P6346Purinergic receptor Y6 (P2Y6) deficiency impairs left ventricular function
Authors: Ahmed I , Merz J , Dimanski D , Rofa K , Rog-Zielinska E A , Koentges C , Hoppe N , Dufner B , Kohl P , Bugger H , Hilgendorf I , Bode C , Zirlik A , Stachon P 
Published: October 2019

Abstract

Introduction
Cardiomyopathies due to pathological remodelling are the most common myocardial disorders and leading indication for heart transplant in young adults. Cardiomyocytes rely on sessile macrophage phagocytosis for cell homeostasis. Purinergic receptor Y6, selectively binding to UDP, promotes phagocytosis and is involved in several tissue-remodelling processes.
We hypothesize a critical role of P2Y6 in the maintenance of cardiac tissue homeostasis in vivo.

Methods
Echocardiography was performed on male C57Bl6 wild-type (WT) and P2Y6-deficient (KO) mice at different ages up to 20 weeks. In addition, WT and KO mice underwent bone marrow transplantation. For histological analysis, hearts from WT and KO mice were stained with HE, Masson's trichrome, wheat germ agglutinin, CD11b, LC3B and TUNEL. Cardiac ultrastructure of WT and KO hearts was investigated by electron microscopic imaging. Furthermore, uptake of fluorescent pHrodo bioparticles by WT and KO bone marrow derived macrophages (BMDMs), either in presence or absence of specific P2Y6 agonist UDP or full antagonist MRS2578, was measured in vitro.

Results
KO mice were significantly impaired in their LV function by reduced ejection fraction (WT: 57.37±1.27%, n=13, KO: 44.13±1.09%, n=16, p<0.0001), stroke volume (WT+: 39.25±1.94μl, n=13, KO: 33.57±1.94μL, n=16, p<0.05), and cardiac output (WT: 20.79±1.11 mL/min, n=13, KO: 17.84±0.85 mL/min, n=16, p<0.05). A long-term follow-up revealed progressive impairment of the cardiac function (4 w: WT: 62.07±1.11%, n=5, KO: 48.73±1.03%, n=10, p<0.0001; 6 w: WT: 54.29±1.88%, n=5, KO: 44.61±1.4%, n=10, p<0.01; 8 w: WT: 56.43±1.44%, n=5, KO: 44.72±0.89%, n=10, p<0.0001). Echocardiography 6 weeks after bone marrow transplantation demonstrated convalescence upon reconstitution with WT bone marrow (WT in KO: 46.19±2.68%, n=3 vs KO in WT: 38.40±1.26%, n=3). No major differences with regard to morphology, cell size, collagen deposition, leukocyte infiltration or apoptosis were observed in histology. However, LC3B expression was increased in KO mice (mean fluorescent area: WT: 191.1±19.93μm2, n=5, KO: 261.2±18.34μm2, n=10, p<0.05). Electron microscopic imaging revealed a distinctly impaired ultrastructure with T-tubule remodelling, mitochondrial derangement and abnormal numbers of autophagosomes in KO hearts. In vitro, fluorescent particle phagocytosis by BMDMs was completely blocked after treatment with MRS2578.

Conclusion
Global P2Y6 deficiency leads to a progressive cardiomyopathy in mice, mainly characterized by an impaired left ventricular function and ultrastructural irregularities. Its exacerbation seems to be prevented by reconstitution with WT bone marrow. For the underlying mechanism, we conclude a deranged cardiomyocyte homeostasis in KO animals due to defective phagocytic activity of resident macrophages. Potential induction of cardiac clearance via P2Y6 signalling could be a promising therapeutic target in the field of cardiomyopathies.