardiology 2020, 17 (11):732-747. Tveden-Nyborg P, Bergmann TK, Jessen N, Simonsen U, Lykkesfeldt J: BCPT policy for experimental and clinical studies . Basic Clin Pharmacol Toxicol 2021, 128 (1):4-8. Additional Declarations No competing interests reported. Supplementary Files WB.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7111461","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513778482,"identity":"11336408-8ca4-41a4-90ed-cb9c767cf09c","order_by":0,"name":"Chengyu Wang","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chengyu","middleName":"","lastName":"Wang","suffix":""},{"id":513778484,"identity":"d5b0d71e-d8cc-49bb-81d2-23d5e50b07fb","order_by":1,"name":"Bingxun Li","email":"","orcid":"","institution":"Yichang Central People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Bingxun","middleName":"","lastName":"Li","suffix":""},{"id":513778487,"identity":"4df83f62-1508-41a1-9bd1-9d9401dc36a2","order_by":2,"name":"Mingjie Lin","email":"","orcid":"","institution":"Cheeloo College of Medicine, Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Mingjie","middleName":"","lastName":"Lin","suffix":""},{"id":513778490,"identity":"515403ce-c079-47e6-a1d2-a111213f80a6","order_by":3,"name":"Qiaomei Yang","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiaomei","middleName":"","lastName":"Yang","suffix":""},{"id":513778493,"identity":"9c746acb-9e72-49e1-90c9-0cf53206dec0","order_by":4,"name":"Gang Li","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Li","suffix":""},{"id":513778496,"identity":"742614c8-218b-4ece-bf2b-578f12c76bd3","order_by":5,"name":"Xiaoyan Liu","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Liu","suffix":""},{"id":513778498,"identity":"4e664cf1-0824-47a1-9127-fcb50c6dc300","order_by":6,"name":"Qing Zhang","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Zhang","suffix":""},{"id":513778500,"identity":"a1787101-8698-4370-beb1-35cd551e9119","order_by":7,"name":"Shandong Yu","email":"","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shandong","middleName":"","lastName":"Yu","suffix":""},{"id":513778501,"identity":"a5973889-255d-4392-910c-005e5291d157","order_by":8,"name":"Lin Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYFAD9sbGBx9I08JzuNlwBmlaJNLbpDmIUWhwI/nYw697bBK3Sz5skGZgsJPTbSCoJS3dWOZZWuLO2YkNxgUMycZmBwhoMbuRYyYtceBw4obbiQ3JMxgOJG4jrCX/G0TLzYMNh3mI05LDJvkBpOUGY2MzUVrszzwzN2Y4kGa8syexmXGGARF+kWxPfvbwxwEb2e3sx5//+FBhJ0dQCxCwMfMASQMw24CwcrAWxh/EKx4Fo2AUjIKRCAALfEnyF5wStAAAAABJRU5ErkJggg==","orcid":"","institution":"Peking University First Hospital","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-07-13 06:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7111461/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7111461/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91307815,"identity":"d944952d-8aa8-4a43-a689-b977beb60e30","added_by":"auto","created_at":"2025-09-15 06:41:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284617,"visible":true,"origin":"","legend":"\u003cp\u003eIvabradine (IVA) decreased sinus heart rate (SHR) in rabbit isolated hearts. ○: IVA alone (n=8); ●: ATX-II+IVA group (n=8); ■: ACh+IVA group (n=8). *: \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e vs. baseline or ATX-II or ACh alone; #: \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e vs. IVA alone group.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/29306e59aa95b18d9d40f4b0.png"},{"id":91306759,"identity":"d3cf30f0-7421-4d50-8c9f-ad9f22543e75","added_by":"auto","created_at":"2025-09-15 06:33:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1499075,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ivabradine (IVA) on the MAPD\u003csub\u003e90\u003c/sub\u003e (a), ERP (b) and PRR (c) in control or ATX-II- or ACh-treated hearts. *: \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e vs. baseline or ATX-II or ACh alone; #:\u003cem\u003e p\u0026lt;0.05\u003c/em\u003e vs. IVA alone group. MPAD\u003csub\u003e90\u003c/sub\u003e, monophasic action potential duration at which repolarization was 90% completed; ERP, atrial effective refractory period; PRR, postrepolarization refractoriness;\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/bb831057d6a6eb5fe644b78e.png"},{"id":91306768,"identity":"de3b76b3-60aa-443f-966c-878188e7501d","added_by":"auto","created_at":"2025-09-15 06:33:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14096557,"visible":true,"origin":"","legend":"\u003cp\u003eTypical diagram of action potentials in single atrial myocyte of rabbits in absence or presence of reagents.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/b22a8ca7cbe779967dcf8c49.png"},{"id":91306766,"identity":"7f0a542d-6b47-499d-bb9a-fe9f752f0633","added_by":"auto","created_at":"2025-09-15 06:33:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12387854,"visible":true,"origin":"","legend":"\u003cp\u003eIvabradine (IVA) increased the incidence of atrial arrhythmias in ATX-II- or ACh-treated isolated hearts. A: RepresentativeMAP (top) and ECG (bottom) of atrial arrhythmias. B-C: Incidence of atrial arrhythmias in all groups. *: \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e vs. baseline or ATX-II or ACh alone; #: \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e vs. IVA alone group. MAP, monophasic action potential.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/060e4ff3b71ad31fe800c503.png"},{"id":91306765,"identity":"f0bef66a-5c27-4120-8aea-e8691ec5e122","added_by":"auto","created_at":"2025-09-15 06:33:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2231210,"visible":true,"origin":"","legend":"\u003cp\u003eIvabradine (IVA) aggravated the incidence of DADs (delayed afterdepolarization) in ATX-II- or ACh-treated atrial myocytes. A: Representative APs and DADs from atrial myocytes. B-C: Incidence of DADs in all groups. *: \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e vs. baseline or ATX-II or ACh alone; #: \u003cem\u003ep\u0026lt;0.05\u003c/em\u003e vs. IVA alone group.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/7db2a2b28150c76f1136d2f0.png"},{"id":91308169,"identity":"22ffd44a-21b7-45d1-9ca0-a1b77becd551","added_by":"auto","created_at":"2025-09-15 06:49:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14378601,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Ivabradine (IVA) on the Ca\u003csup\u003e2+\u003c/sup\u003e spark. A: Typical pictures of Ca\u003csup\u003e2+\u003c/sup\u003e sparks. B: Ca\u003csup\u003e2+\u003c/sup\u003e spark frequency, amplitude, FWHM (full width at half-maximum) and FDHM (full duration at half-maximum) at baseline and after treatment with 0.03, 0.3, or 3μM IVA in the same cell (n=22). *:\u003cem\u003e p\u0026lt;0.05\u003c/em\u003e vs. baseline or ATX-II/ACh alone.