An adipocyte-immune crosstalk drives paracardial fat inflammation and cardiac dysfunction in heart failure with preserved ejection fraction

Introduction Paracardial fat (pCF) - located superior to the visceral pericardium and around the blood vessels - is emerging as a new metabolically active fat depot which could contribute to cardiac dysfunction in conditions of metabolic stress such as cardiometabolic heart failure with preserved ejection fraction (cHFpEF). Purpose To define the role of pCF in the pathogenesis of cHFpEF and to identify adipocyte-derived effectors linking pCF dysfunction to immune activation and cardiomyocyte injury. Methods A mouse model of cHFpEF was generated by the combination of high-fat diet (60 kcal% fat,) and L-NAME in the drinking water (0.5 g/L) for 15 weeks. Control mice received a control diet (10 kcal% fat) and vehicle in the drinking water. Single-cell nuclei RNA sequencing (snRNAseq) and cell-cell communication analysis were performed in pCF from control and HFpEF mice. Gain and loss-of-function of top-ranking genes (i.e. Neuronatin, NNAT) were performed in in vitro differentiated human adipocytes (ADs) exposed to metabolic stress by palmitic acid (PA) and by lipid transfection of plasmid carrying the selected target. Analysis of downstream pathways was performed by real time PCR, capillary electrophoresis, cell staining and colorimetric assays. The crosstalk between immune cells and cardiomyocytes was investigated by exposing cardiomyocytes to conditioned media collected from NNAT-overexpressing ADs. Results pCF weight and ADs size were increased in cHFpEF as compared to control mice. The transcriptional landscape of pCF revealed two different adipocyte clusters: adipocyte1 (defined by Pparg/Nrg4 markers) and adipocyte2 (Ncam1/Itga7 markers). The adipocyte2 cluster was highly enriched in cells from cHFpEF mice and showed a significant dysregulation of several genes, with NNAT (encoding for Neuronatin) as the top transcript. Pathway-gene enrichment network analysis confirmed the activation immuno-metabolic signaling, integrin-mediated signaling, adipocyte catabolic processes and inflammation in pCF from cHFpEF vs control mice. Interactome analysis unveiled a strong link between adipocytes and immune cell activation. Overexpression of NNAT recapitulated features of ADs dysfunction such as oxidative stress, inflammation and senescence. Proteomic profiling in conditioned media from NNAT-overexpressing ADs unveiled a strong upregulation of pro-inflammatory molecules, namely MCP-3 and MCP-4. Consistently, ADs conditioned media was able to induce macrophage activation as well as cardiomyocyte hypertrophy and dysfunction. Conclusions By investigating the molecular landscape of pCF, we unveiled NNAT as a pivotal driver of ADs dysfunction, immune cell activation and cardiomyocyte damage. Therapeutic modulation of NNAT could prevent pCF remodelling and cardiac dysfunction in patients with cardiometabolic HFpEF.