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Introduction

Atrial fibrillation (AF), the most common sustained cardiac arrhythmia in clinical practice, is able to induce cardiac dysfunction and strokes (1). Oxidative stress contributes to the genesis of AF (2) and oxidative modifications of proteins are found in chronic AF patients (3). Reactive oxygen species (ROS) may result in abnormal Ca2+ handling and changes in mitochondrial function, leading to arrhythmogenesis (4–8). Mitochondria are key regulators of cardiomyocyte energy metabolism and redox state control (8). Mitochondrial dysfunction-elicited ROS production was proposed as the basis of the mitochondrial free-radical theory of aging (9–12). Evidence indicates that mitochondrial dysfunction may directly alter cardiomyocyte excitability and cell-to-cell coupling through regulating the adenosine monophosphate protein kinase, the adenosine triphosphate-sensitive potassium channel and the sarcolemmal sodium channel (13–16). Furthermore, coenzyme (Co)-Q10, an agent beneficial for mitochondrial function, is widely used to treat heart failure and ischemic heart diseases, which are critical risk factors of AF (17–19). However, it is not clear whether Co-Q10 has a role in modifying the effects of mitochondrial dysfunction in atrial arrhythmogenesis.

Pulmonary veins (PVs), subsidiary pacemakers, which contain a mixture of working myocardium and pacemaker cells, are an important source of AF initiation and maintenance (20–22). Sinoatrial node (SAN) dysfunction may enhance PV arrhythmogenesis, which may contribute to the high incidence of AF during sick sinus syndrome (23). A previous study has demonstrated that the right and left atria (RA and LA) have different electrical responses to hypoxia and reoxygenation, a condition that may cause mitochondrial dysfunction (24). Therefore, the aim of the present study was to investigate whether mitochondrial dysfunction differentially regulates electrical activity between SANs and PVs or between the RA and LA.

Materials and methods

Ethics statement

The present investigation was approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center (Taipei, Taiwan; IACUC-15-297) and conformed to the institutional Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Rabbit SAN, PV and atrial tissue preparations

As previously described (2,23), all of the rabbits had ad libitum access to food and water, were maintained in a temperature and humidity-controlled environment (20–22°C; 50–70% humidity) with a 12 h light/dark cycle, and were raised in stainless steel cages. A total of 20 male New Zealand rabbits (Animal Health Research Institute, New Taipei City, Taiwan) weighing 1.5–2.0 kg and aged 3–4 months were anesthetized with an intravenous injection of sodium pentobarbital (100 mg/kg of body weight), followed by an intravenous injection of heparin (1,000 IU/kg of body weight). Subsequently, a midline thoracotomy was performed and the heart and lungs were removed. For dissection of the PVs, the LA was opened by an incision along the mitral valve annulus extending from the coronary sinus to the septum in Tyrode's solution, composed of 137 mM NaCl, 4 mM KCl, 15 mM NaHCO3, 0.5 mM NaH2 PO4, 0.5 mM MgCl2, 2.7 mM CaCl2 and 11 mM dextrose. The PV was separated from the atrium at the level of the LA-PV junction and separated from the lungs at the ending of the PV myocardial sleeves. One end of the preparation, consisting of the PV and atrial-PV junction, was pinned with needles to the bottom of a tissue bath. The other end (distal PV) was connected to a Grass FT03C force transducer with a silk thread. The adventitia or epicardial side of the preparation faced upwards. LA and RA tissues were prepared from the LA (10.0×5.0×0.5 mm) and RA appendages (10.0×5.0×0.5 mm), respectively. For SAN-PV tissue preparations, the SAN with the RA and the right superior PV with the LA were isolated. Tissue preparations were superfused with normal Tyrode's solution and were left to equilibrate for 1 h prior to electrophysiological study.

Electrophysiological and pharmacological studies

Transmembrane action potentials (APs) of the SAN, PVs, RA and LA were recorded using machine-pulled glass capillary microelectrodes filled with 3 M KCl. Preparations were connected to a WPI model FD223 electrometer under a tension of 150 mg. Electrical and mechanical events were simultaneously displayed on a Gould 4072 oscilloscope and Gould TA11 recorder. Signals were digitally recorded with a 16-bit accuracy at a rate of 125 kHz. An electrical stimulus with a 10-msec duration and supra-threshold strength was provided by a Grass S88 stimulator through a Grass SIU5B stimulus isolation unit.