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/87ec86e977ace4871c729ed5.png"},{"id":91306769,"identity":"782da1aa-7fc3-4ab0-bd60-c67f0debd43f","added_by":"auto","created_at":"2025-09-15 06:33:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7046200,"visible":true,"origin":"","legend":"\u003cp\u003eIvabradine (IVA) decreased Ca\u003csup\u003e2+\u003c/sup\u003e transport by SERCA in atrial myocytes (n=11-18), and regulated RyR2, SERCA2 and NCX1 protein expression in atriums (n=4-5). *:\u003cem\u003e p\u0026lt;0.05\u003c/em\u003e vs. baseline. A. Ca\u003csup\u003e2+\u003c/sup\u003e transport by SERCA. B-D. Representative Western blots (top) and relative levels (bottom) of SERCA2, RyR2 and NCX1 proteins normalized to GAPDH and expressed relative to baseline levels are presented. *:\u003cem\u003e p\u0026lt;0.05\u003c/em\u003e vs. baseline. RyR2, ryanodine receptor 2; NCX, Na\u003csup\u003e+\u003c/sup\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e exchanger; SERCA, sarcoplasmic reticulum calcium pump/sarcoplasmic reticulum calcium-ATPases.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/3a7837550ff78296059e7de8.png"},{"id":109615527,"identity":"41fe753f-9e1f-4106-a223-a3da34b7fa77","added_by":"auto","created_at":"2026-05-20 08:27:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":46994917,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/ad22758e-78db-410f-b09d-8309041c45b1.pdf"},{"id":91307832,"identity":"a12015f2-4719-4065-b328-fd06ce8f0341","added_by":"auto","created_at":"2025-09-15 06:41:27","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":25372303,"visible":true,"origin":"","legend":"","description":"","filename":"WB.zip","url":"https://assets-eu.researchsquare.com/files/rs-7111461/v1/b7bdf30dc3eed747fe9bf6f0.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ivabradine causes abnormal intracellular calcium handlings and delayed afterdepolarizations to induce atrial fibrillation in rabbit hearts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDrug-induced cardiac adverse effects are diverse and include both atrial and ventricular proarrhythmias[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although not common, drug induced arrhythmias remain a major safety issue in drug development and in clinical practice and may increase under some pathological or pharmacological, even physiological conditions[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, can be triggered by multiple cardiovascular and non-cardiovascular conditions, which contribute to its pathogenesis through diverse mechanisms of action[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, drug-induced AF received insufficient recognition and is often underestimated in the incidence, despite its potential significance in complicating clinical decision-making. Moreover, the lack of clinical data and inadequate understanding of the underlying mechanisms also limits identification and treatment for drug-induced AF[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIvabradine (IVA), a class 0 antiarrhythmic agent according to the modernized classification of antiarrhythmic drugs[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], selectively inhibits hyperpolarization-activated cyclic nucleotide (HCN) channels to reduce the funny current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e) and sinus heart rate (SHR) and has been used to treat inappropriate sinus tachycardia in addition to or as an alternative to digitalis, β-blockers or calcium channel inhibitors without affecting blood pressure and myocardial contractility[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the effect of IVA on AF remains uncertain[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]: on one hand, clinical research found that the unselective use of IVA was associated with increased risk of developing AF by about 13%[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, experimental models showed that IVA reduced the risk of AF[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, researches also supported the application of IVA in controlling heart rate in AF patients[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Therefore, clinical implications of ivabradine in AF patients might remain controversial[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mechanisms of AF, especially drug induced AF are complex and may be attributed to inflammation and endoplasmic reticulum stress to induce delayed afterdepolarization (DADs). Abnormalities of intracellular calcium concentration ([Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e)-calmodulin kinase (CaMK)II-late sodium current (I\u003csub\u003eNa\u003c/sub\u003e)-intracellular sodium concentration ( [Na\u003csup\u003e+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e) pathway under pathological conditions are reported to promote ventricular arrhythmias and AF[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. An increase in late I\u003csub\u003eNa\u003c/sub\u003e has been shown to increase the proarrhythmic potential of low risk QT prolonging agents and to induce AF[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Because the low incidence of AF, in this study, effects of IVA by interacting with the [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e-CaMKII-late I\u003csub\u003eNa\u003c/sub\u003e-[Na\u003csup\u003e+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e pathway to increase [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e, and to induce DADs and AF under conditions of modulated late I\u003csub\u003eNa\u003c/sub\u003e and vagal activity were investigated in rabbit isolated hearts and atrial myocytes, respectively.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eElectrophysiological study in isolated rabbit hearts\u003c/p\u003e\u003cp\u003eNew Zealand White rabbits (female, 2.5-3.0 kg) were anesthetized using xylazine (16 mg/kg by IM, Huamu; China) and ketamine (40 mg/kg by IM, CAHG; China). Isolated heart perfusion was completed according to the Langendorff system. The concentration-response relationships of IVA on the SHR were tested in spontaneously beating hearts (n = 24), while atrial monophasic APs (MAPs) and arrhythmic events were obtained from hearts paced at a fixed pacing cycle length (CL) after sinoatrial nodes were thermoablated. Hearts were treated with either increasing concentrations of IVA (n = 23) or in the absence and presence of low-concentration anemone toxin-II (ATX-II, n = 18) or acetylcholine (ACh, n = 21), respectively. ATX-II (2 nM) and ACh (0.3 µM) were confirmed not to cause atrial arrhythmias in hearts studied[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMAP, ECG and atrial arrhythmia recordings in isolated hearts\u003c/p\u003e\u003cp\u003eThe atrial MAPs and pseudo-12-lead ECGs were continuously monitored and digitized in real time. The atrial effective refractory period (ERP) and arrhythmic events were induced by the S1S2 programmed stimulations[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The postrepolarization refractory period (PRR) was calculated using the following equation: ERP-MAPD\u003csub\u003e90\u003c/sub\u003e. Atrial tachycardia (AT) was defined as a sequence of three or more consecutive, relatively regular spontaneous atrial beats occurring unexpectedly at a rate exceeding the spontaneous or pacing rate. An episode of AF was defined as a sequence of fast, irregular atrial signals in MAP and ECG recordings with irregular QRS complexes in a 12-channel ECG record. Atrial arrhythmias include AT and AF.