Loss of Mouse P2Y6 Nucleotide Receptor Is Associated with Physiological Macrocardia and Amplified Pathological Cardiac Hypertrophy

Sophie Clouet,Larissa Di Pietrantonio,Evangelos-Panagiotis Daskalopoulos,Hrag Esfahani,Michael Horckmans,Marion Vanorlé,Anne Lemaire,Jean-Luc Balligand,Christophe Beauloye,Jean-Marie Boeynaems,Didier Communi

doi:10.1074/jbc.m115.684118

Abstract

The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced postnatal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy.

Highlights

The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration
These various effects of nucleotides are depending on cell-specific expression of P2Y subtypes involved: mouse P2Y4 receptor is not expressed in cardiomyocytes and is mainly expressed in cardiac endothelial cells, whereas mouse P2Y2 and P2Y6 receptors were found in all tested cardiac cells [2, 3, 6, 7]
Identification of a Physiological Cardiac Hypertrophy in P2Y6-null Mice—We proceeded with the observation of heart anatomy of P2Y6 knock-out (P2Y6 KO) and wild type (WT) mice aged between 8 and 12 weeks

Summary

Experimental Procedures

Ethics Statement—C57Bl6/J P2Y6 knock-out (P2Y6 KO) male mice were obtained as previously described [15]. EdU was revealed with the Click-iT௡ EdU Imaging Kit (Invitrogen, Life Technologies) and cardiomyocytes were stained with troponin T antibody (Sigma) and nuclei with Hoechst (Thermo Scientific, Waltham, MA). Ventricles were stored in TRIzol௡ reagent (Life Technologies) for RNA extraction or in Tissue-Tek௡ OCTTM for histological examination. RNA Sequencing Analysis—Indexed cDNA libraries were obtained using the TruSeq Stranded mRNA sample preparation kit (Illumina) following the manufacturer’s recommendation. Genes with counts per million (cpm) Ն1 in KO RNA and with cpm KO/cpm WT ratio Ն2 were selected and analyzed with Enrichnet software, which sorted genes regulated in KO mice in function of KEGG pathways in its database. Cells were fixed with 4% paraformaldehyde and stained with Alexa F488conjugated WGA (Molecular Probes, Life Technologies) and eF570-conjugated Ki67 (eBiosciences, San Diego, CA) antibodies. Comparison between groups were realized using one-way analysis of variance and/or t tests (Mann-Whitney test for non-parametric data), or two-way analysis of variance test

Results

Percentage difference between

Discussion