For the SAN-PV interaction study, transmembrane APs of the PVs and SANs were recorded within 3 mm of the distal part of the PV myocardial sleeve and the SAN by simultaneously using machine-pulled glass capillary microelectrodes filled with 3 M KCl, which were connected to a WPI model FD223 electrometer. Tissue was superfused at a constant rate (3 ml/min) with Tyrode's solution saturated with a 97% O2/3% CO2 gas mixture. The temperature was maintained at 37°C and the preparations were left to equilibrate for 1 h prior to initiation of the electrophysiological study. Electrical events were simultaneously displayed on a Gould 4072 oscilloscope and a Gould TA11 recorder. Signals were digitally recorded with a 16-bit accuracy at a rate of 125 kHz. Trifluorocarbonylcyanide phenylhydrazone (FCCP; a mitochondrial uncoupling agent) at 10, 100 and 300 nM with and without Co-Q10 (at 10 µM) was perfused for 20 min to test the pharmacological responses of the PV and SAN in the intact SAN-PV preparation. Spontaneous activity was defined as the constant occurrence of spontaneous APs in the absence of any electrical stimuli.

AP amplitude (APA) was obtained from the resting membrane potential or maximum diastolic potential to the peak of AP depolarization. AP durations (APDs) at repolarization of 20, 50 and 90% of the APA were measured as the APD20, APD50 and APD90, respectively. Spontaneous activity was defined as the constant occurrence of spontaneous APs in the absence of any electrical stimuli.

Statistical analysis

Data are presented as the mean ± standard error of the mean. A repeated one-way analysis of variance with post-hoc Tukey's test was used to compare the effects of FCCP on the RA and LA. The effects of FCCP and Co-Q10 on the PV and SAN were compared by a Wilcoxon signed-rank test or a paired t-test, depending on the outcome of the normality test. P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of FCCP on the electrical activity in isolated PVs and SANs

FCCP (10, 100 and 300 nM) significantly decreased the SAN spontaneous rate in a concentration-dependent manner compared with the control (P<0.01; Fig. 1). As exhibited in Fig. 2, FCCP at 100 and 300 nM significantly decreased PV spontaneous rates compared with the control and FCCP at 10 nM. In addition, FCCP (100 nM) significantly reduced the beating rate to a greater extent in the SAN than in the PV (34±4.9 vs. 16.3±3.2%; n=6; P<0.05; Fig. 3).

Figure 1. Effects of FCCP on isolated SANs. (A) Effect of application of 10, 100 and 300 nM FCCP on SANs. (B) Average beating rate of SANs before and after FCCP (10, 100 and 300 nM) application. Data are presented as the mean ± standard error of the mean (n=6). FCCP, trifluorocarbonylcyanide phenylhydrazone; SANs, sinoatrial nodes.

Figure 2. Effects of FCCP on isolated PVs with spontaneous activity. (A) Effect of application of 10, 100 and 300 nM of FCCP on PVs. (B) Average beating rate of PVs with spontaneous activity before and after FCCP (10, 100 and 300 nM) application. Data are presented as the mean ± standard error of the mean (n=6). FCCP, trifluorocarbonylcyanide phenylhydrazone; PVs, pulmonary veins.

Figure 3. Difference in beating rates before and after 100 nM FCCP-application on PVs with spontaneous activity and the SAN. Data are presented as the mean ± standard error of the mean (n=6). FCCP, trifluorocarbonylcyanide phenylhydrazone; SANs, sinoatrial nodes; PVs, pulmonary veins.

In the presence of Co-Q10 (10 µM), as exhibited in Fig. 4A and B, FCCP (100 nM) significantly reduced PV spontaneous beating activity (2.3±0.2 to 1.1±0.4 Hz; n=5; P<0.05) and SAN spontaneous beating activity (2.7±0.2 to 1.54±0.3 Hz; n=6; P<0.05) compared with Co-Q10 alone. In addition, in the presence of Co-Q10, FCCP (100 nM) reduced the beating rates in the PV and SVN to a similar extent (51.8±12.7 vs. 41.3±10.5%) compared with Co-Q10 alone.