\u003c/p\u003e\u003cp\u003eRecordings of action potentials (APs) and triggered activity in single atrial myocytes\u003c/p\u003e\u003cp\u003eAtrial myocytes were enzymatically isolated from New Zealand White rabbits as described in a previous study[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Quiescent and Ca\u003csup\u003e2+\u003c/sup\u003e-tolerant atrial myocytes (n = 28) were selected for recording APs. Atrial myocytes were bathed and perfused (2–3 mL/min) in a bath solution containing the following reagents (in mM; Sigma-Aldrich, MA, USA): 144 NaCl, 5.6 KCl, 1.2 MgCl\u003csub\u003e2\u003c/sub\u003e, 5 HEPES, 1.8 CaCl\u003csub\u003e2\u003c/sub\u003e and 11 Glucose at pH = 7.4 titrated with NaOH and maintained at 22–24°C. The patch pipette solution contained the following reagents (in mM; Sigma-Aldrich, USA): 110K-aspartate, 30 KCl, 5 NaCl, 10 HEPES, 0.1 EGTA, 5 Mg-ATP, 5 creatine phosphate, and 0.05 cAMP) at pH = 7.2 titrated with KOH. APs were induced in current-clamp mode at 1 s pacing cycle lengths. EADs were elicited by changing the stimulation frequency to 0.25 Hz, and DADs were determined following a baseline pacing CL of 9 s and 15 beats with a stimulation frequency of 2.5 Hz.\u003c/p\u003e\u003cp\u003eDAD arises from the resting potential after full repolarization of an action potential and it may reach threshold for activation, while the EAD arises on the shoulder of a preceding action potential plateau and it is favored by slow preceding activation rate and prolonged action potentials[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRecording of Ca\u003csup\u003e2+\u003c/sup\u003e sparks in atrial myocytes using confocal imaging\u003c/p\u003e\u003cp\u003eAtrial myocytes (n = 22) were bathed in an external solution composed of (in mM; Sigma-Aldrich, USA), 135 NaCl, 5.4 KCl, 1.0 MgCl\u003csub\u003e2\u003c/sub\u003e, 1.8 CaCl\u003csub\u003e2\u003c/sub\u003e, 10 glucose, 0.33 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and 10 HEPES, pH = 7.4, with NaOH and incubated in 10 µM Fluo-4 AM (Thermo Fisher Scientific; USA) for 20 minutes. A laser-scanning confocal microscope system (TCS SP5; Leica; Germany) was applied to acquire the Ca\u003csup\u003e2+\u003c/sup\u003e sparks. Sparks were analyzed with SparkMaster[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and the number, frequency, amplitude, full width at half-maximum (FWHM) and full duration at half-maximum (FDHM) of the detected sparks were obtained. A threshold criterion for spark detection of 3.8 was chosen for data analysis.\u003c/p\u003e\u003cp\u003eRecording of Ca\u003csup\u003e2+\u003c/sup\u003e transport by SERCA in atrial myocytes\u003c/p\u003e\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e transport by SERCA was estimated from the rate constants (r) of single exponential curves fitted to the decaying part of the electrically and caffeine-evoked Ca\u003csup\u003e2+\u003c/sup\u003e transients, according to Choi and Eisner [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Cardiomyocytes (n = 11–18) were paced by an electrical field at 1.0Hz. The decay rate constant (r1) of electrically evoked Ca\u003csup\u003e2+\u003c/sup\u003e transients reflect Ca\u003csup\u003e2+\u003c/sup\u003e transport from cytoplasm to SERCA and outside the cell. The constant (r2) of decline of caffeine (20 mM)-evoked Ca\u003csup\u003e2+\u003c/sup\u003e transient reflects Ca\u003csup\u003e2+\u003c/sup\u003e transport outside to the myocytes. Finally, Ca\u003csup\u003e2+\u003c/sup\u003e transport by SERCA was estimated by subtracting r2 from r1 (r\u003csub\u003eSERCA\u003c/sub\u003e=r1-r2), and the relative contribution to relaxation of the Ca\u003csup\u003e2+\u003c/sup\u003e transporters was calculated according to the following formula: SERCA contribution= (r1-r2)/r1[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWestern blotting\u003c/p\u003e\u003cp\u003eLeft atrial tissues (n = 3–6) were collected after isolated heart perfusion and homogenized using a tissue lyser. The levels of RyR2 (LS-C93425, LifeSpan BioSciences, USA), SERCA2 (4388s, Cell Signaling Technology, USA) and NCX1 (5507-I-AP, Proteintech, USA) were determined by Western blotting as described in our previous study[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStatistical analyses\u003c/p\u003e\u003cp\u003eData are reported as the Mean ± SEM. Statistical analyses were performed using IBM SPSS Statistics (version 20.0, IBM, New York, USA). The concentration-response relationships were analyzed using GraphPad Prism for Windows (version 6.02, GraphPad Software, Inc., San Diego, CA). When control/baseline and treatment values were obtained from the same heart/cell, the significance of the differences in the measures before and after interventions was determined by repeated measures one-way ANOVA followed by the Newman-Keul test. The χ\u003csup\u003e2\u003c/sup\u003e test and Fisher’s test were used to compare the incidences of atrial arrhythmias and trigger activities. Differences were considered significant at \u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIVA decreased SHR in isolated rabbit hearts\u003c/p\u003e\u003cp\u003eIVA (\u0026ge;\u0026thinsp;0.1 \u0026micro;M) reduced the HR in a concentration-dependent manner (10 \u0026micro;M IVA reduced the HR by 83.24\u0026thinsp;\u0026plusmn;\u0026thinsp;7.30 bpm), and 10 \u0026micro;M IVA prolonged the PR interval, QRS width and QT interval (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline; Figure. 1 and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in heart treated with IVA alone (n\u0026thinsp;=\u0026thinsp;8). ATX-II (n\u0026thinsp;=\u0026thinsp;8) and ACh (n\u0026thinsp;=\u0026thinsp;8) caused a small decrease in HR by 4.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59 and 10.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89 bpm, respectively. However, in the continued presence of either ATX-II or ACh, 10 \u0026micro;M IVA caused a smaller decrease in HR by 47.03\u0026thinsp;\u0026plusmn;\u0026thinsp;9.07 bpm and 49.14\u0026thinsp;\u0026plusmn;\u0026thinsp;4.34 bpm, respectively (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ATX-II or ACh alone, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of ivabradine on ECGs in isolated hearts at the sinus heart rate\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"15\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"14\" nameend=\"c15\" namest=\"c2\"\u003e\u003cp\u003eIvabradine Concentration (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003e3.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c13\" namest=\"c12\"\u003e\u003cp\u003e6.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e10.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eHeart Rates (bpm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e161.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e147.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.77*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e138.62\u0026thinsp;\u0026plusmn;\u0026thinsp;6.65*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e128.36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.63*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003e103.