Stromal Cardiac Cells: Role in Fibrosis and Tissue Regeneration
Description
This repository contains primary echocardiography and flow cytometry data. Spatial transcriptomics data will be deposited at Gene Expression Omnibus (GEO) as soon as the analyses are completed. Representative confocal microscopy images will be included in the manuscript; raw imaging data are available from the corresponding author upon reasonable request.
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Animal Model: Transgenic mice (3 months old, male/female) with PDGFRα cell ablation via Tamoxifen (Sigma Aldrich, # T5648; 2mg IP injection). The animal model to reproduce the data are PdgfrαMerCreMer mice, defined as MCM in this paper, were obtained from RIKEN (Japan), accession number CDB0674K, http://www.clst.riken.jp/arg/mutant%20mice%20list.html. Mice were generated by knocking the tamoxifen inducible Cre-ER cDNA into the Pdgfrα locus, as described by Ding, G., Tanaka, Y., Hayashi, M., Nishikawa, S. & Kataoka, H. PDGF receptor alpha+ mesoderm contributes to endothelial and hematopoietic cells in mice. Dev Dyn 242, 254-268 (2013). MCM mice were kept in hemizygous breeding and crossed with DTA-flox and R26R-eYfp. 2) DTA-flox mice (Jackson Laboratories; strain B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J, JAX stock # 009669) have Cre-inducible expression of the human DTA that renders cells susceptible to ablation following recombination. Ischemia reperfusion model (I/R): Three-four months old male mice were anesthesized using 5% isoflurane and oxygen until loss of righting reflex and then maintained at 2% isoflurane. A 0.5 cm median cervical skin incision was performed, and the lobes of the thyroid gland will be separated to expose the trachea. Trachea was intubated with a cannula (Harvard Apparatus) connected to a ventilator, which was set at 260 μl/stroke (tidal volume), and 130 strokes per minute (ventilation rate). A cut through the skin and thoracic muscles was performed between the 3rd and 4th intercostal space. I/R was induced by ligation of the left descendent coronary artery around a PE-10 tubing for 20 minutes followed by reperfusion. The chest cavity was closed by sewing shut the incision with 6-0 silk suture. The mouse was extubated after resuming normal breathing pattern. For post-operative analgesia, Ethiqua XR was administered at 3.25 mg/kg subcutaneously (SC) before the animal is ambulatory and then provided every 48 hrs if required. Control mice were sham operated with chest opening and no artery ligation. Baseline mice were not operated Echocardiography: Data acquired using the VisualSonics Vevo 2100 system. Measurements (EF, FS, LV mass) were calculated using VevoLab software from M-mode images. Flow Cytometry: Hearts were dissociated with Liberase DH from Roche 30 minutes at 37C in a thermoshacker at 800rpm. Cells were stained with APC-conjugated Anti-Mouse PDGFRα (Clone APA5, Fisher #50-112-9423). Gating/Analysis: Dead cells excluded via DAPI. Analysis performed using Cytobank. The gating strategy focused on the PDGFRα+ stromal population. Data Processing: Statistical analysis and plotting were performed in GraphPad Prism V.10.
Institutions
- Icahn School of Medicine at Mount SinaiNew York, New York
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Funders
- University of PaviaLombardy, PaviaGrant ID: European Union – NextGenerationEU through the Italian Ministry of University and Research (MUR) under the PNRR (Piano Nazionale di Ripresa e Resilienza)