Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Paul Kubes ( pkubes@ucalgary.ca ). This study did not generate new unique reagents.

Pericardial fluid samples (N = 5) were obtained from consenting patients undergoing valve replacement surgery (4- aortic valve replacement (all male, ages 67, 70, 65, and 69), 1-bicuspid aortic valve patient (female, age 42)) at the Foothills Medical Centre (Calgary, Alberta). All pericardial fluid samples were collected in a sterile environment on ice. Experiments involving human tissue usage were approved by the Conjoint Health Research Ethics Board at the University of Calgary and conform to the Declaration of Helsinki.

C57BL/6J mice were purchased from Jackson Laboratories. Gata6reporter mice () were kindly provided by Dr. Hadjantonakis (Memorial Sloan Kettering). Ccr2gene targeted mice () were kindly provided by Richard M. Ransohoff (Lerner Research Institute, Cleveland Clinic, Cleveland) and Israel F. Charo (University of California San Francisco, San Francisco). Gata6 floxed mice () were kindly provided by Dr. Medzhitov from Yale University and bred in-house with Lyz2mice. Lyz2;Gata6were subsequently bred with Gata6to generate Creand Crelittermates. 10-14 week old male were used for experiments. All mice were house under specific pathogen-free, double-barrier unit at the University of Calgary. Mice were fed autoclaved rodent feed and water ad libitum. Farm pigs were obtained from Britestone Farming Co. 10-12 week old pigs were used. All protocols used were in accordance with the guidelines drafted by the University of Calgary Animal Care Committee and the Canadian Council on the Use of Laboratory Animals.

Method Details

In vivo interventions Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending (LAD) coronary artery either with (conventional) or without (modified) excising and tearing the outer pericardial tissue layer. For the procedure, mice were anesthetized using isoflurane (2% isoflurane with oxygen as carrier gas), intubated and ventilated using a VentElite Small Animal Ventilator Ventilator (Harvard Apparatus). The chest wall was shaved and cleaned with ethanol and iodine prior to a left thoracotomy in the fourth intercostal space. The left ventricle was landmarked and the LAD was ligated with monofilament 8-0 suture (Ethicon). The chest and skin were closed with a 5-0 Vicryl suture (Ethicon) and air in the thorax was evacuated via a pleural catheter. The same surgeon performed all procedures in a blinded fashion. For bone marrow chimeras generation, 6 week old male C57BL/6J mice were lethally irradiated (2x 525cGY) and subsequently reconstituted with Gata6 H2B-Venus bone marrow cells for 8 weeks. Weber, 2015 Weber G.F. Immune targeting of the pleural space by intercostal approach. The intercostal approach of the pleural space (ICAPS) method was carried out as previously noted (). For the procedure, mice were anesthetized using isoflurane (2% isoflurane with oxygen as carrier gas) and placed on their left side with legs and arms fixed in place with the use of adhesive tape. The right antero-lateral thoracic area was shave and cleaned with ethanol and baxedin prior to performing a 3 cm long incision. A fluorescent bead loaded catheter syringe is then guided into the intercostal space. Once in the pleural space the total volume (50 μl, 5 μL of fluoresbrite fluorescent microsphere-1 μm(Polysciences, Inc.) + 45 μL saline) is slowly injected and the catheter is removed in one motion. Wound closure was subsequently performed using staples and pain medication (buprenorphine, 0.05 mg/kg) was administered subcutaneously.

Cardiac Function assessment Cardiac function was assessed via both non-invasive echocardiology and invasive pressure volume loop assessment. Functional assessment was performed prior to surgery (baseline) and 28 days post-MI. For PV-loop analysis, mice were anesthetized using isoflurane (4% induction, 2% maintenance), intubated and ventilated with a VentElite Small Animal Ventilator Ventilator (Harvard Apparatus). The neck was shaved, cleaned and excised to expose the right carotid artery. The carotid was occluded distally and a 1F conductance catheter (Millar Instruments) was gently advanced down the carotid artery into the left ventricle chamber. After recording baseline pressure volume measurements, an abdominal occlusion of the vena cava was performed to obtain a family of loops with varying afterload and preloads. After recording was complete, a parallel conductance value was obtained by jugular vein injection of hypertonic saline and blood was withdrawn to calibrate the conductance catheter. Animals were euthanized and tissues collected for subsequent analysis. Data were analyzed using the PV loop analysis module in Labchart (ADI Instruments). Transthoracic echocardiography was performed under light anesthesia (isofluorane) at baseline and 28 days post myocardial infarction. Images were acquired using a 30 MHz linear transducer and analyzed with Vevo 770 software (Visual Sonics). LV ejection fraction was calculated using end-diastolic (ED) and end-systolic (ES) volumes (LV ejection fraction = (LVED volume–LVES volume)/LVED volume x100%).

