2025-11-07
ENA first public
ERC000011
ENA-CHECKLIST
Spanish National Center for Cardiovascular Research
INSDC center name
public
INSDC status
ERS20600866
SRA accession
E-MTAB-14275:Sample_ND_3_agosto_21
Submitter Id
ArrayExpress
broker name
non-cardiomyocytes
cell_type
not collected
collection date
pig
common name
Protocols: Hearts were harvested and dissected, single cell suspension was prepared by enzymatic digestion. Tissue was minced to ~1 mm3 cubes and digested in 3 mL of DMEM containing 450 U/mL collagenase I, 60 U/mL DNase I, and 60 U/mL hyaluronidase (all Sigma- Aldrich, Munich, Germany) for 60 min at 37º C. The resulting cell suspension was filtered through a 70-μm nylon mesh (Corning, USA) to remove undigested tissue fragments, and pelleted by centrifugation (400 × g for 5 min at 4ºC). Cells were re-suspended in fluorescence activated cell sorting (FACS) buffer (phosphate-buffered saline [PBS] with 1% foetal calf serum + 0.1% bovine serum albumin) before antibody labeling and FACS sorting. For preparation of porcine cell samples for scRNA-seq analysis, viable and nucleated (Sytox Blue- DRAQ5+) endothelial (CD31+ CD45-), leukocyte (CD45+) and other non myocytes (CD31- CD45-) were sorted in FACS Aria II cell sorter (BD biosenses). Then, cells were resuspended in PBS supplemented with 0.04% bovine serum albumin. Cell viability was then checked using an automated Countess III cell counter with software v1.0.296.782 (Thermo Fisher Scientific) in all single cell suspension composed of approximately 20% endothelial cells, 70% leukocytes and 10% other non-myocytes. Yucatan-Large White crossbred pigs were used to generate the long-lasting lone AF model (N=19) and sham-operated controls (N=8). A dual chamber pacemaker (Accent DR-RF, St. Jude Medical, CA, USA) was surgically implanted in the subcutaneous tissue of the right side of the neck. Atrial and ventricular leads were (Tendril STS, Abbott, MN, USA) inserted through the jugular vein and then positioned in the right atrial appendage and right ventricular apex or septum using active fixation electrodes. Ten days after the pacemaker implantation and animal recovery, a second procedure was performed to ablate de atrio-ventricular node. The latter aimed to prevent rapid atrio-ventricular node conduction and heart failure upon starting the AF induction protocol. The ablation procedure was performed using an 8-mm tip ablation catheter (Blazer II XP, Boston Scientific Corporation, Marlborough, USA), which was introduced through an 8-French introducer (ENGAGE Introducer Sheaths, Abbot, MN, USA) in the right femoral vein. Then, the ablation catheter was positioned at the atrio-ventricular node under fluoroscopic guidance and radiofrequency energy was delivered using a temperature control mode with power set at 70W and a cut-off temperature of 70ºC. Complete atrio-ventricular block was documented using intracardiac electrograms and surface ECG recordings. Then, the atrial pacing protocol was activated to induce AF. The atrial pacing protocol consisted of a 30-second burst pacing at 20 Hz and twice diastolic threshold, followed by a 6-second sensing period. If the pacemaker detected sinus rhythm restoration during the sensing period, high-rate atrial pacing was automatically reinitiated to induce a new AF episode. Atrial electrograms during AF episodes were recorded and stored using the automatic switch-mode algorithm of the pacemaker device. The same procedures were performed in sham-operated controls, but the high-rate atrial pacing protocol was not activated and the pacemaker was programmed in DDDR mode after atrio-ventricular node ablation. The procedures were performed after pre-medication (intramuscular ketamine 20 mg/kg and midazolam 0.5 mg/kg), anesthesia induction (intravenous fentanyl 0.010 mg/kg). After endotracheal intubation, pigs were mechanically ventilated using a volumetric ventilator (tidal volume: 10 ml/kg, respiratory rate: 14 breaths per minute, maximum inspiratory pressure: 25 mm H2O, intermittent positive end expiratory pressure: 4 mm H2O). Anesthesia was maintained using 2% sevoflurane. Electrocardiogram, oxygen and carbon dioxide levels were monitored throughout the procedure. Some pigs underwent also a procedure of ischemia-reperfusion (3-hour ischemia followed by 1 hour or continuous monitoring during reperfusion) at the proximal left circumflex artery. The procedure follows a similar approach to previous reports of ischemia-reperfusion to generate infarct-related substrates.39 Briefly, the procedure was performed using percutaneous access thorough an 8-French introducer (ENGAGE Introducer Sheaths, Abbot) in the right femoral artery. A bolus of intravenous heparin was administered (300 IU/kg) at the beginning of the procedure. Then, a 5-French AL-2 guide catheter (Cordis, FL, USA) was progressed until the left coronary trunk to assess the coronary anatomy with two angiographic projections. Then, the catheter tip was positioned into the proximal circumflex artery with the use of a 0.014-inch guidewire. Thereafter, an angioplasty balloon (3.5-mm diameter balloon, Medtronic, MN, USA) was positioned into the proximal circumflex artery and further inflated 462 to generate ischemia in both the lateral-basal part of the left ventricle and the left atrium. Appropriate balloon inflation and artery occlusion were confirmed on the coronary angiogram. The artery was occluded for 3 hours before reperfusion. Intravenous amiodarone (300 mg/h) was administered throughout the procedure to decrease the risk of ventricular arrhythmia. The procedure was performed using the same pre-medication and anesthesia protocol described for pacemaker implantation and atrio ventricular node ablation. Eight weeks after ischemia-reperfusion in the circumflex artery the animals underwent the same protocol described for the lone persistent AF model. Single-cell suspensions were loaded into a Chromium Next GEM Chip G (10x Genomics) to capture a maximum of 10,000 single cells per port utilizing the Chromium Controller (10x Genomics). scRNA-seq libraries were prepared using the Chromium Next GEM Single-Cell 3′ Kit v3.1 (10x Genomics). Individual libraries were diluted to a concentration of 10 nM and pooled for sequencing.
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
3_agosto_21
individual
not applicable
isolate
Sus scrofa
organism
Sus scrofa
organism
heart
organism part
atrial fibrillation + Myocardial Infarction
phenotype
NonDriver
region
Sus scrofa
scientific_name
male
sex
Yucatan-Large White
strain
Sample_ND_3_agosto_21
title