Cold and hot fibrosis define clinically distinct cardiac pathologies
Fibrosis is a pathology of excessive scarring and a major unmet medical need. Identifying key simplifying principles is needed to better understand fibrosis and may yield new therapeutic approaches. Fibrosis is driven by myofibroblasts that interact with macrophages. We developed a cell-circuit model that predicted two types of fibrosis - 'hot fibrosis' that includes both macrophages and myofibroblasts, and 'cold fibrosis' dominated by myofibroblasts alone. Here, we tested these concepts using an in-vivo pathological model for cardiac fibrosis resulting from myocardial infarction (MI). We show that MI progresses into cold fibrosis in both mice and pigs. We used the cell-circuit model to find a fragility of cold fibrosis - the autocrine loop which supports myofibroblast growth. We identified the growth factors in the autocrine loop and demonstrate that one of these factors- TIMP1- can stimulate cardiac myofibroblast proliferation in vitro. Inhibiting TIMP1 in-vivo reduced fibrosis in adult mice after MI. This study demonstrates the concept of cold fibrosis following acute MI and the feasibility of our circuit-to-target approach to better understand and treat fibrosis. Overall design: Landrace pigs, 3 months old (male and female) ~22-35 kg, were purchased from a local farm. Animal care and all experimental procedures were performed in strict accordance with the German and National Institutes of Health animal legislation guidelines and were approved by the local animal care and use committees. Experimental approach: Briefly, a balloon was placed in the LAD distal to the bifurcation of the first diagonal branch and inflated with 6 atm. Correct localization of the coronary occlusion and patency of the first diagonal branch was ensured by injection of contrast agent. The percutaneous transluminal coronary angioplasty balloon was deflated after 60 minutes of ischemia; the onset of reperfusion was documented angiographically. At the onset of reperfusion, animals were treated with 33 µg/Kg (in 5mL of saline) of rhAgrin (#6624-AG, R&D Biosystems) or saline alone as control. Pig heart samples were collected at day 3 and 28 following the MI. Hearts were harvested and stained by Tetrazolium chloride (TTC) to visualize scar/infarct. Left ventricles were dissected transversely to 5, 1cm thick slices, and each slice was further sectioned into 4/8 sections. Sections were annotated as infarct zones (positive for TTC) and remote zones (TTC negative). Sections were frozen on dry ice for further analysis and were later subjected to RNA purification/ histological staining. Total RNA was extracted from 28 frozen pig samples in a paired manner (Remote and Infarct zones from the same animal were used to obtain optimal internal control [rhAgrin day 3 (n = 4), Saline day 3 (n = 3), rhAgrin day 28 (n = 4), Saline day 28 (n = 3)]. Following acquisition, frozen samples were crushed into fine powder using a mortar and pestle and further processed using miRNeasy Mini Kit (Qiagen, Cat# 217004) according to RNA-seq libraries were prepared at the Crown Genomics institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science. Libraries were prepared using the INCPM-mRNA-seq protocol. Briefly, the polyA fraction (mRNA) was purified from 500ng of total input RNA followed by fragmentation and the generation of double-stranded cDNA. After Agencourt Ampure XP beads cleanup (Beckman Coulter), end repair, A base addition, adapter ligation, and PCR amplification steps were performed. Libraries were quantified by Qubit (Thermo fisher scientific) and TapeStation (Agilent). Sequencing libraries were constructed with barcodes to allow multiplexing. Sequencing was done on a NovaSeq600 instrument (Illumina) using an S1 100 cycles kit, allocating approximately ±35.7million single-end 100-base-pair reads per sample (single read sequencing). Fastq files for each sample were generated by the usage bcl2fastq v2.20.0.422.  manufacturer's guidelines. Replicates of high RNA integrity (RIN), determined by TapeStation (Agilent) (RIN=8.2±0.1) were processed for RNA-seq library preparation.