Experimental animals and exercise protocols

All mice were housed at the Division of Comparative Medicine animal facility in accordance with the standards of the Canadian Council on Animal Care. Ethical approval for all animal experiments was granted by the University of Toronto. In preliminary studies we found that CD1 strain of mice (Charles River Laboratories) instinctively swim against a current in our swimming apparatus, a behaviour that was found to be absent in other strains. In studies using mice with whole-body TNFα disruption (c57b, Taconic model #1921) or expressing an NFκB-Luc reporter (c57b, Jackson Laboratories stock #006100), a single back-cross with CD1 mice was sufficient to manifest instinctive swimming behaviour. Mice swam in water containers (30 cm diameter) with a steady water current (325 l min−1) generated by a central water pump at 34 °C. Swimming training began with 30-min swimming sessions (twice daily, separated by 4 h) that were increased to 90 min by adding 10 min per day. Training then continued for 6 weeks. For running exercise, mice trained on a treadmill apparatus (Harvard Apparatus) at 35 cm s−1 with a 30° incline. Running training began with 30-min sessions (once daily) that were increased by 10 min per day to 120 min. In the first 3–4 sessions, electric stimulation (0.2 mA) was used to promote running during training; thereafter, mice were encouraged to run using a soft brush pressed against their posterior region, to reduce the potential stress of continued electric shocking. Male mice were randomly assigned to exercised or sedentary groups at 6 weeks of age. No mice were excluded from the study based on inability to train. Control animals were placed in water containers without water current for 5 min, or placed on the treadmill for 5 min at 5 cm s−1.

Mice treated with etanercept (Enbrel) were injected subcutaneously daily at 2.5 mg kg−1 for the entire 6-week exercise training. Mice treated with SB203580 (AbMole Bioscience) were injected i.p. at 4 mg kg−1. All experimental assessments were conducted 24 h after the final exercise session. The identity of the groups (exercised versus sedentary) was concealed to the investigators. Sample sizes were determined using power calculations at 95% confidence interval and 80% power using comparable data from previously published results67.

VO 2 consumption measurements

Oxygen consumption was determined using Oxymax oxygen monitoring (Columbus instruments, Columbus, OH). For swimming, mice were placed in a sealed container containing water pumps to generate current (34 °C). Maximal O 2 consumption was determined using the swimming apparatus with two simultaneous pumps generating current on opposite ends. We have found that mice in this set-up consume the greatest amount of O 2 (11260±563 ml kg−1 h−1), which was much greater than values that could be obtained using exhaustive treadmill exercise with speeds of up to 50 cm s−1 and 30° incline (9384±643 ml kg−1 h−1).

Echocardiography

Mice were anaesthetized using 1.5% isoflurane oxygen mixture. Mice were placed on a heating pad and body temperature was maintained between 36.9 and 37.3 °C for the duration of the measurements. Transthoracic M-mode echocardiographic examination was conducted using a Vevo 7700 system (VisualSonics) equipped with ultrasonic linear transducer scanning heads operating at 30 MHz. The left ventricular long axis view was used for measurement of chamber size and wall diameter. Data analysis was performed using the VisualSonics data analysis suite.

Electrocardiography

Surface ECG measurements were conducted on mice anaesthetized using a 1.5% isoflurane oxygen mixture with sub-dermal platinum electrodes placed in lead II arrangement. Body temperature was maintained at 36.9 to 37.3 °C. To record heart rates from unanaesthetized, unrestrained animals, radiofrequency emitting ECG units (Data Sciences International) were implanted in the intraperitoneal cavity and the electrodes placed in lead II arrangement subdermally. After 1 week of recovery, mice were exercised according to the standard regimen and heart rate (HR) was recorded weekly over a 48-h period. Data were analysed using Ponemah Physiology Platform software. To assess pharmacological inhibition of autonomic nerve activity, HRs of anaesthetized mice were monitored before and after intraperitoneal injection of 2 mg kg−1 BW of atropine sulphate and 10 mg kg−1 BW of propranolol hydrochloride (Sigma-Aldrich) to block the parasympathetic and sympathetic branches of the autonomic nervous system.

