The long non-coding RNA Heat4 is elevated in heart failure patients and mediates anti-inflammatory functions thereby promoting vascular regeneration
Authors: Kneuer J M , Winkler M , Meinecke T , Moebius-Winkler M N , Weiss R , Haas J , Garfias-Veitl T , Von Haehling S , Keller T , Thiele H , Lurz P , Speer T , Laufs U , Boeckel J N 
Published: October 2022

Abstract

Background and purpose
Activation of the immune system correlates with the severity and the prognosis of patients with heart failure (HF). Here, we aim to identify and characterize long non-coding RNAs (lncRNAs) as a potential mechanistic link between the activation of the immune system and the pathophysiology of HF.

Methods and results
Using next-generation sequencing we found a yet uncharacterized lncRNA to be significantly upregulated in peripheral blood mononuclear cells of ischemic cardiomyopathy patients compared to controls, which we named Heat4 – Heart-disease associated transcript 4 (N=4; 2.05-fold increase; p&lt;0.05). In the blood, monocytes show the highest expression of Heat4 and here in particular the non-classical monocytes compared to classical monocytes (N=4; 3.37-fold; p&lt;0.05). Matching the known anti-inflammatory properties of this monocyte subpopulation we found that overexpression of Heat4 in monocytes resulted in decreased levels of inflammation (TNFα: −38.6%; p&lt;0.05). Accordingly, a knockdown of Heat4 increased levels of inflammatory cytokine expression (TNFα: +4.14-fold; p&lt;0.05). Non-classical monocytes are known to maintain vascular homeostasis by patrolling the endothelium in search of injury. Indeed, overexpression of Heat4 in human monocytes increased vascular regeneration after injury of the carotid artery in NOD-SCID mice (N=6; +1.85-fold compared to injection of control monocytes; p&lt;0.05). We found Heat4 enriched in the cytoplasm of monocytes compared to the nuclear fraction. Using biotin-labelled RNA probes containing 2$'$O-Me-RNA oligonucleotides we performed RNA antisense affinity selection and subsequent mass spectrometry to identify proteins interacting with Heat4. We found two proteins, namely IP1 and IP2, enriched in the Heat4 fraction (+1.20 and +1.45-fold, respectively compared to the control probe). Knockdown of IP1 resulted in reduced induction of inflammatory gene expression (IL-6: −49.2%; p&lt;0.05) after stimulation of monocytes with TNFα. Mechanistically, overexpression of Heat4 resulted in reduced extracellular levels of the IP1/IP2 heterodimer (IP1/IP2: −23.6%; p&lt;0.05) as determined by ELISA.

Conclusion
The lncRNA Heat4 is elevated in the blood of patients with HF. Heat4 limits the extent of the inflammatory response of non-classical monocytes and leads to a faster regeneration after vascular injury. Heat4 is located in the cytoplasm of monocytes interacting with the pro-inflammatory proteins IP1/IP2 and repealing their extracellular release. Modulating Heat4 levels may represent a novel strategy for treatment of cardiovascular diseases with impaired vascular functions.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Projektfoerderung im Bereich der Herzmedizin, Leipzig