Optical Mapping of Atrial Electrophysiological and Calcium Handling Abnormalities in a Rat Model of Sterile Pericarditis

Summary

This protocol describes dual optical mapping of voltage and calcium transients in rat sterile pericarditis hearts to assess inflammation-induced atrial remodeling underlying postoperative atrial fibrillation.

Abstract

Postoperative atrial fibrillation (POAF) frequently arises after cardiac surgery and is associated with atrial inflammation and remodeling. The sterile pericarditis (SP) model reproduces this condition in rats, providing a valuable platform to investigate inflammation-induced atrial electrophysiology. This protocol describes the preparation of Langendorff-perfused rat hearts after SP induction and the use of simultaneous dual optical mapping to record transmembrane voltage and calcium transients. Hearts are stained with the voltage-sensitive dye RH237 and the calcium indicator Rhod-2 AM, while contractility is suppressed using blebbistatin to minimize motion artifacts. High-speed imaging enables spatiotemporal mapping of atrial action potentials and calcium dynamics during controlled pacing protocols. Quantitative parameters include action potential duration, conduction velocity, calcium transient duration, time to peak, and alternans magnitude. The method allows precise assessment of atrial conduction heterogeneity, electro-calcium coupling, and arrhythmia vulnerability under inflammatory stress. This visualized approach provides a reproducible and versatile platform for studying POAF mechanisms and testing pharmacological or genetic interventions that target atrial remodeling.

Introduction

Postoperative atrial fibrillation (POAF) is a common complication following cardiothoracic surgery, characterized by transient episodes of atrial fibrillation (AF) that typically peak within 2-4 days post-surgery. While POAF is often self-limited, it is associated with increased risk of stroke, longer hospital stays, and elevated healthcare costs¹. Inflammatory stress in the atrial myocardium is considered a key contributor to its pathogenesis².

The sterile pericarditis (SP) model is a well-established small animal model for studying POAF^3,4. In this model, epicardial talc application induces localized pericardial inflammation that mimics postoperative changes. Previous studies using this model have demonstrated structural remodeling, altered gap junction expression, and calcium-handling abnormalities that predispose to arrhythmogenesis^5,6,7. However, conventional electrophysiological techniques provide limited spatiotemporal information, making it difficult to capture conduction heterogeneity and electro-calcium coupling dynamics across the intact atrial surface.

Optical mapping enables simultaneous high-resolution imaging of transmembrane voltage and calcium transients (CaT) across the atrial surface, enabling precise visualization of atrial conduction, repolarization, and calcium signaling⁸. In this protocol, we describe dual optical mapping in Langendorff-perfused SP rat hearts. This approach allows quantification of conduction velocity, action potential duration (APD), CaT properties, and alternans under controlled pacing protocols. By integrating electrical and calcium-handling assessment in an inflamed atrial preparation, the method provides a powerful tool for studying the mechanistic basis of POAF and for testing targeted therapeutic interventions.

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) under approval ID [2024]IACUC Number 4241 and conducted in accordance with institutional and national guidelines. Throughout the study, strict adherence to animal welfare guidelines was maintained, with clearly defined humane endpoints: Rats exhibiting persistent lethargy, sudden weight loss exceeding 20%, respiratory distress, inability to eat or drink normally, or severe postoperative complications were immediately euthanized using an overdose of isoflurane (5% with oxygen flow at 1 L/min) to minimize suffering.

1. Preparation of solutions and reagents

1. Prepare Modified Tyrode's solution (in mM): 128.2 NaCl, 4.7 KCl, 1.3 CaCl₂, 1.05 MgCl₂, 1.19 NaH₂PO₄, 20 NaHCO₃, 11.1 Glucose. Bubble with 95% O₂/5% CO₂, adjust pH to 7.38-7.42, and warm to 37 ± 1 °C.
2. Blebbistatin: Dissolve in DMSO (5 mg/mL), store at -20°C, light-protected. Use at 10 µM final concentration.
3. RH237 (voltage-sensitive dye): Dissolve in DMSO (2 mg/mL), store at −20 °C, light-protected. Use at 2 µM final concentration.
4. Rhod-2 AM (calcium indicator): Dissolve in DMSO (1 mg/mL); store at −20 °C, light-protected. Use at 0.89 µM final concentration.
5. Pluronic F-127 (Surfactant): 20% solution in DMSO (Commercially available reagents).
6. Sodium pentobarbital (1%): Dissolve 0.1 g in 10 mL of saline for anesthesia (40 mg/kg, i.p.).
   NOTE: Handle dyes and DMSO stocks with appropriate PPE; protect blebbistatin and dyes from light. Follow biosafety and chemical safety rules.