Heart-rate variability analysis

Minimum anaesthesia deepness was strictly monitored and maintained at equivalent levels between sedentary and exercised mice using the toe pinch-pedal reflex. Surface ECG recordings were marked for R-R intervals using P3 Plus (Data Sciences International), and the raw text files of the inter-beat-intervals (IBI) were input for analysis using Kubios HRV software (University of Eastern Finland). Frequency bands of 0–0.1 Hz and 0.1–5 Hz for low and high frequency components, respectively, were determined in a series of preliminary experiments (Supplementary Fig. 9) using atropine (2 mg kg−1) and propranolol (10 mg kg−1). An interpolation rate of 20 Hz, with 256 point window was used to generate frequency domain plots. The absolute power from the frequency domain plot was used for analysis. A minimum of 5 min of data were used for the analysis.

Assessment of electrical properties and arrhythmia vulnerability of hearts

Mice were anaesthetized using 1.5% isoflurane and oxygen mixture, and a 2.0 French octapolar recording/stimulation electrophysiology catheter (CI’BER Mouse, Numed) was inserted into the right jugular vein and advanced into the right ventricle. The his bundle signal was used for consistent positioning of the catheter (see Supplementary Fig. 4). Appropriate leads were used to deliver programmed stimulation to either the right atria or right ventricle. All stimulations were delivered at a voltage magnitude of 1.5 times capture threshold, with 1 ms pulse duration. Refractory periods were determined by delivering seven pulses at 20 ms below the R-R interval followed by extrastimulation. The S2 coupling interval was initially delivered at below capture (~15 ms) and increased by 5 ms increments until capture. The coupling interval was reduced by 1–2 ms again until loss of capture. Our protocols for arrhythmia were based on previously published protocols using burst pacing with intervals less than atrial effective refractory in both exercise and sedentary mice (Supplementary Table 4). For arrhythmia induction, 27 pulses (of 2 ms duration) at 40 ms intervals were applied to the right atrium or ventricle and reduced to 20 ms intervals by 2 ms decrements, repeated three times. If arrhythmia events were not induced, 20 trains (applied every 1.5 s) of 20 pulses (2 ms duration) with an interpulse interval of 20 ms duration were used. Sustained arrhythmias were defined as reproducible episodes of rapid, chaotic and continuous atrial or ventricular activity, lasting longer than 10 s (examples in Supplementary Fig. 6).

Optical mapping and intracellular action potential recording

Heparinized mice were anaesthetized using isoflurane and killed via cervical dislocation. The thorax was opened by midsternal incision, and the heart was excised into warm (35 °C) Tyrode’s solution (in mmol/l): 140 NaCl, 5.4 KCl, 1.2 KH 2 PO 4 , 1 MgCl 2 , 1.8 CaCl 2 , 5.55 D-glucose, 5 HEPES and 10 U ml−1 heparin (pH 7.4). The heart was pinned to a Sylgard coated petri dish with insect pins to reveal the dorsal atrioventricular connective tissue. The pericardium and any other residual lung and connective tissue were carefully excised from the heart. A small lining of adipose tissue guided incisions along the atrioventricular connective tissue until complete separation of the atria from the ventricles was achieved. Atria were then pinned so as to reveal the mitral and tricuspid valves. Fine scissors were inserted into the superior and inferior vena cava, and incisions were made in a straight path to ‘open’ the atria. The atria were then turned to reveal the pulmonary veins, and residual lung tissue was removed. Atria were then transferred and continuously superfused using 35 °C carbogenized (95% O 2 & 5% CO 2 ) Krebs solution (in mmol l−1): 118 NaCl, 4.2 KCl, 1.2 KH 2 PO 4 , 1.5 CaCl 2 , 1.2 MgSO 4 , 2.3 NaHCO 3 , 20 D-glucose, 2 Na-pyruvate (pH measured at 7.35–7.4).

