Haematopoietic and cardiac GPR55 synchronize post-myocardial infarction remodelling

While classical cannabinoid receptors are known to crucially impact on myocardial infarction (MI) repair, a function of the cannabinoid-sensitive receptor GPR55 herein is poorly understood. We investigated the role of GPR55 in cardiac physiology and post-MI inflammation and remodelling. Global GPR55−/− and wildtype (WT) mice were basally characterized or assigned to 1, 3 or 28 days permanent MI and subsequently analysed via pro-inflammatory and pro-hypertrophic parameters. GPR55−/− deficiency was basally associated with bradycardia, increased diastolic LV volume and sarcomere length and a subtle inflammatory phenotype. While infarct size and myeloid cell infiltration were unaffected by GPR55 depletion, acute cardiac chemokine production was prolonged post-MI. Concurrently, GPR55−/− hearts exhibited a premature expansion of pro-reparative and phagocytic macrophages paralleled by early up-regulation of extracellular matrix (ECM) factors 3 days post-MI, which could be mimicked by sole haematopoietic GPR55 depletion. Moreover, global GPR55 deficiency mitigated MI-induced foetal gene re-programming and cardiomyocyte hypertrophy, culminating in aggravated LV dilatation and infarct expansion. GPR55 regulates cardiac homeostasis and ischaemia responses by maintaining adequate LV filling and modulating three crucial processes post-MI: wound healing kinetics, cardiomyocyte hypertrophy and maladaptive remodelling.


Fig. S6 Summary of experimental findings
Under naïve conditions GPR55-/-deficiency is associated with bradycardia, increased diastolic LV volume and sarcomere length, cardiomyocyte size, heart weight and an immune response resembling the stretch-induced inflammation. The latter is reflected by elevated neutrophil and macrophage counts and enhanced chemokine and Mmp-9 expression in the heart. After MI, GPR55 deficiency initially attenuates, yet subsequently prolongs acute burst in LV chemokine expression and increases cardiac abundance of pro-reparative CD206+ and MERTK+ macrophages. This in turn is accompanied by potentiated up-regulation of ECM factors such as collagen and MMP-9. Simultaneously, absence of GPR55 mitigates reexpression of the fetal gene programme and exacerbates LV remodelling culminating in an amplified functional decline post MI. Taken together, our study alludes for the first time in vivo toward a contributory role of GPR55 to development of LV volume-overload, synchronization of post-MI wound healing and regulation of LV remodelling.

Fig. S7 Scheme of LV partition for different analyses
Myocardium below the ligation knot (and at the respective LV level in no MI and sham groups) was split for histology or flow cytometry and concurrent gene expression analyses. Herein, midventricular tissue was used for flow cytometry or histology, respectively, the apical LV was assigned to gene expression analyses.

Tissue sampling and processing
At each study endpoint, mice were euthanized by overdosed xylazine/ketamine (120/10 mg/kg body weight) anaesthesia. EDTA blood was collected from the right ventricle and split for flow cytometry (50 µl) and plasma collection. After LV puncture and perfusion with PBS, the heart, lungs, spleen, liver, left kidney, femoral bone marrow and left tibia were excised.
Wet and dry weight of lungs, liver and the left kidney were determined to assess tissue congestion. Tibia length was measured to serve as intervention independent normalization factor for heart weight. The myocardium was either entirely used for determination of infarct size or split for gene expression and histological analyses or for gene expression and flow cytometry analyses, respectively, depending on the post-MI time-point and read-outs (see Supplementary Fig. S7). Spleens were split longitudinally for mRNA and flow cytometry analyses. Bone marrow from one femur was used for flow cytometry. Tissue slices for histological analyses were fixed in 4% paraformaldehyde within one minute after excision of the heart and stored at 4°C. Tissue for qPCR was immediately frozen in liquid N 2 and stored at -80°C. Tissue for flow cytometric analyses was directly transferred into PBS and stored on ice until further processing.

Flow cytometry antibodies and gating strategies
All obtained cell suspensions were rinsed, re-suspended in PBS with 1% BSA, subjected to

Plasma albumin and volume assessment
Plasma albumin levels were determined via the Abcam Mouse Albumin ELISA kit ab207620.
Plasma volume was quantified after centrifugation of 400 µl whole EDTA blood at 7500 rpm for 10 min and calculated as plasma volume per ml blood.