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<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<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<0.05). Accordingly, a knockdown of Heat4 increased levels of inflammatory cytokine expression (TNFα: +4.14-fold; p<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<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<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<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
Background and purpose Cardiogenic shock (CS) remains the leading cause of death in acute myocardial infarction (AMI), with high mortality rates of 40–50%. The long non-coding RNA (lncRNA) Heat4 is associated with the inflammatory response of non-classical monocytes. Previous experimental work shows that this mechanism may be important in heart failure (HF) and during regeneration after vascular injury. Here, we investigate the association of Heat4 with survival in patients with chronic HF and assessed its regulation in AMI and CS. Methods and results Heat4 was elevated in the blood of HF patients compared to age-matched non-failing controls (+5.2-fold; HF: N=63; Controls: N=38; p<0.05). Heat4 showed a positive correlation with systemic inflammation (hsCRP; r=0.41; p<0.05) and was negatively associated with LVEF (r=−0.45; p<0.001). Heat4 blood levels showed good discriminatory power for prevalence of HF (AUC = 0.734; p<0.05) and mortality prediction after 4-year follow-up (AUC = 0.789; HF: Death N=32; Controls: Death N=0; p<0.05). Furthermore, Heat4 was elevated in the blood of patients with AMI compared to controls (+1.85-fold; AMI: N=42; Controls: N=23; p<0.05). Heat4 showed a very strong induction in patients suffering from CS (+284.5-fold; CS: N=4; Controls: N=5; p<0.05). In agreement with an anti-inflammatory signaling, Heat4 showed a dynamic regulation in patients with CS with a 284.5-fold increase during acute shock and a decrease 24 hours after revascularization (−82.3% compared to day of revascularization). This regulation was validated in an independent second cohort. Conclusion The lncRNA Heat4 is upregulated in the blood of patients with chronic heart failure, acute myocardial infarction and cardiogenic shock. In CS, Heat4 is dynamically regulated. These data set the stage to further assess Heat4 blood levels as a strategy for risk stratification and potential treatment target in HF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Projektfoerderung im Bereich der Herzmedizin, Leipzig
Background and purpose Activation of the immune system correlates with the severity and the prognosis of patients with heart failure (HF). This study aims to identify and characterize long non-coding RNAs (lncRNAs) as a potential mechanistic link between the pathophysiology of HF and the activation of the immune system. Methods and results Next-generation sequencing (NGS) studies identified a 2.05-fold increase of the lncRNA Heat4 in the blood of patients with HF compared to controls, which was validated in a larger cohort (HF: N=63; Controls: N=38; p<0.05). Interestingly, the lncRNA Heat4 is encoded in the well-known immune receptor locus CD300, together with 8 CD300-receptors which are associated with activation of the immune system. To determine the cellular origin of Heat4 in blood, we performed MACS and identified Heat4 to be enriched in non-classical monocytes compared to classical monocytes (3.37-fold, p<0.05). The expression of Heat4 in non-classical monocytes was further validated by single-cell RNA sequencing. Overexpression of Heat4 in monocytes decreased levels of pro-inflammatory cytokines such as TNFα (38.6% reduction, p<0.05). Conversely, the knockdown of Heat4 resulted in elevated levels of pro-inflammatory cytokines, including IL6 (10.83-fold, p<0.05) and TNFα (4.14-fold, p<0.05). In a larger cohort including patients with HF, Heat4 was able to determine the prevalence of heart failure by AUC=0.734 (p<0.05). Moreover, in a 4-year follow-up of the same cohort, Heat4 predicted mortality by AUC=0.789 (HF: N=63, Dead=32; Controls: N=38, Dead=0; p<0.05). Conclusion The long non-coding RNA Heat4 is elevated in the blood of HF patients. Mechanistically, Heat4 limits the extent of the inflammatory response of non-classical monocytes. Therefore, Heat4 may provide a regulatory link between inflammation and HF. FUNDunding Acknowledgement Type of funding sources: None.
Background and Purpose: Activation of the immune system correlates with the severity and the prognosis of patients with heart failure (HF). This study aims to identify and characterize long non-coding RNAs (lncRNAs) as a potential mechanistic link between the pathophysiology of HF and the activation of the immune system. Methods and Results: Using next-generation sequencing, we identified the lncRNA Heat4 to be 2-fold upregulated in the blood of patients with HF compared to controls (N=4; p<0.05). Heat4 induction was validated in an independent second patient cohort via qPCR (HF: N=63; Controls: N=38; p<0.05). Magnetic-activated cell sorting revealed non-classical monocytes as the primary cellular source of Heat4 (N=4; 3.37-fold compared to classical monocytes; p<0.05). This finding was verified by single-cell RNA sequencing. Overexpression of Heat4 in monocytes decreased inflammation, as indicated by a reduction of Interleukin 1β (IL1β) expression (N=6; 38.71% reduction). Accordingly, reduction of Heat4 caused upregulation of IL1β levels (N=5; 1.51-fold; p<0.05). In vivo , 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<0.05). Conclusion: The long non-coding RNA Heat4 is elevated in the blood of HF patients. Mechanistically, Heat4 limits the extent of the inflammatory response of non-classical monocytes and leads to a faster regeneration after vascular injury. Therefore, lncRNAs such as Heat4 may provide novel targets for future heart failure treatments.
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