For optical mapping measurements, isolated atrial preparations were stained with 10 μM voltage-sensitive dye Di-4-ANEPPS (Sigma-Aldrich) for 10 min and then superfused continuously with carbogenized Krebs solution (35 °C). The flow rate, volume and temperature of the solution was kept constant throughout and in-between the experiments. The pH of the bath solution in the perfusion vessels was monitored to ensure that the pH was between 7.35 and 7.4. To calculate conduction velocity, atria were paced at 90 ms interval from the left atrial appendage, and images were captured at 1408 frames s−1 using a high speed camera (Photometrics, AZ, USA). Activation maps were generated to calculate conduction velocity. To induce atrial arrhythmias, the same stimulation protocols that were used in vivo using intracardiac catheter were applied. Sustained arrhythmias were defined as a reproducible episode of rapid, chaotic atrial activation lasting longer than 10 s. Images were acquired using ImagePro Plus software (Mediacy) and analysed using ImageJ or Scroll (custom software)68, which was modified to perform phase analyses.

For intracellular action potential recordings, pipettes were pulled from borosilicate glass using a Flaming/Brown pipette puller (Sutter Instrument Company). Pipettes were filled with 3 M KCl solution and the resistance was between 30 and 50 MΩ. Intracellular recordings of atrial myocytes (in tissue, not isolated) were acquired by placing the microelectrodes into the left atrial appendage. ClampFit software (Axon) was used for analysis.

Isolation of atrial myocytes and I Ca measurement

Left atrial myocytes were obtained from 6-week exercised and age-matched sedentary (CD-1, male 14 weeks old) mice. Mice were anaesthetized with 2.5% isoflurane, and hearts were removed rapidly and retrograde perfused with Ca2+-free Tyrode’s solution (mM): 137 NaCl, 5.4 KCl, 1.0 MgCl 2 , 10 D-Glucose, 10 HEPES, pH 7.4 at 37 °C through the aorta for 10 min. After perfusing the heart with collagenase (1.0 mg ml−1, Worthington) and elastase (0.2 mg ml−1, Worthington) for 8–10 min, the left atrial appendage was dissected and stored in Kraftbruhe (KB) buffer (mM): 120 potassium glutamate, 20 KCl, 20 HEPES, 1.0 MgCl 2 , 10 D-glucose, 0.5 K-EGTA, 0.1% bovine serum albumin, pH 7.4. Single myocytes were obtained by gently triturating the left atrial appendage using a polished glass pipette with a 5 mm opening for 6–10 min. Single atrial myocytes were stored in KB buffer at 4 °C until use. For recording Ca2+ currents (I Ca ), single left atrial myocytes were superfused with bath solution containing (mM): 140 NaCl, 4 CsCl, 1 MgCl 2 , 1.5 CaCl 2 , 10 HEPES, 10 D-glucose (pH 7.3) for 10 min and only Ca2+ tolerant cells were used for recording. I Ca was recorded using whole-cell patch clamp (Axopatch 200B and Clampex 8 software, Axon Instrument, CA, USA) at room temperature. The pipette resistance ranged between 4 and 6 MΩ when filled with internal solution, containing (in mM): 135 CsCl, 6 NaCl, 1 MgCl 2 , 3 MgATP, 10 HEPES and 10 EGTA (pH to 7.2 with CsOH). I Ca was recorded under voltage-clamp mode with 80% compensation of cell capacitance and series resistance. After membrane rupture, cell capacitance was measured by integrating capacitance transients initiated by 5 mV steps from holding potential of −50 mV and was used to normalize the magnitude of recorded currents. For recording I Ca , cells were held at −75 mV and stepped to −40 mV for 50 ms to inactivate voltage-gated Na+ currents before stepping to test potentials from +50 mV to −40 mV decremented by 10 mV. For recordings acetylcholine-activated K+ currents (I k,Ach ), single left atrial myocytes were superfused with bath solution containing (mM): 140 NaCl, 4 KCl, 1 MgCl 2 , 1.2 CaCl 2 , 10 HEPES, 10 D-glucose (pH 7.3) for 10 min and only Ca2+ tolerant cells were used for recording. To block K ATP 10 μM Glybenclamide (Sigma) was added to the bath solution. Two different concentrations of carbachol (CCh, Sigma) were delivered to cells through 0.1 mm internal-diameter tubing sitting on top of the cells. The pipette resistance ranged between 4 and 6 MΩ when filled with internal solution, containing (in mM): 120 potassium aspartate, 20 KCl, 1 MgCl 2 , 3 MgATP, 0.1Na 2 GTP, 10 HEPES and 10 EGTA (pH 7.2). Cells were held at −70 mV and stepped to +30 mV for 120 s to ensure inactivation of all voltage-gated currents before addition of CCh for additional 120 s. I K,Ach current density was measured by subtracting baseline current before addition of CCh at +30 mV from maximal current induced by CCh addition normalized to cell size.