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.04*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e91.50\u0026thinsp;\u0026plusmn;\u0026thinsp;6.08*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e81.14\u0026thinsp;\u0026plusmn;\u0026thinsp;5.15*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003ePR interval (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e42.84\u0026thinsp;\u0026plusmn;\u0026thinsp;3.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e39.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e41.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e44.12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.77*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003e48.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.53*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e54.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e61.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.50*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eQRS width (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e62.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e62.16\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e60.13\u0026thinsp;\u0026plusmn;\u0026thinsp;8.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e61.49\u0026thinsp;\u0026plusmn;\u0026thinsp;6.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003e62.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e76.22\u0026thinsp;\u0026plusmn;\u0026thinsp;15.19*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e80.46\u0026thinsp;\u0026plusmn;\u0026thinsp;7.33*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eQT interval (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003e212.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003e211.02\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e213.44\u0026thinsp;\u0026plusmn;\u0026thinsp;6.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u003cp\u003e210.56\u0026thinsp;\u0026plusmn;\u0026thinsp;5.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003e216.26\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c14\" namest=\"c13\"\u003e\u003cp\u003e218.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u003cp\u003e220.16\u0026thinsp;\u0026plusmn;\u0026thinsp;6.94*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"15\"\u003e*: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIVA changed the atrial MAPD\u003csub\u003e90,\u003c/sub\u003e EPR and PRR in paced hearts\u003c/p\u003e\u003cp\u003eIVA, at high concentrations of 10 \u0026micro;M, prolonged the MAPD\u003csub\u003e90\u003c/sub\u003e, ERP and PRR when hearts were paced at a CL of 570 ms before and after IVA infusion from 50.59\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55, 92.50\u0026thinsp;\u0026plusmn;\u0026thinsp;5.23 and 44.29\u0026thinsp;\u0026plusmn;\u0026thinsp;3.03 ms to 58.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21, 126.25\u0026thinsp;\u0026plusmn;\u0026thinsp;7.14 and 68.57\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52 ms, respectively (n\u0026thinsp;=\u0026thinsp;8, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). When the hearts were paced at a CL of 350 ms, IVA (10 \u0026micro;M) significantly prolonged the atrial MAPD\u003csub\u003e90\u003c/sub\u003e, ERP and PRR from 47.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72, 85.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45 and 40.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45 ms to 62.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.01, 120.80\u0026thinsp;\u0026plusmn;\u0026thinsp;6.09 and 61.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.68 ms (n\u0026thinsp;=\u0026thinsp;13, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline; Figure. 2A).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eATX-II and ACh caused either prolongation or shortening of the MAPD\u003csub\u003e90\u003c/sub\u003e, ERP and PRR in heats were paced at a CL of 350 ms, respectively (n\u0026thinsp;=\u0026thinsp;8 and 8, respectively, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). IVA (1\u0026ndash;10 \u0026micro;M) prolonged the MAPD\u003csub\u003e90\u003c/sub\u003e in 0.3 \u0026micro;M ACh-treated hearts (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ACh alone; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-a) but shortened the MAPD\u003csub\u003e90\u003c/sub\u003e in 2 nM ATX-II-treated hearts (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ATX-II alone; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-a). For the ERP and PRR, IVA (1\u0026ndash;10 \u0026micro;M) showed prolonging effects in all hearts (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ATX-II or ACh alone; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-b-c and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-b-c). These results suggest that the effect of IVA on atrial electric parameters depends on the hearts\u0026rsquo; substrate in rabbits.\u003c/p\u003e\u003cp\u003eEffects of IVA on the APs of atrial myocytes\u003c/p\u003e\u003cp\u003eIVA (0.3 \u0026micro;M) reduced the AP amplitude (APA) and maximum upstroke velocity of the AP (V\u003csub\u003emax\u003c/sub\u003e) (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and shortened the APD\u003csub\u003e30\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;12, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline) without changing the resting membrane potential (RMP). A high concentration of IVA (10 \u0026micro;M) reduced the RMP and shortened the APD\u003csub\u003e50\u003c/sub\u003e and APD\u003csub\u003e90\u003c/sub\u003e (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of ivabradine on action potentials in control atrial myocytes\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e\u003cp\u003eIvabradine Concentration (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMP (mV)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-78.72\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-78.37\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-77.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-76.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-72.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPA (mV)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e139.02\u0026thinsp;\u0026plusmn;\u0026thinsp;5.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e136.58\u0026thinsp;\u0026plusmn;\u0026thinsp;6.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e128.03\u0026thinsp;\u0026plusmn;\u0026thinsp;5.37*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e125.62\u0026thinsp;\u0026plusmn;\u0026thinsp;5.24*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e107.64\u0026thinsp;\u0026plusmn;\u0026thinsp;6.27*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e (V/s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e187.47\u0026thinsp;\u0026plusmn;\u0026thinsp;14.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e165.89\u0026thinsp;\u0026plusmn;\u0026thinsp;16.