Elektra-Qki and Alien-Wt1 lncRNA-protein interaction controls myocardial ion channel expression and epicardial EMT during heart development in mice

Figures

• 

  Fig. 1. Expression and cellular distribution of Elektra and Alien lncRNAs during embryonic development and adulthood. Panel (A). Volcano plot showing differentially expressed lncRNAs identified by RNA-seq analysis of PE (E9.5) and EE (E10.5) samples. Alien and Elektra are highlighted as two of the most significantly altered lncRNAs, being downregulated and upregulated in EE, respectively. Panels (B, C). qPCR analysis showing tissue expression (n = 3) of Elektra and Alien at E13.5 (B) and adult stage (C). Elektra is highly expressed in the kidney at E13.5 and restricted to the heart in adults. Alien shows high expression in the lung, stomach and liver in both embryonic and adult stages. Panel (D). Percentage of Elektra-positive cells during cardiac development stages (E10.5 to P30) using SCRINSHOT analysis (n = 4), whose expression is higher during early embryogenesis and reduces over time with transient increases at postnatal day 2 (P2) and postnatal day 30 (P30). Panel (E). Percentage of Alien-positive cells during cardiac development stages (E10.5 to P30) (n = 4). Alien expression is higher at E13.5, decreases by early postnatal stages (P2 and P7), and reactivates at P21 and P30. The…

• 

  Fig. 2. Elektra and Alien lncRNAs have distinct subcellular localization and cell-type-specific transcriptional regulation in cardiac cell lines. Panel (A). RT-qPCR analyses of Elektra and Alien expression in HL1 cardiomyocytes, MEC1 and EPIC epicardial cells and MEVEC endocardial cells. Elektra shows the highest expression in MEVEC endocardial cells. Followed by epicardial cells, and lowest in HL1 cells. In contrast, Alien is most highly expressed in epicardial cells. Panel (B). RT-qPCR analyses of nuclear and cytoplasmic distribution. Elektra shows variable localization depending on cell type, while Alien is mainly nuclear in all cell lines. Panel (C). RT-qPCR analyses of Elektra and Alien expression following overexpression of Pitx2c, Mef2c, Srf and Nkx2.5 in HL1 and MEC1 cells (n = 3; each biological replicate corresponds to a single transfection assay). Panel (D). RT-qPCR analyses of Elektra and Alien expression following siRNA-mediated silencing of Pitx2c, Mef2c, Srf and Nkx2.5 in HL1 and MEC1 cells (n = 3). These data reveal cell-type-specific transcriptional responses. Statistical analysis: Fig. 2A, one-way ANOVA followed by Tukey’s multiple-comparisons test; Fig. 2B, unpaired…

• 

  Fig. 3. Distinct protein interaction networks of Elektra and Alien in cardiac cell types. Panel (A). Schematic representation of the RNA pull-down (PD) and mass spectrometry (MS) workflow used to identify lncRNA-associated proteins in HL1 cardiomyocytes, MEVEC endocardial cells and MEC1 epicardial cells. PD experiments were performed in biological triplicates (n = 3). Panel (B). Number of unique Elektra-associated proteins identified in HL1 cells compared to Gapdh and Input conditions. Panel (C). Subcellular classification of Elektra-binding proteins in HL1 cells, showing enrichment in the cytoplasm. Panel (D). GO enrichment analysis of Elektra interactors in HL1 cells, highlighting RNA binding, RNA splicing, ion channel regulation and membrane-related functions. Panel (E). Number of unique Alien-associated proteins identified in MEC1 epicardial cells compared to Gapdh and Input conditions. Panel (F). Subcellular distribution of Alien-associated proteins, with a predominance in the cytoplasm. Panel (G). GO analysis of Alien interactors in MEC1 epicardial cells, showing enrichment in EMT, cytoskeletal remodeling, RNA processing and mitochondrial translation. Schemes were created using…

• 

  Fig. 4. Elektra silencing alters cardiac ion channel expression through Qki-mediated regulation. Panel (A). RT-qPCR analyses of Elektra enrichment in Qki RIP assays from HL1 cardiomyocytes compared to IgG control. Panel (B). RT-qPCR validation of Elektra silencing and overexpression using siRNA and pcDNA3.1 vector, respectively, in HL1 cells (n = 3). Panel (C). Representative images for immunofluorescence analysis of Qki protein levels after Elektra loss- and gain-of-function. Increased Qki levels are observed upon Elektra inhibition (n = 3). Panel (D). RT-qPCR analyses of calcium, sodium and potassium channel gene expression in HL1 cells following Elektra silencing (n = 3). Notably, lncRNA knockdown leads to upregulation of Qki, Ryr2, Atp2a2 and Ncx1, and downregulation of Cacnb3 and Casq2. Panel (E). RT-qPCR analyses of calcium, sodium and potassium channel gene expression in HL1 cells following Elektra overexpression (n = 3). Elektra overexpression causes opposite effects for some genes. Both treatments increased Kcnn3 expression. Panel (F). RT-qPCR analysis for Qki silencing validation in HL1 cardiomyocytes (n = 3). Panel (G). RT-qPCR analysis showing that knockdown of Qki leads to a…