2. Equipment

1. Langendorff system with temperature control (37 ± 1 °C), reservoir, bubble column, and peristaltic pump (flow 10-12 mL/min).
2. Imaging: High-speed CMOS camera (≥ 1 kHz frame rate; ≥ 80 × 80 pixels field of view for atrium), LED excitation source (530 ± 25 nm), dichroic beamsplitter (~630 nm), emission filters (Rhod-2 ~590 nm; RH237 >700 nm), lens with adjustable aperture, optical table with vibration isolation. Excitation Light Source: LED laser with a maximum current of 5 A, green light with a maximum power of 2.2 W.
3. Stimulation: Constant-voltage stimulator, bipolar pacing electrodes.
4. Physiology: Submersible ECG electrodes; thermistor probe; black background substrate (rubber) to reduce ventricular light contamination; fine surgical instruments; ventilator for rats. Detailed inventory is available in the Table of Materials.

3. Induction of sterile pericarditis (SP) in rats

1. Anesthetize male Sprague-Dawley rats (200-220 g) with 1% sodium pentobarbital (40 mg/kg, i.p.). Confirm anesthesia depth (loss of pedal reflex).
2. Intubate the rat using a polyethylene or metal cannula under direct visualization. Advance the tube carefully into the trachea, positioning just above the carina, and secure it to prevent displacement. Connect to a ventilator set at a tidal volume of 2 mL/100 g, 55 breaths/min, I:E = 1:1. Confirm effective ventilation by observing symmetrical chest expansion and stable respiration.
   NOTE: Avoid deep or shallow placement to prevent bronchial intubation or accidental extubation.
3. Prepare the surgical field. Perform a small parasternal thoracotomy (2nd intercostal space near the sternum). Gently expose the heart.
4. Lift the thymus to visualize both atria. Carefully open the pericardium over the atrial surfaces with fine forceps.
5. Evenly apply sterile talc over the left and right atrial surfaces using a fine spatula (talcum powder dosage: 50 mg/100 g rat body weight). Avoid spilling onto great vessels/ventricles. Replace the thymus over the atria to limit talc migration.
6. Reposition the chest muscles to cover the intercostal surgical incision. After disinfection with 0.5% povidone-iodine, suture the muscles and skin to close the thoracic cavity, and administer penicillin (160,000 U/100 g, i.p.) postoperatively.
7. Sham: Thoracotomy without pericardiotomy or talc application.
   NOTE: Apply a thin, uniform talc layer only to atrial epicardium; excessive talc or ventricular contamination increases mortality and can confound atrial-specific readouts. Previous studies have confirmed that aseptic pericarditis surgery in rats causes severe atrial myocarditis with marked fibrosis and elevated inflammatory factors, including IL-6, IL-1β, and TGF-β1^3,9.

4. Heart isolation and Langendorff perfusion

1. On day 3 post-surgery, anesthetize the rats with isoflurane (2-3%) via a nose cone and administer systemic heparin (250 U/Kg, i.p.) to prevent intravascular clot formation that could obstruct coronary circulation and compromise Langendorff perfusion. This time point was selected based on our previous in vivo findings that AF inducibility peaks 3-4 days after SP³, enabling the capture of the acute, inflammation-driven arrhythmogenic phase while avoiding later adhesions that interfere with dye loading and optical recordings.
   NOTE: Before you begin, prepare fresh; equilibrate gas and temperature. Align optics and confirm filters/dichroic. Configure acquisition software (1-2 kHz, exposure 1 ms), and perform calibration with a fluorescent card.
2. Perform a rapid median thoracotomy to expose the thoracic cavity. Excise the heart en bloc with a portion of the aortic arch, and immediately immerse it in ice-cold, oxygenated Tyrode's solution to arrest metabolism and preserve tissue viability. Carefully dissect the surrounding connective tissue while maintaining a 0.5 mm aortic length for cannulation. Secure the aorta onto the Langendorff cannula using 4-0 silk sutures, ensuring an airtight seal and that the lower edge of the catheter aligns with the upper border of the atrium. Begin retrograde perfusion with oxygenated Tyrode's at a constant flow rate of 10-12 mL/min, maintaining the perfusate temperature at 37 ± 1 °C. For reproducibility, the rat aortic cannula specifications are as follows: outer diameter: 1.4 mm; inner diameter: 1.0 mm; total length: 3-4 cm; insertion depth: approximately 0.5-1.0 cm into the aortic root; tip shape: slightly tapered; material: stainless steel or blunted metal tubing. The cannula must be rigid enough to withstand perfusion pressures (typically 60-80 mmHg) without bending, and the tip should avoid obstructing the coronary ostia.
3. Observe the heart for immediate reperfusion effects. Successful perfusion is indicated by prompt and brisk washout of residual blood from the coronary vasculature, restoration of a uniform pink myocardial color, and resumption of spontaneous rhythmic contractions. Minimal myocardial edema and a stable heart rate (typically 300-400 bpm) are required before further experimentation. Simultaneously record the electrocardiogram of the heart. Allow the heart to equilibrate for at least 20 min under constant perfusion before initiating optical mapping protocols.