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e175.12\u0026thinsp;\u0026plusmn;\u0026thinsp;14.73*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e163.59\u0026thinsp;\u0026plusmn;\u0026thinsp;12.16*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e147.53\u0026thinsp;\u0026plusmn;\u0026thinsp;12.00*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003csub\u003e30\u003c/sub\u003e (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e131.06\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e122.91\u0026thinsp;\u0026plusmn;\u0026thinsp;5.94*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e111.00\u0026thinsp;\u0026plusmn;\u0026thinsp;9.92*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e89.60\u0026thinsp;\u0026plusmn;\u0026thinsp;8.02*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e88.91\u0026thinsp;\u0026plusmn;\u0026thinsp;13.22*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003csub\u003e50\u003c/sub\u003e (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e154.20\u0026thinsp;\u0026plusmn;\u0026thinsp;14.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e150.98\u0026thinsp;\u0026plusmn;\u0026thinsp;13.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e150.06\u0026thinsp;\u0026plusmn;\u0026thinsp;10.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e130.28\u0026thinsp;\u0026plusmn;\u0026thinsp;11.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100.29\u0026thinsp;\u0026plusmn;\u0026thinsp;17.94*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003csub\u003e90\u003c/sub\u003e (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e222.36\u0026thinsp;\u0026plusmn;\u0026thinsp;12.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e227.55\u0026thinsp;\u0026plusmn;\u0026thinsp;13.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e222.56\u0026thinsp;\u0026plusmn;\u0026thinsp;13.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e203.72\u0026thinsp;\u0026plusmn;\u0026thinsp;17.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e178.93\u0026thinsp;\u0026plusmn;\u0026thinsp;16.76*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e*: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline. RMP: resting membrane potential; APA: action potential amplitude; V\u003csub\u003emax\u003c/sub\u003e: maximum upstroke velocity of the action potential; APD\u003csub\u003e30\u003c/sub\u003e: action potential duration at which repolarization was 30% completed; APD\u003csub\u003e50\u003c/sub\u003e: action potential duration at which repolarization was 50% completed; APD\u003csub\u003e90\u003c/sub\u003e: action potential duration at which repolarization was 90% completed.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eATX-II (1 nM) slowed the V\u003csub\u003emax\u003c/sub\u003e and prolonged the APD\u003csub\u003e30\u003c/sub\u003e, APD\u003csub\u003e50\u003c/sub\u003e and APD\u003csub\u003e90\u003c/sub\u003e (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline, n\u0026thinsp;=\u0026thinsp;8, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) without changing the RMP and APA. In the presence of ATX-II, the administration of IVA (0.3 and 3 \u0026micro;M) shortened the APD\u003csub\u003e30\u003c/sub\u003e, APD\u003csub\u003e50\u003c/sub\u003e and APD\u003csub\u003e90\u003c/sub\u003e \u003cem\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ATX-II alone). However, the administration of ACh alone increased the RMP, decreased the APA, and shortened the APD\u003csub\u003e30\u003c/sub\u003e, APD\u003csub\u003e50\u003c/sub\u003e and APD\u003csub\u003e90\u003c/sub\u003e values of atrial myocytes (n\u0026thinsp;=\u0026thinsp;8, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), respectively. In the continued presence of ACh, IVA (0.3 and 3 \u0026micro;M) maximally prolonged the ACh-induced shortening of the APD\u003csub\u003e30\u003c/sub\u003e, APD\u003csub\u003e50\u003c/sub\u003e and APD\u003csub\u003e90\u003c/sub\u003e (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ACh alone). The present results indicate that IVA affects the depolarization of APs in atrial cells while influencing the whole AP process in ATX-II- and ACh-pretreated cells.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffects of ivabradine on action potentials in Ach or ATX-II pretreated rabbit atrial myocytes\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eATX-II (1 nM)\u0026thinsp;+\u0026thinsp;ivabradine\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003eACh (0.3 \u0026micro;M)\u0026thinsp;+\u0026thinsp;ivabradine\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 \u0026micro;M ivabradine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.3 \u0026micro;M ivabradine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 \u0026micro;M ivabradine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 \u0026micro;M ivabradine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.3 \u0026micro;M ivabradine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3 \u0026micro;M ivabradine\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMP (mV)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-78.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-79.34\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-77.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-65.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.61*#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-66.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.15#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-74.87\u0026thinsp;\u0026plusmn;\u0026thinsp;4.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPA (mV)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e138.56\u0026thinsp;\u0026plusmn;\u0026thinsp;6.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e137.62\u0026thinsp;\u0026plusmn;\u0026thinsp;6.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e128.83\u0026thinsp;\u0026plusmn;\u0026thinsp;6.12#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e123.78\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13*#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e124.55\u0026thinsp;\u0026plusmn;\u0026thinsp;8.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e107.87\u0026thinsp;\u0026plusmn;\u0026thinsp;11.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eV\u003csub\u003emax\u003c/sub\u003e (V/s)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e167.63\u0026thinsp;\u0026plusmn;\u0026thinsp;21.58*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e162.89\u0026thinsp;\u0026plusmn;\u0026thinsp;23.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e160.24\u0026thinsp;\u0026plusmn;\u0026thinsp;23.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e149.55\u0026thinsp;\u0026plusmn;\u0026thinsp;15.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e143.51\u0026thinsp;\u0026plusmn;\u0026thinsp;14.