Figure 4. Effects of Co-Q10 and FCCP on SAN and PV with spontaneous activity. (A) SANs with spontaneous activity after Co-Q10 (10 µM) and Co-Q10 (10 µM) + FCCP (100 nM) administration, and average beating rate of SANs with spontaneous activity following Co-Q10 (10 µM) and Co-Q10 (10 µM) + FCCP (100 nM) administration. (B) PVs with spontaneous activity after Co-Q10 (10 µM) and Co-Q10 (10 µM) + FCCP (100 nM) administration, and average beating rate of PVs with spontaneous activity following Co-Q10 (10 µM) and Co-Q10 (10 µM) + FCCP (100 nM) administration. Data are presented as the mean ± standard error of the mean (n=6). Co-Q10, coenzyme-Q10; FCCP, trifluorocarbonylcyanide phenylhydrazone; SANs, sinoatrial nodes; PVs, pulmonary veins.

Effects of FCCP in the intact PV-SAN electrical connection

As demonstrated in Fig. 5, FCCP (100 nM) decreased rates in intact SAN-PV preparations; however, FCCP reversed SAN-to-PV electrical conduction to PV-to-SAN conduction in 5 of 8 (62.5%) preparations.

Figure 5. Effects of 100 nM FCCP on intact SAN-PV preparations. Schematic drawings of simultaneous recordings at the SAN and PV in intact SAN-PV preparations at the baseline (upper panel) and after application of 100 nM FCCP (lower panel). FCCP at 100 nM induced a conduction direction shift from SAN-to-PV at the baseline to PV-to-SAN (n=8). FCCP, trifluorocarbonylcyanide phenylhydrazone; SANs, sinoatrial nodes; PVs, pulmonary veins.

Effects of FCCP on the electrical activities of the RA and LA

As exhibited in Fig. 6, 100 nM FCCP significantly shortened the APD20, APD50 and APD90 (P<0.05) and decreased the contractility in the LA, whereas 100 nM FCCP only shortened the APD20 to a greater extent in the RA.

Figure 6. Effects of 100 nM FCCP on electrophysiological characteristics of the LA and RA. (A) APs at the baseline and after 100 nM FCCP. Average data of APs and contractility in the LA before and after administration of 100 nM FCCP (n=5). (B) APs at the baseline and after 100 nM FCCP. Average data of APs and contractility in the RA before and after administration of 100 nM FCCP (n=6). Data are presented as the mean ± standard error of the mean. FCCP, trifluorocarbonylcyanide phenylhydrazone; RA, right atrium; LA, left atrium; APs, action potentials; RMP, resting membrane potential; APA, action potential amplitude; APD20, AP duration measured at 20% repolarization of the amplitude; APD50, AP duration measured at 50% repolarization of the amplitude; APD90, AP duration measured at 90% repolarization of the amplitude.

Discussion

Cardiac mitochondrial function has important roles in cardiomyocyte energy metabolism and redox state control, and has emerged as a target to decrease arrhythmias (6). Hypoxia, which may lead to mitochondrial dysfunction, has been demonstrated to significantly alter cardiac electrophysiology (24). In the present study, it was observed that decreases in PV and SAN spontaneous activities occurred after FCCP treatment, with a high probability of reverse overdrive in PV and SAN electrical interactions. These findings suggest that mitochondrial dysfunction may modulate PV and SAN electrophysiological properties and enhance PV arrhythmogenesis through a greater reduction of SAN rates.

Hypoxia is able to decrease the rate of spontaneous impulse initiation in SAN fibers by decreasing the slope of diastolic depolarization (25). Similarly, the present study demonstrated that mitochondrial dysfunction is able to decrease PV and SAN spontaneous activities. As mitochondrial dysfunction may lead to an ATP deficiency, the ATP-sensitive potassium (KATP) channel may subsequently be influenced and remain open, which may lead to decreasing pacemaker activity that is noted in hypoxic conditions.