5. Motion suppression and venting

1. Add blebbistatin to the perfusate (final 10 µM). Perfuse for ~10 min until contractions cease.
2. Insert a hypotensive catheter into the left ventricle via the pulmonary vein through the mitral valve to decompress the ventricle, thereby reducing intracavitary pressure while preserving atrial morphology and signal integrity. For reproducibility, the catheter specifications are: outer diameter: 1.4 mm; inner diameter: 1.0 mm; total length: 1.5 cm; insertion depth into the heart: 1.0 cm. Do not open atria to preserve atrial geometry and signals.

6. Dye loading

1. Heart perfusion: After cardiac arrest, perfuse the heart at room temperature at a constant flow rate of 10-12 mL/min.
2. Calcium indicator staining: Add Rhod-2 AM (final concentration 0.89 µM) and 20 µL of 20% Pluronic F-127 to 100 mL of perfusion solution. Perfuse the heart with this solution for approximately 30 minutes, until the atria and ventricles exhibit uniform red fluorescence.
3. Voltage-sensitive dye staining: Add RH237 (final concentration 2 µM) to 100 mL of perfusion solution. Perfuse the heart for ~5 min until the atria and ventricles appear pink.
4. Rewarming and washout: Transfer the heart back to the original perfusion system. Rewarm to physiological temperature and perfuse for 5-10 min to wash out unabsorbed dye.
   NOTE: Weak CaT signals often reflect insufficient Rhod-2 loading; extend loading by 10-15 min and confirm Pluronic is fresh. If motion persists, confirm blebbistatin potency and light protection.

7. Imaging geometry and signal isolation

1. Place the heart in a 37 °C imaging bath. Orient the left atrium (LA) toward the camera. Align the primary and secondary LED light sources toward LA.
2. Support the heart to prevent drift; gently spread the atrium and secure it with acupuncture needles for optimal visualization. Cover the ventricles with black rubber sheets to minimize ventricular light pollution.
3. Position bipolar pacing electrodes on the right atrium (RA). Place ECG electrodes near the atria for large P-wave capture.

8. Acquisition and pacing protocols

1. Launch the mapping software(OMapRecord4.0 software). Set frame rate to 1-2 kHz; adjust LED load to3-4 A; field of view to encompass the entire LA. Adjust gain/exposure to avoid saturation in either channel. Optimize camera gain and exposure to avoid saturation while maximizing signal-to-noise ratio. Imaging parameters: exposure 1 ms; max camera frequency 30 kHz; sampling rate 12.5 kHz; resolution 2048 × 2048 pixels; ROI frame rate up to 3.5 kHz; spatial resolution 70 µm. These settings ensure high-fidelity AP and CaT recordings.
2. S1S1 pacing (baseline): RA pacing at 7 Hz (pulse width 2-4 ms; twice diastolic threshold). Acquire ≥ 5 s movies for APD/CaT duration (CaD), time-to-peak, and conduction velocity.
3. S1S2 protocol (refractoriness/AF): Deliver 30 S1 beats at 7 Hz, then a single S2; decrease S1-S2 interval from 140 ms in steps (10/5/1 ms) to refractoriness. Record CaT recovery (A2/A1), effective refractory period (ERP), AF inducibility/duration.
4. Incremental pacing (alternans): Increase RA pacing from 8 to 18 Hz in 1-2 Hz steps. Record ≥ 5 s at each rate to quantify Ca-ALT, APD-ALT, and DIS-ALT. Save simultaneous dual-channel movies and synchronized ECG/stim traces for analysis.
   NOTE: Maintain constant temperature and perfusion throughout pacing; hypothermia reduces conduction and exaggerates alternans artifacts.

