The anti-inflammatory LncRNA Heat4 interacts with the pro-inflammatory protein IP1 in non-classical monocytes promoting vascular regeneration

Background and Aims: Using next-generation-sequencing, we identified the lncRNA Heat4 which is significantly upregulated in patients with ischemic cardiomyopathy compared to controls. Here, we aim to characterize Heat4's function and identify potential interaction-partners. Methods: We performed poly-A-fractionation, qPCR and single-cell-sequencing to study Heat4’s function in vitro and used a carotid-injury model for in vivo-studies. Pulldown of Heat4 with subsequent mass spectrometry was used to identify potential Heat4-interaction-partners. Results: Heat4 is located in the cytoplasm of non-classical monocytes where it is stabilized by its poly-A-tail (+5.30-fold-enrichment in poly(A+)-fraction; p<0.05). Matching the known anti-inflammatory properties of non-classical monocytes, Heat4 mediates anti-inflammatory functions in vitro (Heat4-overexpression: -38.6% TNFα-RNA-reduction; p<0.05) and promotes vascular regeneration in vivo(carotid arteries of NOD-SCID mice regenerated faster after injection of human monocytes with Heat4-overexpression compared to control-monocytes (% regenerated area: +1.85-fold-enrichment; N=6; p<0.05)). In addition, an increased expression of the non-classical monocyte marker CD16 was found in monocytes after Heat4-overexpression (+2.37-fold-enrichment; p<0.05). We identified IP1 and IP2 as Heat4-interaction-partners (+1.20 and +1.45-fold-enrichment in Heat4-fraction compared to control-probe). While IP1 was validated as Heat4-interaction-partner, IP2 was probably only detected as part of the known heterodimer with IP1. Lower IP1- than IP1/IP2-concentrations were found in both monocyte cell lysate and supernatant. Heat4-overexpression resulted in reduced extracellular levels of the IP1/IP2-heterodimer (IP1/IP2: -23.6%; p<0.05) but not IP1 alone. Conclusions: The lncRNA Heat4 is elevated in the blood of patients with heart failure and limits the inflammatory response of non-classical monocytes. Modulating Heat4 levels may represent a novel strategy for treatment of cardiovascular diseases with impaired vascular functions.