However, in intact PV-SAN preparations, the present study demonstrated that FCCP (100 nM) altered the electrical conduction from SAN-to-PV to PV-to-SAN, which may have arisen from a greater decrease in SAN rates by FCCP with a resulting overdrive suppression from PVs. This finding suggests an increased vulnerability of SANs to an ATP deficiency compared with PVs. Evidence suggests that sinus node dysfunction is able to facilitate the conditions for AF occurrence by increasing ectopy and dispersion of refractoriness (23,26,27). Intact SAN electrical activity is able to suppress arrhythmogenesis from PVs through a constant overdrive of the PVs. The reverse overdrive of the PV on the SAN caused by FCCP may facilitate the occurrence of PV arrhythmogenesis and contribute to mitochondrial dysfunction-related atrial arrhythmogenesis.

The results of the present study revealed that Co-Q10 (10 µM) may modulate mitochondrial dysfunction. The presence of Co-Q10 led to similar FCCP-induced rate reductions in SANs and PVs, which suggests that the FCCP-induced PV-overdrive-SAN conduction shift is attenuated by Co-Q10. A previous study demonstrated that the use of Co-Q10 as adjuvant treatment in patients with heart failure may attenuate the incidence of AF (18), which may occur in part through the protective role of Co-Q10 against mitochondrial dysfunction-induced PV arrhythmogenesis, as revealed in the present study. Co-Q10 promoted recovery of ATP following reoxygenation, which suggests that exogenous Co-Q10 may facilitate resynthesis of ATP in functionally impaired mitochondria. Generation of APs in SAN cells is able to be maintained by a small quantity of ATP (28), which may be produced by exogenous Co-Q10. A previous study demonstrated that Co-Q10 did not prevent decreases in ATP in tissues in the initial period of hypoxia at 30–60 min; however, the ATP content at 120 min of hypoxia in the presence of Co-Q10 was higher than that of the control (28), which may partially explain the failure of Co-Q10 to prevent FCCP-induced PV and SAN rate reductions.

In the present study, FCCP at 100 nM shortened the APD and decreased contractility slightly in the RA and significantly in the LA. The influence of the mitochondrial energetic status on APs is mediated largely by KATP channels in the membrane. These findings are consistent with previous studies, whereas hypoxia or ischemia progressively shortens the APD caused by the opening of KATP channels (24). Discrepant effects of hypoxia on AP shortening between the RA and LA were observed in a rabbit model. Shortening the APD in the RA and LA provides a basis for AF persistence through facilitating the generation of atrial reentry circuits. The differential response of the RA and LA to FCCP may increase dispersions of the APD and may facilitate the maintenance of AF. Although the mechanisms underlying differences between the RA and LA are not clear, it is possible that higher expression levels of heat shock protein 70 in the RA may result in the lower sensitivity of the RA to FCCP (24).

There were some limitations to the present study. Firstly, administration of FCCP may produce a non-physiological condition of mitochondrial dysfunction. Secondly, an acute effect of mitochondrial dysfunction caused by FCCP application was observed in the present study, which may differ from the chronic effect of mitochondrial dysfunction. Finally, the present study used young, healthy tissue preparations and so results may differ in pathological settings.

In conclusion, mitochondrial dysfunction regulates electrophysiological characteristics of the PV, SAN, RA and LA, which may have a role in the pathophysiology of atrial arrhythmogenesis.

Acknowledgements

The present study was financially supported by grants from the Ministry of Science and Technology (grant nos. MOST103-2314-B-038-041-MY2, MOST103-2314-B-281-005-MY2, MOST103-2314-B-281-006, MOST103-2314-B-038-055, NSC102-2314-B-016-029-MY2, NSC102-2325-B-010-005 and NSC102-2628-B-038-002-MY3), Taipei Medical University (grant no. TMU101-AE1-B31), Taipei Medical University-Wan Fang Hospital (grant nos. 101-wf-eva-11, 101-wf-phd-01, 103swf05, 103-wf-eva-02, 104swf02 and 104-wf-eva-01) and Chi-Mei Medical Center (grant nos. 104CM-TMU-07 and CMNDMC10410) of Taiwan.

References