Deep molecular phenotyping of paracardial fat unveils biological signals implicated in cardiometabolic heart failure with preserved ejection fraction

Abstract Background Cardiometabolic HFpEF (cHFpEF) is a multisystemic syndrome where inflammatory and metabolic signals generated in several organs contribute to adverse cardiac remodeling and high mortality rates. Paracardial fat (pCF) - located superior to the visceral pericardium and around the blood vessels - is emerging as a metabolically active adipocyte depot which could contribute to myocardial dysfunction and cHFpEF features. Purpose The present study investigates the role of pCF in the pathophysiology of cHFpEF. Methods We employed a mouse model of cHFpEF, characterized by the combination of highFat diet (60 kcal% fat,) and LNAME 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) was performed by using the 10x Chromium analyzer. Cell-cell communication analysis was performed by using CellChat software. Gain and loss-of-function of top-ranking genes were performed in human adipocytes exposed to metabolic stress by palmitic acid (PA). Analysis of downstream pathways and secretome changes were also performed upon modulation of target genes. Results pCF weight was increased in cHFpEF as compared to control mice (Figure). The transcriptional and cellular landscape of pCF was performed by high-resolution snRNAseq analysis and 20’397 cells from control and cHFpEF mice were captured. At the chosen clustering resolution, the analysis 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 including Nnat (encoding for Neuronatin), Mcam, C4b and Acaca (Acetyl-CoA Carboxylase Alpha). Pathway-gene enrichment network analysis confirmed the activation immuno-metabolic signaling, integrin-mediated signaling, adipocyte catabolic processes and inflammation in pCF cHFpEF vs. controls. Interactome analysis unveiled a strong link between adipocytes and immune cell activation in HFpEF vs control pCF specimens. Overexpression of Nnat and gene silencing of Acaca recapitulated adipocyte dysfunction by increasing lipid peroxidation and endoplasmic reticulum (ER) stress. Of note, the secretome of Nnat-overexpressing adipocytes led to macrophage activation thus suggesting a detrimental cross-talk between pCF adipocytes and immune cells. Conclusions By investigating the molecular landscape of pCF, we unveiled new biological signals implicated in pCF dysfunction, adipocyte inflammation and immune cell activation. Modulation of the identified targets could help prevent the secretion of pro-inflammatory mediators and cardiac dysfunction in cardiometabolic HFpEF.