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Abstract

Background: Pathological cardiac overload triggers maladaptive myocardial remodelling that predisposes to the development of heart failure. The contribution of long non-coding RNAs (lncRNAs) to intercellular signalling during cardiac remodelling is largely unknown. In this study, two novel lncRNAs, Gadlor1 and Gadlor2, which are enriched in endothelial-cell-derived extracellular vesicles (EVs) were described. The functional role of endogenous Gadlor lncRNAs was investigated in intra-cardiac communication during cardiac remodelling upon pressure overload. Methods and Results: Analysis of the different cardiac cell types revealed that Gadlor1 and Gadlor2 expression were enriched in endothelial cells (EC) compared to fibroblast (FB) and cardiomyocytes (CM). Interestingly, the abundance of Gadlor lncRNAs was even more pronounced in EC-derived EVs, suggesting a potential role in paracrine signalling. The effect of Gadlor knock-out (Gadlor-KO) and Gadlor overexpression during cardiac pressure overload induced by transverse aortic constriction (TAC) was analysed by echocardiography, histological analyses and bulk RNA sequencing from isolated cardiac cells. Gadlor1 and Gadlor2 are upregulated in failing mouse hearts as well as in the myocardium and serum of hypertrophy patients. Interestingly, secreted Gadlor lncRNAs from ECs were mainly taken up by CM, and to a lesser extent by cardiac FBs. Gadlor-KO mice exhibited reduced cardiomyocyte hypertrophy, diminished myocardial fibrosis and improved cardiac function, but paradoxically, suffered from sudden death during prolonged pressure overload. Gadlor overexpression, in turn, triggered hypertrophy, fibrosis and cardiac dysfunction. Mechanistically, Gadlor1 and Gadlor2 inhibited angiogenic gene expression in ECs, while promoting the expression of pro-fibrotic genes in cardiac FBs. In CMs, Gadlor1 and Gadlor2 upregulate mitochondrial and pro-hypertrophic genes, but downregulate angiogenesis and inflammatory genes. GLYR1 and CaMKII were identified as Gadlor1/2 interaction partners by RNA antisense purification coupled with mass-spectrometry (RAP-MS). These binding partners could in part 8 explain the changes observed in gene expression of Gadlor-KO CMs, cardiomyocyte hypertrophy and perturbed calcium dynamics, respectively. Phosphorylation of phospholamban (p-PLN) by CaMKII at threonine-17 alters the activity of the SERCA pump and therefore calcium reuptake into the sarcoplasmic reticulum. A significant decrease in pThr17-PLN in Gadlor-KO heart tissue samples, and a considerable increase in pThr17-PLN in Gadlor1/2 overexpressing heart tissue samples compared to WT after 2 weeks of TAC suggested that Gadlor lncRNAs promote CaMKII activity. Indeed, reduced or increased CaMKII activation could explain in large parts ameliorated or exaggerated maladaptive remodelling during Gadlor knock-out or overexpression, respectively. Conclusions: Gadlor1 and Gadlor2 are novel lncRNAs, which are upregulated in cardiac pathological overload and are secreted from endothelial cells within EVs. Gadlor1 and Gadlor2 induce cardiac dysfunction, cardiomyocyte hypertrophy and myocardial fibrosis by acting on multiple cardiac cells, affecting cellular gene expression and by affecting calcium dynamics in cardiomyocytes, which take up the Gadlor1/2 by EV-mediated transfer from endothelial cells. Targeted inhibition of Gadlor lncRNAs in endothelial cells or fibroblasts might serve as a therapeutic strategy in the future.