Cell isolation, Flow Cytometry, Cell sorting Mouse pericardial lavage was performed in anesthetised animals by a single injection of 100 μL of sterile saline into the pericardial cavity via PE-10 catheter and subsequent retrieval. Human pericardial fluid was collected during cardiac surgery using a syringe prior to opening the pericardial cavity. Pericardial samples were subsequently centrifuged at 1500 rpm for 5 min at 4°C and cell pellets was subsequently processed for flow cytometry. The murine heart ventricular tissue was excised, minced and subsequently digested in 450 U/mL collagenase I(Sigma), 125 U/mL collagenase XI(Sigma), 60 U/mL DNase I(Roche) and 60 U/mL hyaluronidase (Sigma) PBS for 1 hour at 37°C on an orbital shaker. For cell sorting experiments enzyme concentrations were doubled and digestion was performed for 30 min. Homogenates were initially passed through a 70 μm cell stainer and spun down at 60 g for 5 min at 4°C to remove cardiac parenchymal cells. Supernatant was collected and passed through a 40 μm cell stainer for a single cell suspension. For remainder of experiments, residual red blood cells were lysed using ACK (Invitrogen). The cells were blocked using anti-mouse CD16/32 antibody (2.4G2 clone, BioXcell) or human FcγR binding inhibitor (eBioscience) for 20-30 min. Cells were then stained for 30 min with specified markers (Key Resources Table). Appropriate isotype control antibodies were used to confirm positive signals. Non-viable cells were identified using viability dye efluor 780 (eBioscience) or ghost dye™ red 710 (TONBO Biosciences). For intracellular staining the Foxp3 staining kit (Thermo Fisher) was used. Samples were run using BD FACS Canto flow cytometer and analyzed using FlowJo software (Tree Star). Neutrophils were identified as CD11b+ Ly6Ghi Ly6Cint. Classical monocytes were identified as CD11b+ Ly6G-Ly6Chi. Cardiac macrophages were identified as CD11b+ Ly6Clo CD64+ MHCII+/−. Gata6+ pericardial macrophage (GPCM) were identified as CD11bhiCD102+MHCII-Gata6+. Pericardial MHCII+ myeloid and DC populations were identified as CD11b+ CD102- MHCII+ CD11c- and CD11b+CD102- MHCII+CD11c+, respectively. For cell sorting experiments, cardiac macrophages were identified as viable CD45+ CD11b+ CD64+ Ly6Clo and cavity macrophages from the pericardium, pleura, and peritoneum were identified as viable CD45+ CD11b+ CD102+.

