Abstract 118 Long non-coding RNAs Gadlor1 and Gadlor2 act as paracrine mediators of intra-cardiac communication to affect cardiac remodelling and trigger arrhythmia during pressure overload

Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02087-y
Long non-coding RNAs Gadlor1 and Gadlor2 act as paracrine mediators of intra-cardiac communication to affect cardiac remodelling and trigger arrhythmia during pressure overload
M. Keles¹, S. Grein¹, N. Froese², F. A. Trogisch¹, R. Wardman¹, S. Hemanna¹, G. M. Dittrich¹, A. Martin Garrido¹, M. Szaroszyk², J. Heineke¹
¹Department of Cardiovascular Physiology, ECAS (European Center for Angioscience), Mannheim Faculty of Medicine, Heidelberg University, Mannheim; ²Kardiologie und Angiologie, Medizinische Hochschule Hannover, Hannover;
Introduction. Studies have shown that lncRNAs contribute to cardiac pathophysiology. Recently, we have identified two novel lncRNAs, Gadlor1 and Gadlor2, which are enriched in endothelial-cell (EC) derived extracellular vesicles (EVs). We aim to investigate the functional role of endogenous and secreted Gadlor lncRNAs in intra-cardiac communication during cardiac remodelling. Methods and Results. Analysis of the different cardiac cell types revealed that Gadlor expression is enriched in ECs compared to fibroblasts (FB) and cardiomyocytes (CM). Interestingly, the abundance of Gadlors is even more pronounced in EC-derived EVs suggesting a role in paracrine signalling. To investigate the effect of endogenous Gadlors, mice with a systemic knock-out of both Gadlors (KO) were generated. Unchallenged KO mice were indistinguishable from their wild-type littermates (WT) based on baseline physiological and echocardiographic analysis. Gadlor levels were significantly increased in mouse cardiac tissue after transverse aortic constriction (TAC), as well as in the myocardium and serum of heart failure patients. Gadlor deletion markedly ameliorated cardiac hypertrophy following TAC as shown by a less pronounced increase in cardiomyocyte area and hypertrophy-associated genes (Nppa and Nppb) compared to WT. Additionally, KO mice exhibited less TAC-induced cardiac dysfunction based on echocardiography (Ejection fraction (EF): 41.7 vs. 30.7%, KO vs. WT, p<0.0001) and protected from pulmonary congestion. Moreover, myocardial capillarization during pressure-overload was substantially increased in KO mice after TAC compared to WT mice, suggesting a preserved endothelial function. Interestingly, KO mice were strongly protected from extracellular matrix deposition and showed significantly less induction of fibrosis-related genes (Col1a1 and Col3a1), indicating an improved fibrotic response. RNA sequencing from cardiac cells isolated after 2-weeks of TAC confirmed significant upregulation of angiogenesis and cell-cycle associated genes in ECs, as well as a downregulation of matrix gene expression in FBs in KO mice. Unexpectedly, KO mice after persistent long-term pressure-overload (8-weeks TAC) showed a higher mortality rate compared to WT despite maintaining better systolic function (EF: 26.5 vs 18.1%, KO vs. WT, p<0.05). To investigate potential arrhythmic behaviour in KO mice to explain the sudden death, we performed contractility and calcium transient measurements (Multicell HT System). In isolated adult KO-CMs after TAC, a considerable delay in diastolic sarcoplasmic calcium re-uptake was observed compared to WT-CMs. Investigations are ongoing to decipher the molecular mechanism of how Gadlors affect cardiac calcium homeostasis. To understand the role of Gadlor lncRNAs in intra-cardiac communication, we transferred Gadlor overexpressing EVs to cultured CMs and FBs, respectively, which revealed CMs as the main Gadlor recipient cell-type. Additionally, co-culture studies of CMs isolated from KO mice with WT-ECs confirmed the transfer of endogenous Gadlors within EVs, since the expression of both Gadlors detectable in KO-CMs after 48-hours of co-culture. Conclusion. Gadlors are mainly expressed in EC-derived EVs and upregulated in failing human hearts and a pre-clinical model of cardiac hypertrophy. Deletion of Gadlors protects from cardiac fibrosis and preserves cardiac function, although it might trigger arrhythmia in persisting high pressure overload.
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