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e134.32\u0026thinsp;\u0026plusmn;\u0026thinsp;13.64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003csub\u003e30\u003c/sub\u003e (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e159.67\u0026thinsp;\u0026plusmn;\u0026thinsp;13.07*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e126.35\u0026thinsp;\u0026plusmn;\u0026thinsp;22.08*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e91.13\u0026thinsp;\u0026plusmn;\u0026thinsp;26.66*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e91.99\u0026thinsp;\u0026plusmn;\u0026thinsp;17.60*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e97.43\u0026thinsp;\u0026plusmn;\u0026thinsp;15.73*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e101.49\u0026thinsp;\u0026plusmn;\u0026thinsp;17.61*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003csub\u003e50\u003c/sub\u003e (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e179.97\u0026thinsp;\u0026plusmn;\u0026thinsp;20.69*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e152.70\u0026thinsp;\u0026plusmn;\u0026thinsp;23.14*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e122.71\u0026thinsp;\u0026plusmn;\u0026thinsp;25.47*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e121.31\u0026thinsp;\u0026plusmn;\u0026thinsp;11.11*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e142.04\u0026thinsp;\u0026plusmn;\u0026thinsp;17.85*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e131.85\u0026thinsp;\u0026plusmn;\u0026thinsp;13.47*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAPD\u003csub\u003e90\u003c/sub\u003e (ms)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e260.94\u0026thinsp;\u0026plusmn;\u0026thinsp;19.26*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e227.76\u0026thinsp;\u0026plusmn;\u0026thinsp;25.16*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e190.71\u0026thinsp;\u0026plusmn;\u0026thinsp;29.52*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e126.41\u0026thinsp;\u0026plusmn;\u0026thinsp;10.48*#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e146.47\u0026thinsp;\u0026plusmn;\u0026thinsp;8.89*#\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e140.18\u0026thinsp;\u0026plusmn;\u0026thinsp;5.15*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003e*: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.\u003c/em\u003e05 vs. baseline or ATX-II or ACh alone, #: \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ivabradine alone group. RMP: resting membrane potential; APA: action potential amplitude; V\u003csub\u003emax\u003c/sub\u003e: maximum upstroke velocity of the action potential; APD\u003csub\u003e30\u003c/sub\u003e: action potential duration at which repolarization was 30% completed; APD\u003csub\u003e50\u003c/sub\u003e: action potential duration at which repolarization was 50% completed; APD\u003csub\u003e90\u003c/sub\u003e: action potential duration at which repolarization was 90% completed.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIVA caused atrial arrhythmias in hearts and DADs in atrial myocytes\u003c/p\u003e\u003cp\u003eAtrial arrhythmias, were not observed before IVA administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), but these were induced by programmed stimulations in the presence of IVA in 6 of 23 hearts (26.1%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline) and 10 of 13 hearts (76.9%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline) in hearts paced at CL of 350 and 570 ms, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, in hearts paced at 350 ms and pretreated with either ATX-II or ACh (no atrial arrhythmia), the incidence of IVA induced atrial arrhythmias significantly increased in 8 of 18 (44.4%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ATX-II alone) and 13 of 21 (61.9%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. ACh alone) hearts, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The atrial single-cell patch-clamp tests indicated that IVA induced DADs but not EADs, with incidences of 41.7% (5/12), 62.5% (5/8) and 50.0% (4/8) in control, ATX-II-treated and ACh-treated cells, respectively (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIVA altered the properties of Ca\u003csup\u003e2+\u003c/sup\u003e sparks and Ca\u003csup\u003e2+\u003c/sup\u003e transport function with regulating related protein expression\u003c/p\u003e\u003cp\u003eCompared to baseline (no IVA, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), IVA (0.3 \u0026micro;M) increased the spark frequency, amplitude and FWHM of the Ca\u003csup\u003e2+\u003c/sup\u003e spark by 1.9-, 4.2- and 2.7-fold (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), respectively, and the FDHM remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results suggest that IVA mainly affects the spatial characteristics but not the temporal properties of Ca\u003csup\u003e2+\u003c/sup\u003e sparks. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, SERCA transporting function (r\u003csub\u003eSERCA\u003c/sub\u003e) was depressed by 0.3 \u0026micro;M and 3 \u0026micro;M but not 0.03 \u0026micro;M IVA from 77.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27% to 62.86\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93% and 57.59%\u0026plusmn;2.56% (n\u0026thinsp;=\u0026thinsp;11\u0026ndash;18, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline) in atrial myocytes, along with decreasing SERCA2 expression by \u0026ge;\u0026thinsp;0.1 \u0026micro;M IVA in atrium in a concentration-dependent manner (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The expression of RyR2 and NCX1 was increased in hearts treated with 1\u0026ndash;10 \u0026micro;M and 10 \u0026micro;M IVA (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;6, \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e vs. baseline, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main findings of this study include the following: (1) the intrinsic heart rate reduction by IVA was attenuated in hearts with either increased vagal activity or late I\u003csub\u003eNa\u003c/sub\u003e; (2) IVA changed the MAPD\u003csub\u003e90\u003c/sub\u003e, and prolonged ERP and PRR in the atria and decreased the APA and V\u003csub\u003emax\u003c/sub\u003e in myocytes at relatively low therapeutic concentrations (\u0026ge;\u0026thinsp;0.1 \u0026micro;M) and lengthened the QRS and QT intervals in the high concentration range (\u0026gt;\u0026thinsp;3 \u0026micro;M) in isolated hearts; (3) the modulation by IVA of the atrial MAPD\u003csub\u003e90\u003c/sub\u003e and APD was condition dependent, i.e., it prolonged the MAPD\u003csub\u003e90\u003c/sub\u003e/APD in ACh-treated hearts but shortened the MAPD\u003csub\u003e90\u003c/sub\u003e/APD in ATX-II-treated hearts or cells; (4) IVA (0.03-10 \u0026micro;M) induced a greater incidence of atrial arrhythmias either at slow HR or in the presence of ATX-II or ACh as well as DADs in atrial myocytes; and (5) IVA increased the frequency, amplitude, and FWHM of calcium sparks, depressed Ca\u003csup\u003e2+\u003c/sup\u003e transport by SERCA, upregulated RyR2 and NCX1 protein expression, and downregulated SERCA2 protein expression, leading to intracellular Ca\u003csup\u003e2+\u003c/sup\u003e inhomeostasis.