9. Data processing and quantification

1. Analyze optical signals using OMapScope 4.0 software. A 3×3 Gaussian spatial filter was applied to reduce noise while preserving signal morphology by averaging each pixel with its neighbors. Activation times are defined as the point of maximal dF/dt during the upstroke, from which conduction velocity is calculated by dividing the linear distance between the earliest and latest activation sites by the corresponding activation time difference. APD_70/90 was defined as the time from the maximal dF/dT of the action potential to 70% or 90% repolarization of the AP amplitude. CaD_70/90 was defined as the time from the peak of the calcium transient (Ca²⁺) to 70% or 90% decay. Alternans were quantified as follows: Ca-ALT was calculated as 1 minus the ratio of smaller to larger mean CaT amplitude, APD-ALT as the difference between the longer and shorter APD₈₀ values, and DIS-ALT as spatially out-of-phase alternans separated by nodal lines. The RyR recovery ratio was calculated as the ratio of two consecutive CaT peaks (smaller/larger).

10. Critical parameters and acceptance criteria

1. Perfusion: 10-12 mL/min, 37 ± 1 °C; stable sinus or paced rhythm; LA well-perfused.
2. SNR: Upstroke dF/F ≥ 3 for both channels; minimal photobleaching over acquisition window.
3. Motion: Negligible residual motion after blebbistatin; stable image registration across frames.
4. Temperature: Continuous monitoring; exclude datasets with bath < 36 °C or > 38 °C during acquisition.

11. Statistical Analyses

1. All statistical analyses were performed using GraphPad Prism 9. Data are presented as mean ± SEM. The normality of data distribution was assessed using the Shapiro-Wilk test. For data with a normal distribution, differences between the two groups were analyzed using an unpaired two-tailed Student's t test; for non-normally distributed data, the Mann-Whitney U test was applied. Categorical variables were analyzed using the χ² test. A P < 0.05 was considered statistically significant.

Results

AF Inducibility
Ectopic activity and reentry are essential mechanisms contributing to POAF initiation and perpetuation², The reentry wavelength (λ) is determined by the product of ERP and conduction velocity (CV), i.e., λ = ERP × CV. In sham-operated hearts, S1S2 pacing evoked stable atrial responses without arrhythmia (Figure 2A). In contrast, SP-treated hearts frequently exhibited abnormal responses, including atrial ectopy (

Discussion

This protocol describes dual optical mapping of APs and CaTs in isolated rat hearts following SP, an inflammation-driven model of POAF. Simultaneous high-resolution recording of Vm and Ca²⁺ signals across the intact atrial surface under programmable pacing allows assessment of APD, time to peak, conduction velocity, spatial heterogeneity of repolarization, CaD, CaT amplitude, CaT time to peak, and regional Ca handling variability. It also enables detection and quantification of frequency-dependent alternans, i...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Blebbistatin	Abcam	ab120425
CaCl2	Sigma-Aldrich	10043-52-4
CMOS camera	MappingLab	OMS-PCIE-2002
ECG system	Chengdu Techman	BL-420F
FE32 -Meter	Mettler Toledo	30254108
Filters/dichroic mirrors	MappingLab	RH237 >700 nm; CaT 590 nm
Glucose	Sigma-Aldrich	921-60-8
Imgae bath	MappingLab	N/A
KCl	Sigma-Aldrich	7447-40-7
Langendorff perfusion system	Radnoti / Custom	N/A
LED source (530 ± 25 nm)	MappingLab	N/A
MgCl2	Sigma-Aldrich	7786-30-3
NaCl	Sigma-Aldrich	7647-14-5
NaH2PO4	Sigma-Aldrich	10049-21-5
NaHCO3	Sigma-Aldrich	144-55-8
OMapRecord4.0 software	MappingLab	N/A
OMapScope4.0 software	MappingLab	N/A
Peristaltic pump	Cole-Parmer	Masterflex® L/S®
Pluronic F-127	Invitrogen	P3000MP
RH237 (voltage dye)	Thermo Fisher / Invitrogen	S1109
Rhod-2 AM (Ca indicator)	Thermo Fisher / Invitrogen	R1245MP
Small Animal Ventilators	YUYANBIO	V-100
Sodium pentobarbital	Sigma-Aldrich	P3761
Sprague-Dawley rats (200–220 g, male)	Local certified vendor	N/A
Sterile talc powder	Sigma-Aldrich	T1586
Stimulator	MappingLab	LEDC-2002
Water bath (temp-controlled)	Ningbo Xinzhi	SC-15