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.
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
Introduction: The versatility of the human genome is increased by the process of alternative mRNA splicing. Impaired splicing of the cardiac transcriptome is involved in the pathophysiology of heart failure. Especially, mutations in cardiac-specific splicing factors such as RBM20 cause severe forms of cardiomyopathy. Aim: We aimed to identify novel cardiomyopathy-associated splicing factors in the human heart using a score of myocardial tissue specificity including 53 human tissues and disease-associated expression changes in hearts of dilated cardiomyopathy (DCM) patients and controls. Methods & Results: We found the splicing factor Slm2 to be significantly upregulated on the mRNA and protein level in the human myocardium of patients diagnosed with DCM (1.88 and 5.3-fold, both P<0.05). Reduction of Slm2 in vivo resulted in DCM in zebrafish and sarcomere irregularity and altered calcium cycling in human cardiomyocytes. Sequencing of RNAs bound to Slm2 isolated from diseased human hearts, revealed the interaction of Slm2 with important mRNA transcripts encoding for sarcomere constituents, such as troponin I, troponin T, tropomyosin and titin. In the failing human myocardium, Slm2 bound to an alternative titin mRNA variant including retained introns. In detail, Slm2 interacted with exons of the titin mRNA encoding for the PEVK region, which mediates flexibility and contractile properties of the heart muscle. Using a splice reporter assay, which contains parts of titin’s PEVK region, we found Slm2 mediating the splicing process of intron retention. Conclusion: Slm2 is a novel splicing factor in the diseased human myocardium, maintaining cardiomyocyte integrity and function by binding and splicing essential sarcomere constituents including titin.
Background and purpose Post-transcriptional RNA editing is an important mechanism in the development of human diseases. RNA editing can affect RNA stability and alternative splicing. The aim of our study was to characterize RNA editing and its impact on alternative RNA splicing in the healthy and failing human heart. Methods and results Human heart samples of heart failure (HF) patients (n=20) and controls (n=10) were analyzed using RNA sequencing with subsequent analysis of RNA editing. We identified adenosine-to-inosine (A-to-I) editing as the major form of RNA editing in human hearts, being reduced in HF patients. Consistently, we found the editing enzyme ADAR2 reduced in HF patients. A-to-I RNA editing predominantly occurred in intronic regions of protein-coding genes, specifically in repetitive, primate-specific Alu elements which can affect RNA splicing. Indeed, we found 173 circular RNAs (circRNAs) regulated by alternative mRNA splicing in the failing heart. Loss of ADAR2 led to reduced RNA editing concomitant with an increase of circRNA, while overexpression reduced circRNA expression and enhanced RNA editing. Conclusion A-to-I editing is the major type of RNA editing in the human heart, being reduced in HF. We demonstrate a primate-specific alternative RNA splicing mechanism mediated by RNA editing in human hearts. The findings may be relevant to diseases with reduced RNA editing such as cancer, neurological and cardiac diseases. FUNDunding Acknowledgement Type of funding sources: None.
Introduction: Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional modification process regulating RNA stability and alternative splicing. A-to-I RNA editing is conducted by the enzymes ADAR1 and ADAR2 and mainly targets Alu elements, primate-specific elements which have been associated with the formation of circular RNA (circRNA). Although differential expression of circRNAs has been studied in heart failure (HF), the extent of A-to-I RNA editing and consequences in the human heart remain largely unknown. Methods and Results: We analyzed RNA editing in human heart samples of HF (n=20) patients and controls (n=10) using RNA sequencing. We found a reduction of A-to-I RNA editing in intronic Alu elements of protein-coding genes in HF patients compared to controls. The majority (96%) of regulated circRNAs were upregulated. The predicted back-splice sites (BSS) of 20 circRNAs were validated by qPCR. The circRNA candidates correlated with RNA editing (R=0.47, P=0.02). Among the upregulated circRNAs, we identified two circular transcripts (circAKAP13) derived from the AKAP13 gene, which showed reduced A-to-I RNA editing in HF (-70.7%, n=20). In HF, ADAR2 was reduced (-68.2%) and ADAR1 was increased (7.41±0.13 -fold) on protein level (n=3-6). The knockdown of ADAR1 did not alter circRNA levels, whereas the knockdown of ADAR2 led to significantly upregulated levels of circAKAP13 (1.88±0.42 -fold, n=6). Consistently, ADAR2 overexpression reduced circAKAP13 expression (-41%, n=3). Using two mini-genes containing exons 15-19 of the AKAP13 gene and flanking Alu elements, we found convergent Alu elements enhancing circAKAP13 expression. Conclusion: In conclusion, these data describe the A-to-I RNA editome in the human heart for the first time. Reduced A-to-I RNA editing in HF patients is associated with elevated circRNA levels. We propose a primate-specific splicing mechanism mediated by A-to-I RNA editing in the human heart. These findings contribute to a better mechanistic understanding of A-to-I RNA editing in cardiac diseases.
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