\u003c/p\u003e\u003cp\u003eThe intrinsic SHR was reduced by IVA at relevant clinical concentrations (about 0.02\u0026ndash;0.05 \u0026micro;M [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]), which is conformed to that IVA inhibited I\u003csub\u003ef\u003c/sub\u003e and slowed HR [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Interestingly, in isolated hearts treated with ATX-II or ACh to increase late I\u003csub\u003eNa\u003c/sub\u003e or vagal activity, respectively, the amplitude of HR blunted by IVA was reduced, which might result from the slower basal HR by drugs or cardiac pathological conditions. The MAPD\u003csub\u003e90\u003c/sub\u003e, ERP and PRR prolongation evoked by high concentrations of IVA alone were in consistent with previous research in rabbit hearts [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which could be attributed to the I\u003csub\u003eKr\u003c/sub\u003e blockade (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.8 \u0026micro;M) at concentrations greater than the therapeutic range [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], suggesting a potential of QT prolongation and the tendency to develop TdP if it was overdosed [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIVA caused atrial arrhythmias with low incidence in control heart but increased incidence in hearts with either a slow HR, or in heart with augment late I\u003csub\u003eNa\u003c/sub\u003e or increased vagal activity. IVA induced a higher incidence of atrial arrhythmias in hearts paced at a CL of 570 ms than those paced at a CL of 350 ms (76.9% vs. 26.1%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In hearts with increased late I\u003csub\u003eNa\u003c/sub\u003e by ATX-II or simulate vagal activity by using ACh, IVA changed MAPD\u003csub\u003e90\u003c/sub\u003e, lengthened the ERP and PRR at high concentrations range, and induced atrial arrhythmias in 44.4% and 61.9% respectively, when hearts were paced at a fixed CL of 350 ms, suggesting that the risk of atrial arrhythmia induced by IVA was increased under these conditions. The risk of AF increased by 24% in patients treated with IVA in clinical studies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Wu et al. reported that an increase in late I\u003csub\u003eNa\u003c/sub\u003e by ATX-II potentiated the proarrhythmic activity of low-risk arrhythmic drugs [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The prolongation of the MAPD caused by drugs that purely inhibit I\u003csub\u003eKr\u003c/sub\u003e is synergistically increased in hearts treated with late I\u003csub\u003eNa\u003c/sub\u003e enhancers [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, drugs that potentially inhibit late I\u003csub\u003eNa\u003c/sub\u003e cause an increase (such as pentobarbital) or sometimes a shortening (such as ranolazine) of the MAPD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eI\u003csub\u003ef\u003c/sub\u003e is a kind of Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e mixed current mainly involved in the automatic depolarization of sinoatrial node cells in phase 4 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. IVA decreased the amplitude and V\u003csub\u003emax\u003c/sub\u003e of APs without affecting the AP duration at relatively low concentrations, which might be attributed to the inhibitory effect on the I\u003csub\u003eNa\u003c/sub\u003e in atrial myocytes. IVA mainly affected the APD and triggered activities, i.e., DADs, of atrial cells pretreated with either ACh or ATX-II. When IVA was applied to ATX-II-/ACh-treated cells, the APD\u003csub\u003e30\u003c/sub\u003e, APD\u003csub\u003e50\u003c/sub\u003e and APD\u003csub\u003e90\u003c/sub\u003e were either shortened or prolonged, indicating that IVA could also affect I\u003csub\u003eK1\u003c/sub\u003e and I\u003csub\u003eKACh\u003c/sub\u003e under certain conditions without affecting the RMP, APA and V\u003csub\u003emax\u003c/sub\u003e in the low therapeutic concentration range [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Finally, IVA increased DADs but not EADs in both the absence and presence of either ACh or ATX-II in atrial myocytes. DADs are related to intracellular calcium overload and abnormal Ca\u003csup\u003e2+\u003c/sup\u003e handling associated with an increase in Na\u003csup\u003e+\u003c/sup\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e exchange [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e instability occurs in AF and contributes to atrial arrhythmias and the maintenance of AF[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These mechanisms may be attributed to changes in the Ca\u003csup\u003e2+\u003c/sup\u003e release flux as the Ca\u003csup\u003e2+\u003c/sup\u003e gradient crosses the SR membrane or to luminal Ca\u003csup\u003e2+\u003c/sup\u003e-dependent RyR regulation. Diastolic Ca\u003csup\u003e2+\u003c/sup\u003e sparks are spontaneous bouts of localized inter-RyR Ca\u003csup\u003e2+\u003c/sup\u003e-induced Ca\u003csup\u003e2+\u003c/sup\u003e release (CICR) that are likely triggered by a rare stochastic opening of a single RyR channel. A spark occurs if the RyR Ca\u003csup\u003e2+\u003c/sup\u003e flux amplitude mediated by that rare channel opening is sufficient to drive inter-RyR CICR. IVA mainly enhanced the frequency, amplitude and FWHM (spatial characteristics) with little effect on the FDHM (temporal properties) of Ca\u003csup\u003e2+\u003c/sup\u003e sparks. The results of this study indicated that IVA increased Ca\u003csup\u003e2+\u003c/sup\u003e release and that Ca\u003csup\u003e2+\u003c/sup\u003e-based arrhythmogenic substrates may contribute to the initiation of AF caused by IVA.\u003c/p\u003e\u003cp\u003eCollectively, IVA-induced AF might be related to atrial DADs and activation of Ca\u003csup\u003e2+\u003c/sup\u003e sparks. In addition, we found that SERCA function was decreased by 0.3 \u0026micro;M IVA in rabbit atrial myocytes. Decreased SERCA expression was also observed in our study (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The predominant resource of increased intracellular Ca\u003csup\u003e2+\u003c/sup\u003e is yet to be fully determined in this study and is worth further investigation. When [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e increase due to spontaneous Ca\u003csup\u003e2+\u003c/sup\u003e release events, a Ca\u003csup\u003e2+\u003c/sup\u003e-based membrane current is activated during diastole. This arrhythmogenic transient inward current (I\u003csub\u003eti\u003c/sub\u003e), which is carried by the sarcolemma NCX, is responsible for DAD generation. Enhanced late I\u003csub\u003eNa\u003c/sub\u003e is one of the causes of increased [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e because it increases [Na\u003csup\u003e+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e and then [Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ei\u003c/sub\u003e through NCX to facilitate DAD formation; therefore, it was used in this project to augment the proarrhythmic risk[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] of IVA. The results of this study indicated that ivabradine-induced AF is more, feasible in slow rate and further verifications are warranted.