Endothelial derived, secreted long non-coding RNAs Gadlor1 and Gadlor2 aggravate cardiac remodeling

Pathological cardiac remodeling predisposes individuals to developing heart failure. Here, we investigated two co-regulated long non-coding RNAs (lncRNAs), termed Gadlor1 and Gadlor2, which are upregulated in failing hearts of patients and mice. Cardiac overexpression of Gadlor1 and Gadlor2 aggravated myocardial dysfunction and enhanced hypertrophic and fibrotic remodeling in mice exposed to pressure overload. Compound Gadlor1/2 knockout (KO) mice showed markedly reduced myocardial hypertrophy, fibrosis, and dysfunction, while exhibiting increased angiogenesis during short and prolonged periods of pressure overload. Paradoxically, Gadlor1/2 KO mice suffered from sudden death during prolonged overload, possibly due to cardiac arrhythmia. Gadlor1 and Gadlor2, which are mainly expressed in endothelial cells (ECs) in the heart, where they inhibit pro-angiogenic gene expression, are strongly secreted within extracellular vesicles (EVs). These EVs transfer Gadlor lncRNAs to cardiomyocytes, where they bind and activate calmodulin-dependent kinase II, and impact pro-hypertrophic gene expression and calcium homeostasis. Therefore, we reveal a crucial lncRNA-based mechanism of EC-cardiomyocyte crosstalk during heart failure, which could be specifically modified in the future for therapeutic purposes.


Carotid flow measurement
To evaluate the degree of aortic constriction during TAC surgery, the peak velocity of flow in the right and left common carotid arteries (RCCA and LCCA) were measured 2 days after surgery with PW-Doppler at the level of carotid bifurcation to calculate the ratio of RCCA/LCCA flow.

EV Transmission Electron Microscopy (TEM)
Visualization of EVs was performed with TEM.After centrifugation, pellets were resuspended in the minimum possible volume of residual liquid and 25 % aqueous glutaraldehyde was added to a final concentration of 1 %.After fixation overnight, samples were mixed 1:1 (v/v) with 4 % agar at 40 °C.After hardening, the agar blocks were cut into cubes of 1 mm in size.Further preparation of samples and electron microscopy were performed as described, before being imaged with a FEI Morgagni 268 transmission electron microscope (FEI, Eindhoven, Netherlands) operated at 80 kV using a Veleta CCD camera (Olympus Soft Imaging Solutions). 1

RNAScope in situ Hybridization
In situ hybridization was performed to localize Gadlor1 (Mm-LOC118567341-C3), Gadlor2 (Mm-AK038629-C1, Cat no: 404391) and Cdh5 (Mm-Cdh5-C2, Cat no: 312531) mRNA expression on mouse heart tissue slides with RNAscope Multiplex Fluorescent v2 kit.The assay was performed according to the manufacturer's protocol.Briefly, OCT-embedded tissue slides (7 μm thickness) were used in the assay, fixed with 4% PFA and then stepwise dehydration was performed with increasing concentrations of ethanol.Tissue slides were subsequently treated with hydrogen peroxide (10 min at RT) and protease IV (30 min at RT) in the humidity control incubator.Slides were incubated for 2 hours with the mixture of probes to hybridize with the corresponding RNAs.Next, the signal was amplified with RNAScope Multiplex FL v2 AMP 1-3 reagents and then HRP channels were developed sequentially.Finally, DAPI was used to counterstain the slides, and each was mounted with ProLong Gold Antifade Mountant (Fisher Scientific).

Proximity Ligation Assay (PLA)
We performed proximity ligation assay (PLA) with minor modifications to a previously described protocol to confirm the interaction of Gadlor1 and Gadlor2 lncRNAs with CaMKII. 2 Briefly, specific single-stranded DNA probes, identical to those used in RAP-MS, were designed to be complementary to Gadlor1 and Gadlor2 RNA, each with a 3' biotin tag.
For each target, five specific probes were pooled based on their region within the target RNA.
Primary antibodies (Rabbit-anti CamkIIΔ) were diluted in Duolink antibody diluent and incubated overnight at 4°C.After washing with Duolink buffer A, Duolink probes (Rabbit MINUS and Mouse PLUS) were applied to the slides for 1 hour at 37°C.Slides were washed in buffer A, treated with Duolink ligase mix for 30 minutes at 37°C, followed by a polymerase/ amplification mix for 100 minutes at 37°C.Finally, slides were washed in buffer B, rinsed in 0.01% buffer B, and mounted in Vectashield Mounting Medium with DAPI.The specificity of PLA was confirmed by omitting the primary antibody.

Gadlor1_ExonIntron AGCACATTGTACTCCCATGTT GGCAGTTTGGAAGGTGATCA
Gadlor2_ExonIntron TGGGAAGTAGAGAGCTATTGTTC TCCTATGTGTGCACTGTCCA Table S5: List of Gadlor1 and Gadlor2 probes used for RNA antisense purification (RAP).A. Venn diagram of upregulated genes in different cardiac cell types (CM: red, EC: grey, FB: blue) of Gadlor-KO mice compared to WT after 2-weeks TAC.The numbers indicating the number of genes in corresponding cell types.Genes were filtered as fold-change greater or equals to 1.2 for each condition.B-E.Gene ontology (GO) analysis of indicated intersections representing the overlapping upregulated genes in shown cell type of Gadlor-KO compared to WT littermates.F. Venn diagram of downregulated genes in different cardiac cell types (CM: red, EC: grey, FB: blue) of Gadlor-KO mice compared to WT after 2-weeks TAC.G-J.Gene ontology (GO) analysis of indicated intersections representing the overlapping downregulated genes in shown cell type of Gadlor-KO compared to WT littermates.Bar plots showing selected GO-terms for each condition.