Voltage-activated K+ currents were recorded in bath buffer containing (mM): 140 NaCl, 4 KCl, 1 MgCl 2 , 1.2 CaCl 2 , 10 HEPES, 10 D-glucose and 0.3 CdCl 2 , pH 7.4. The pipette resistance ranged from 3 to 5 MΩ when filled with solution containing (mM): 120 K-aspartate, 20 KCl, 6 NaCl, 1 MgCl 2 , 3 MgATP, 10 HEPES, 10 EGTA, pH 7.4. Voltage-gated K+ currents were measured by 20 s depolarization to +60 mV (close to reversal potential for sodium) from holding potential of −70 mV. Voltage-gated K+ currents were analysed by fitting the decay phase of the outward K+ currents to a three-exponential equation (equation (1)) to dissect the three components of outward K+ current comprising of Ifast (I to ), intermediate (Kv1.5 encoded I K,slow1 ) and slow (Kv2-encoded I K,slow2 ).

where A f , A i and A s are the amplitudes of the fast, intermediate and slow kinetic components decaying with time constants of τ f , τ i and τ s respectively. C is the amplitude of the steady-state, non-inactivating component. All data analysis was performed using ClampFit software (Axon).

Histology

Hearts were first perfused with saline containing 1% KCl followed by 4% paraformaldehyde in 0.1 M phosphate buffer. Hearts were embedded in paraffin and 5-μm thin sections were stained with Picrosirius red for visualization of collagen. Confocal laser scanning microscopy was used to image Picrosirius red stained sections (491 nm laser, 750 nm band pass emission), and imageJ software69 was used for quantification. Perivascular and endocardial fibrosis were not included in the analysis for the quantification of % fibrosis. For quantitative measurement of macrophage infiltration, antibodies against mouse Mac-3 (1:200, BD Pharminogen, Cat. # 553322) were used with the streptavidin-biotin diaminobenzidine chromogen detection method (Vector Laboratories). Mac-3-positive cells were counted in three different sections (100 μm apart) of the left atrial appendage or left ventricle per replicate and standardized to the total tissue area of each slice. Images were acquired using Metamorph software (Molecular Devices), and data analysis was performed using ImageJ software. Mast cells were stained for using Toludine Blue at the appropriate PH. Mast cells were identified by metachromatic granules, counted and normalized to total tissue area. For quantitative measurement of connexin staining, heart sections were stained against connexin 40 (Abcam ab16585, 1:100 dilution) and 43 (Abcam ab11370, 1:100 dilution). Alexa Fluor 488 conjugated secondary antibody (Life technologies, 1:500 dilution) was used for connexin detection. Threshold method was used to determine number of pixels corresponding to connexin 40/43 staining and presented as value normalized to area of atria.

