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A TRANSGENIC APPROACH TO STUDY THE EFFECT OF CARDIAC FIBROBLAST-SPECIFIC ABLATION OF IL-1 SIGNALLING ON MYOCARDIAL REMODELLING
  1. KE Hemmings,
  2. SA Bageghni,
  3. KE Porter,
  4. MJ Drinkhill,
  5. JFX Ainscough,
  6. NA Turner
  1. Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Clarendon Way Leeds, UK

Abstract

Introduction Cardiac fibroblasts (CF) play a critical role in the repair of the heart following MI, but aberrant remodelling can lead to fibrosis and heart failure. We have previously demonstrated that interleukin-1 (IL-1), a key proinflammatory cytokine released following MI, is a potent modulator of CF function. The aim of this study was to develop a mouse model with fibroblast-specific ablation of IL-1 signalling through deletion of Myd88.

Methods We generated a mouse line with tamoxifen-inducible Cre-recombinase under control of the fibroblast-specific Col1a2 promoter, and loxP sites flanking exon 3 of the Myd88 gene, an essential component of the IL-1 receptor signalling complex. Mice were administered tamoxifen (75–100 mg/kg; i.p; 5 days) to induce Myd88 deletion. DNA was obtained from ear notches and RNA from cultured CF. Myd88 DNA deletion efficiency and mRNA expression levels were determined by real-time RT-PCR. All data are expressed as mean±SEM %GAPDH.

Results Tamoxifen induced a 32% reduction in Myd88 DNA (Cre- 0.172±0.017 (n=7) vs. Cre+ 0.117±0.015 (n=9); P=0.049) and a 63% reduction in CF Myd88 mRNA expression (Cre- 1.64±0.27 (n=4) vs. Cre+ 0.60±0.25 (n=4); P=0.023). Expression of DDR2 mRNA, a fibroblast marker, was unaffected (Cre- 0.91±0.06 vs. Cre+ 0.77±0.10; P=0.279) indicating the specificity of Myd88 deletion. Increasing tamoxifen dose (100 mg/kg) did not enhance deletion efficiency.

Conclusions We have successfully reduced expression of Myd88 in CF from tamoxifen-treated Cre+ animals. To obtain greater deletion efficiency we propose using a PGK-Cre line to globally delete one Myd88 allele in the germline prior to tamoxifen-induced ablation in CF.

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