• 

  Fig. 5. Elektra modulated Pitx2c and Wnt signaling, affecting cardiac ion channel and sarcomeric gene expression. Panel (A). RT-qPCR analyses of Pitx2c, Wnt11 and Wnt8a expression in HL1 cardiomyocytes after silencing and overexpression of Elektra (n = 3). Elektra knockdown led to upregulation of Pitx2c and Wnt11, while Elektra overexpression did not modulate their expression. Panels (B-D). RT-qPCR analyses showing the effect of Pitx2c overexpression (B), Wnt8a overexpression (C), and Wnt11 overexpression (D) on Elektra and Qki expression in HL1 cardiomyocytes (n = 3). Pitx2c and Wnt8a overexpression upregulated Elektra and downregulated Qki, whereas Wnt11 overexpression did not affect Elektra levels. Panel (E). RT-qPCR analysis of Pitx2c expression following Qki knockdown in HL1 cells (n = 3), showing decreased Pitx2c levels. Panel (F). Representative images of immunofluorescence analysis of MF-20 and pHH3 protein expression in HL1 cardiomyocytes after Elektra inhibition (n = 3), showing reduced MF-20 signal compared to controls, whereas no significant changes were observed for pHH3. Panel (G). RT-qPCR analyses of Myh6 and Myh7 after Elektra inhibition (n = 3), which are significantly…

• 

  Fig. 6. Alien modulates EMT-related gene expression and epicardial cell migration. Panel (A). RT-qPCR analysis of Alien enrichment following RNA immunoprecipitation (RIP) with Wt1-specific antibody versus IgG control in MEC1 epicardial cells. Panels (B, C). RT-qPCR analyses showing Wt1 and Alien expression after Wt1 knockdown with siRNA at 48 h (B) and 72 h (C) post-transfection (n = 3), demonstrating increased lncRNA levels following Wt1 silencing. Panel (D). RT-qPCR analysis of Alien expression at 6 h and 24 h after transfection with ASO1 and ASO2 in MEC1 epicardial cells, showing overexpression with ASO1 and transient knockdown followed by overexpression with ASO2 (n = 3). Panel (E). Representative images of scratch assay of MEC1 epicardial cells at 6 h,12h and 24 h after transfection with ASO1 and ASO2. Note that ASO1 significantly reduces migration, while ASO2 shows no significant effect. Panel (F). RT-qPCR analysis of Alien levels at 6 h and 24 h following ASO2 treatment at 20nM and 80nM concentrations, confirming similar expression pattern in both conditions (n = 3). Panels (G, H). RT-qPCR analysis of EMT-related genes (Wt1, Snai1, Snai2, Cdh1, Cdh2, Cdh5) at 6 h and 24 h…

• 

  Fig. 7. Alien regulates EMT-related gene expression and promotes mesenchymal transition in ex vivo epicardial cells. Panel (A). RT-qPCR analysis of Alien expression at 6 h and 24 h after ASO2 treatment at 20nM and 80nM in epicardial cells isolated from E10.5 mouse ventricular explants (each biological sample (n) corresponds to a pool of 7–10 explants). Note that overexpression is seen at 20nM and transient downregulation followed by overexpression is observed at 80nM. Panels (B, C). RT-qPCR analysis of EMT-related gene expression (Wt1, Snai1, Snai2, Cdh1, Cdh2, Cdh5) at 6 h and 24 h after 20nM ASO2 treatment (n = 3). At 24 h, Alien overexpression leads to upregulation of Wt1, Snai1, Snai2 and Cdh2, with reduced Cdh1 and Cdh5 levels. Panels (D, E). RT-qPCR analysis of EMT-related genes (Wt1, Snai1, Snai2, Cdh1, Cdh2, Cdh5) at 6 h and 24 h after 80nM ASO2 treatment (n = 3). Panel (F). Representative images of collagen gel EMT assay performed ex vivo on epicardial cells from ventricular explants after TGF-β and ASO2 treatment at 20nM and 80nM. While explants do not initiate EMT without any treatment, transfection with TGF-β and ASO2 at 20nM induces EMT, in contrast to ASO2 at 80nM, for…

• 

  Fig. 8. Proposed working model of Elektra and Alien function during cardiac development. Schematic representation summarizing the functional roles of Elektra and Alien in distinct cardiac lineages. In myocardial cells, Elektra interacts with Qki and modulates Pitx2c > Wnt signaling, thereby regulating the expression of ion channels and sarcomeric genes, contributing to electrical and contractile homeostasis. In epicardial cells, Alien interacts with Wt1 and regulates EMT-associated genes, influencing cell migration and promoting a partial epithelial-mesenchymal transition. Schemes were created using Biorender (https://www.biorender.com/)