\u003c/p\u003e\u003cp\u003eDrug-induced AF may have diverse mechanisms. Adequate understanding of the mechanisms underlying the increased risk of drug-induced AF is critical for the prevention and management of this kind of AF. The results of this study indicated that intracellular Ca\u003csup\u003e2+\u003c/sup\u003e inhomeostasis-associated triggered activities and the reduction of HR by IVA might have synergistic effects to increase the risk of IVA-induced AF. Further study will be needed to determine how IVA induces Ca\u003csup\u003e2+\u003c/sup\u003e handling abnormalities and the characteristics of IVA-induced AF under different pathophysiological conditions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIVA reduced sinus rate, evoked Ca\u003csup\u003e2+\u003c/sup\u003e sparks and caused DAD-driven trigger activities to induce or increase the risk of AF in a condition-dependent manner. Slower HR, enhancement of vagal activity and late I\u003csub\u003eNa\u003c/sub\u003e facilitated the proarrhythmic effects of IVA.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIVA, ivabradine; HCN, hyperpolarization-activated cyclic nucleotide; AF, atrial fibrillation; AP, action potential; ATX-II, anemone toxin-II; ACh, acetylcholine; SHR, sinus heart rate; MAP, monophasic AP; MAPD, MAP duration; MPAD\u003csub\u003e90\u003c/sub\u003e, MAP duration at which repolarization was 90% completed; ERP, atrial effective refractory period; PRR, post-repolarization refractoriness; RMP, resting membrane potential; APA, AP amplitude; Vmax, maximum upstroke velocity of the AP; DAD, delayed afterdepolarization; EAD, early afterdepolarization; FWHM, full width at half-maximum; FDHM, full duration at half-maximum; RyR, ryanodine receptor; NCX, Na\u003csup\u003e+\u003c/sup\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e exchanger; SERCA, sarcoplasmic reticulum calcium pump/sarcoplasmic reticulum calcium-ATPases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal used in this study were purchased from Beijing Fangyuanyuan Breeding Farm and conformed to the Basic \u0026amp; Clinical Pharmacology \u0026amp; Toxicology policy for experimental and clinical studies [36] and was approved by the Institutional Animal Care and Use Committee of Peking University First Hospital (201879).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read this manuscript and agreed publication in its current version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was funded by the National Natural Science Foundation of China (Grant Nos. 82370312).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.W. and L.W. conceived and designed the study. C.W., B.L., M.L., Q.Y., G.L., X.L., Q.Z., and S.Y. performed the experiments and collected the data. C.W., B.L., M.L., and L.W. analyzed and interpreted the data. C.W. and L.W. drafted the manuscript. All authors (C.W., B.L., M.L., Q.Y., G.L., X.L., Q.Z., S.Y., L.W.) critically reviewed, edited, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN/A\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTisdale JE, Chung MK, Campbell KB, Hammadah M, Joglar JA, Leclerc J, Rajagopalan B: \u003cstrong\u003eDrug-Induced Arrhythmias: A Scientific Statement From the American Heart Association\u003c/strong\u003e. \u003cem\u003eCirculation \u003c/em\u003e2020, \u003cstrong\u003e142\u003c/strong\u003e(15):e214-e233.\u003c/li\u003e\n\u003cli\u003eLei M, Wu L, Terrar DA, Huang CL: \u003cstrong\u003eModernized Classification of Cardiac Antiarrhythmic Drugs\u003c/strong\u003e. \u003cem\u003eCirculation \u003c/em\u003e2018, \u003cstrong\u003e138\u003c/strong\u003e(17):1879-1896.\u003c/li\u003e\n\u003cli\u003eLei M, Wu L, Terrar DA, Huang CL: \u003cstrong\u003eThe modernized classification of cardiac antiarrhythmic drugs: Its application to clinical practice\u003c/strong\u003e. \u003cem\u003eHeart Rhythm \u003c/em\u003e2025.\u003c/li\u003e\n\u003cli\u003eNattel S, Heijman J, Zhou L, Dobrev D: \u003cstrong\u003eMolecular Basis of Atrial Fibrillation Pathophysiology and Therapy: A Translational Perspective\u003c/strong\u003e. \u003cem\u003eCirc Res \u003c/em\u003e2020, \u003cstrong\u003e127\u003c/strong\u003e(1):51-72.\u003c/li\u003e\n\u003cli\u003eDobrev D, Heijman J, Hiram R, Li N, Nattel S: \u003cstrong\u003eInflammatory signalling in atrial cardiomyocytes: a novel unifying principle in atrial fibrillation pathophysiology\u003c/strong\u003e. \u003cem\u003eNature reviews Cardiology \u003c/em\u003e2023, \u003cstrong\u003e20\u003c/strong\u003e(3):145-167.\u003c/li\u003e\n\u003cli\u003eTamargo J, Villacast\u0026iacute;n J, Caballero R, Delp\u0026oacute;n E: \u003cstrong\u003eDrug-induced atrial fibrillation. 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arrhythmia, action potential duration, heart rate, late sodium current, vagal tone, Ca2+ homeostasis ","lastPublishedDoi":"10.21203/rs.3.rs-7111461/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7111461/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eObjective\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe present paper is to determine the effects and underlying mechanisms of ivabradine (IVA) on atrial fibrillation (AF).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eElectrophysiological changes were determined using Langendorff-perfused hearts and patch-clamp techniques. Parameters of Ca\u003csup\u003e2+\u003c/sup\u003e handling were evaluated by using calcium imaging and western blotting.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIVA (0.1\u0026ndash;10 \u0026micro;M) slowed HR in a concentration-dependent manner in isolated hearts of rabbit. IVA induced atrial arrhythmias in 26.1% and 76.9% of hearts paced at a basic cycle length of 350 and 570 ms, respectively. In hearts pretreated with either acetylcholine (ACh) or anemone toxin-II (ATX-II) which caused no inducible atrial arrhythmias, adding to IVA administration caused atrial arrhythmias in 61.9% (13/21) and 44.4% (8/18) of hearts, respectively. In atrial myocytes, IVA induced DADs by 41.7%, 62.5% and 50.0%, respectively, in the absence and presence of either ACh or ATX-II. IVA increased the frequency, amplitude and full width at half-maximum (FWHM) of Ca\u003csup\u003e2+\u003c/sup\u003e sparks and decreased Ca\u003csup\u003e2+\u003c/sup\u003e transport in association with increased protein expression of RyR2 and NCX1 and decreased SERCA2.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIVA increases atrial proarrhythmic risk in hearts with a slow HR, enhanced vagal tone and increased late sodium current by inducing DADs resulting from an enhanced intracellular Ca\u003csup\u003e2+\u003c/sup\u003e inhomeostasis.\u003c/p\u003e","manuscriptTitle":"Ivabradine causes abnormal intracellular calcium handlings and delayed afterdepolarizations to induce atrial fibrillation in rabbit hearts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-15 06:33:21","doi":"10.21203/rs.3.rs-7111461/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f32b463f-7d30-4990-a104-07061bf0b52e","owner":[],"postedDate":"September 15th, 2025","published":true,"recentEditorialEvents":[{"type":"decision","content":"Withdrawn","date":"2026-05-20T08:18:35+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-20T08:26:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-15 06:33:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7111461","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7111461","identity":"rs-7111461","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}