Figure S1 :
Figure S1: Gadlor1 and Gadlor2 are non-coding transcripts that are located in the intronic region of the Lsamp gene. A. Position of primer pairs targeting different regions within Gadlor1 and Gadlor2 lncRNAs.B-C.Quantitative RT-PCR analysis of Gadlor1/2 expression in heart and liver tissues with indicated primer pairs.D-F.Systemic deletion of the whole region containing Gadlor lncRNAs did not affect the expression of the neighbor gene Lsamp, which is mainly expressed in brain tissue (Gapdh expression used for normalization).Relative expression of Gadlor1 (D), Gadlor2 (E) and Lsamp (F) were shown in mouse brain, heart and liver tissue samples collected from WT and Gadlor-KO mice (n=4, same samples were indicated in each graph).G. Coding probability of Gadlor1 and Gadlor2 was evaluated with the online Coding Potential Assessment Tool (CPAThttp://lilab.research.bcm.edu/) and compared with coding transcripts and well-known lncRNAs for validation that revealed Gadlor1 and Gadlor2 are non-coding transcripts.H. Fluorescent in situ hybridization of Gadlor1 (cyan), Gadlor2 (red) and Cdh5 RNAs in mouse heart tissue sections of sham and following 8-weeks of TAC surgery.Cdh5 shows endothelial cells (green), nuclei stained with DAPI (blue), and the overlap in 3 channels was indicated with arrows.Dashed rectangles were the zoomed areas showed in separate pictures.Scale bars: 50 m and 10 m, as indicated.I. Gadlor1/2 lncRNA levels were assessed in mouse cardiac ECs at different time-points post-TAC compared to sham samples.Sham group (n=6), 1-week TAC (n=7), 2-week TAC (n=8), Long-term TAC (10-12 weeks, n=7).The housekeeping gene 18S was used for normalization of RT-qPCR data.Data are shown as mean±SEM.*p-value<0.05,**p-value<0.01,***p-value<0.001;****p-value<0.0001

Figure S3 :Figure S4 :
Figure S3: Gadlor lncRNAs are mainly secreted in endothelial cells (EC) derived extracellular vesicles (EVs).A. Scheme depicting the experimental design of EV isolation with ultracentrifugation (UC) from cultured primary cardiac ECs.Characterization of cardiac EC-derived EVs by B. visualizing microvesicles and exosomes with electron microscopy (scale bar: 500 nm) and C. detecting the size in diameter (nm: nanometers) with Nanoparticle Tracking Analysis (NTA).D. Flow cytometry analysis of EV surface markers (CD63-APC, CD9-PerCP-Cy5.5 and CD54-FITC) on EC-derived EVs that were stained against markers (blue curve) and compared to isotype control (red curve).E. Expression of Gadlor1 and Gadlor2 in C166 mouse endothelial cell and in EVs derived from these cells.Data are shown as mean±SEM.Data normality was evaluated with Shapiro-Wilk test and p-values were calculated with Student's t-test for parametric (or Mann-Whitney for nonparametric) assessment.*p-value<0.05,**p-value<0.01,***p-value<0.001.

Figure S5 :
Figure S5: RNA sequencing of Gadlor-KO cardiac FBs showed less induction of fibrosis-associated genes after TAC. A. Heatmap showing differentially regulated genes analysed by bulk RNAseq of isolated cardiac fibroblasts after 2-weeks TAC.B. Bar plots showing the gene-ontology (GO) analysis of upregulated and downregulated genes in Gadlor-KO FBs after 2-weeks TAC (Red: Upregulated in Gadlor-KO, Blue: Downregulated in Gadlor-KO).Exemplary genes were listed for selected GO-terms.C-D.Validation of selected genes from RNAseq data with qRT-PCR in isolated adult cardiac FBs after 2 weeks of TAC (Red: Upregulated in Gadlor-KO, Blue: Downregulated in Gadlor-KO).E. qRT-PCR of selected genes in neonatal rat fibroblasts (NRFB) overexpressing Gadlor lncRNAs after Gadlor1/2 adenovirus treatment (βgal as control) followed by phenylephrine (PE) stimulation (100 μM, 24 hours).Data are shown as mean±SEM.Data normality was evaluated with Shapiro-Wilk test and p-values were calculated with Student's t-test for parametric and Mann-Whitney test for non-parametric assessment.*p-value<0.05.

Figure S6 :Figure S7 :
Figure S6: Visualization of the specific interactions between CaMKII and Gadlor1 and Gadlor2 lncRNAs in situ by proximity ligation assay (PLA).A. Representative images from proximity ligation assays (PLA) in HL1 cardiomyocytes in situ, showing the interaction between CaMKII with endogenous Gadlor1 or Gadlor2 lncRNAs (red).PLA specificity was confirmed by omitting the primary antibody as a negative control.B. Representative images from PLA in HL1 cardiomyocytes in situ, showing the interaction between CaMKII with adenoviral overexpressed Gadlor1 and Gadlor2 lncRNAs (red).Cells were mounted with Vectashield containing DAPI (blue).Scale bar: 25 µm.

Table S2 :
List of primers used for genotyping of Gadlor-KO mouse line.

Table S3 :
List of antibodies/reagents used in cell isolation methodologies and immunofluorescence (IF) staining.