Western blotting

Atria and ventricles were isolated and frozen in liquid nitrogen. Protein was extracted in a cell lysis buffer consisting of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 50 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 2 mM Benzamindine, 1 μg ml−1 leupeptin. After centrifugation for 15 min at 12,000 × g, supernatants were extracted and protein concentration was determined using DCTM Protein Assay kit (1:1,000, #500–0112, BioRad, CA, USA). Forty micrograms of each sample was resolved by 12% sodium dodecyl sulphate–PAGE (SDS–PAGE) and transferred to nitrocellulose membranes. Blots were incubated with primary antibodies: phosphorylated p38 MAPK (1:1,000, PAB15917, Abnova, Taiwan, Supplementary Fig. 16A), endogenous p38 MAPK (1:1,000, #9218, Cell Signaling Technology, USA, Supplementary Fig. 16B), phosphorylated IκBα (1:2,000, 2859, Cell Signaling Technology, Supplementary Fig. 16C), endogenous IκBα (1:1,000, #4814, Cell Signaling Technology, USA, Supplementary Fig. 16D), and with secondary antibodies anti-rabbit (1:3,000, #7074, Cell Signaling Technology, USA) or anti-mouse (1:3,000, #7076, Cell Signaling Technology, USA). Phosphorylation of RyR2 was detected by using specific anti-S2814 RyR2 (1:1,000, Supplementary Fig. 16E), S2808 RyR2 antibody (1:1,000, both in-house generated antibodies) and RyR2 antibody (1:1,000, Thermo scientific, Supplementary Fig. 16F). Phosphorylation of CaMKII was detected by using a specific anti-pT286/287 CaMKII antibody (1:1,000, Cayman Chemical) and anti-CaMKII antibody (Epitomics). GAPDH was detected using an Anti-GAPDH antibody (1:1,0000, Millipore). Oxidation level of RyR2 was measured by oxyblot (OxyBlotTM Protein Oxidation Detection Kit, Millipore). Amersham ECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare, UK) was used for detection, and analysis of bands was performed using densitometry plugin for ImageJ software.

Ca2+ spark activity

Isolated left atrial myocytes were loaded with 1 μM fluo-3 Am at room temperature for 15 min followed by 15 min of desertification by continuously superfusing the cells with Tyrod’s buffer containing 1.2 mM Ca2+. After 15 min of desertification at room temperature, Ca2+ tolerant cells were field stimulated at 1 Hz for 30 s at room temperature and Ca2+ transients were recorded using a Yokagawa spinning disk confocal (491 nm excitation, 510 nm emission) with a high speed camera (Cascade 128+). For measurement of spark activity, the field stimulation was stopped and images were acquired after 2 s and analysed using Sparkmaster plugin for Image J (background fl. U: 10, criteria: 3.8, no of intervals: 1)70.

NFκB activity assays

Transgenic 6- to 8-week old male mice expressing firefly luciferase driven by two copies of the NFκB regulatory element (Jackson Laboratories Stock #006100) were swim exercised for 2 days, two sessions per day for 90 min separated by 4 h. Two hours after the final session atria and ventricles (dissected and quickly rinsed in cold PBS) were flash frozen in liquid nitrogen. Luciferase activity was assessed in tissue homogenates according to the manufacturer’s specification (Promega), and the values were standardized to total protein concentration (Bio-Rad).

Statistical analysis

Data are presented as mean±s.e.m.. Statistical significance was determined using unpaired or paired student’s t-test (two-tailed) as appropriate, a repeated measure one-way ANOVA with Sidak’s multiple comparison test, or a repeated measure two-way ANOVA with Sidak’s multiple comparison test. Welch’s t-test was used when the variance differed between groups (assessed via F-test). The Mann–Whitney U-test was used to compare AF durations as the data were not normally distributed according to a D’Agostino& Pearson omnibus normality test. For comparison of arrhythmic events between different treatment groups, a 2 × 2 contingency table using χ2-test without yates correction was used. P values <0.05 were considered statistically significant.