RNaseq Analysis 5 cells from each group were sorted directly into TRIzol and stored at −80°C. A total of 4 biological replicates were prepared for each macrophage group. RNA was isolated using the Direct-zol RNA isolation kit (Zymo Research) following the included protocol. The harvested RNA was prepared for sequencing using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs). Sample Concentrations were calculated using PicoGreen and multiplexed samples were mixed in equimolar concentration and sent to the NIAMS Sequencing Core (Bethesda, MD) for sequencing on an Illumina HiSeq 3000. Data were demultiplexed and converted to FastQ using Bcl2fastq 2.17.1.14 (Illumina) and mapped to mm10 with Tophat 2.1.1. RPKM counting and normalization were performed using Partek GS 7.18.0723. Principal Component Analysis and Hierarchical clustering were performed on gene level RPKM data using Partek GS. Differential expression analysis was performed using fold change ≥ 2, with a raw p value of < 0.05 and a false discovery rate (FDR) adjusted p value ≤ 0.1 as thresholds. Venn diagrams were generated using Venny 2.1 ( Pericardial cavity, pleural cavity, peritoneal cavity, and cardiac macrophage samples were collected and pooled from multiples naive C57 wild-type mice and 1x10cells from each group were sorted directly into TRIzol and stored at −80°C. A total of 4 biological replicates were prepared for each macrophage group. RNA was isolated using the Direct-zol RNA isolation kit (Zymo Research) following the included protocol. The harvested RNA was prepared for sequencing using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs). Sample Concentrations were calculated using PicoGreen and multiplexed samples were mixed in equimolar concentration and sent to the NIAMS Sequencing Core (Bethesda, MD) for sequencing on an Illumina HiSeq 3000. Data were demultiplexed and converted to FastQ using Bcl2fastq 2.17.1.14 (Illumina) and mapped to mm10 with Tophat 2.1.1. RPKM counting and normalization were performed using Partek GS 7.18.0723. Principal Component Analysis and Hierarchical clustering were performed on gene level RPKM data using Partek GS. Differential expression analysis was performed using fold change ≥ 2, with a raw p value of < 0.05 and a false discovery rate (FDR) adjusted p value ≤ 0.1 as thresholds. Venn diagrams were generated using Venny 2.1 ( http://bioinfogp.cnb.csic.es/tools/venny/index.html ). Metascape ( Metascape.org ) was employed to identify enriched pathways based on the differentially expressed genes between two groups. Pathway gene sets from Gene Ontology (GO) biological processes ( http://www.geneontology.org/ ), KEGG pathway ( https://www.genome.jp/kegg/ ), Reactome molecular pathways ( http://www.reactome.org/ ), and CORUM complexes database ( http://mips.gsf.de/genre/proj/corum/index.html ) were used.

Whole-mount imaging and tissue clearing Prior to sacrifice, mice were anesthetized (10 mg/Kg xylazine hydrochloride and 200 mg/Kg ketamine hydrochloride), intubated and ventilated with a MiniVent ventilator for mice (Model 845, Harvard Apparatus). Thoracotomy was performed and Ef660 conjugated anti-mouse podoplanin antibody (clone, eBioscience) was injected into the pericardial space using a PE10 cathether and allowed to label the pericardial mesothelium for 10-15min. For whole-mount imaging, the animal was subsequently euthanized, the outer pericardial layer removed and the heart excised and placed in 2% PFA containing PBS solution for 30 min. The fixed heart was placed left ventricle down onto a glass coverslip on the inverted microscope stage and kept in place using modeling putty. Image acquisition of the heart was performed using Olympus IX81 inverted microscope, equipped with an Olympus focus drive and a motorized stage (Applied Scientific Instrumentation, Eugene, OR) and fitted with a motorized objective turret equipped with 4x/0.16 UPLANSAPO, 10X/0.40 UPLANSAPO and 20x/0.70 UPLANSAPO objective lenses and coupled to a confocal light path (WaveFx; Quorum Technologies, Guelph, ON) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation, Tokyo, Japan). Laser excitation wavelengths 491-, 561-, and 642nm (Cobolt) were used in a rapid succession together with the appropriate band-pass filters (Semrock). A back-thinned EMCCD 512 × 512 pixel camera was used for fluorescence detection. Volocity software (Perkin Elmer) was used to drive the confocal microscope and for acquisition and analysis of images. Composite stitch images of the heart were acquired combining multiple 10x FOVs and placed together by the Volocity software. Venus+ cells were identified using the “Find object” function within the measurement modality and quantified for the entire area for the heart captured in the stitch. Klingberg et al., 2017 Klingberg A.

Hasenberg A.

Ludwig-Portugall I.

Medyukhina A.

Männ L.

Brenzel A.

Engel D.R.

Figge M.T.

Kurts C.

Gunzer M. Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy. Heart tissue clearing was performed as previously described (). In brief following intrapericardial administration of antibody, mice were perfused using 5mM EDTA containing PBS and 4% PFA containing PBS. Heart was excised, placed in 4% PFA containing PBS for 2 hours followed by sequential dehydration steps (50%, 70%, 99%, 99% EtOH) of 4 hours at 4°C. After dehydration, hearts were place in ethyl cinnamate (Sigma). For imaging, cleared heart were placed LV upward in an ethyl cinnamate containing vessel and topped with a coverslip. Image acquisition of was performed using a Leica TCS SP8 upright resonant scanning dual white-light laser equipped confocal microscope. Fluorescently labeled cells were excited with with a white-light laser at 488, 594 and 642nm and detected using HyD hybrid internal descanned detectors. Leica software was used to drive the confocal microscope, 3D rendering, acquisition of images, and video generation.