Abstract A growing body of evidence suggests that tissue-specific lncRNAs play pivotal roles in the heart. Here, we exploit the synteny between the mouse and human genomes to identify the human lncRNA HSCHARME and combine single-cell transcriptomics, CAGE-seq data, RNA-FISH imaging and CRISPR/Cas9 genome editing to document its role in cardiomyogenesis. By investigating the mechanism of action of HSCHARME in hiPSC-derived cardiomyocytes, we report that the locus produces the major pCHARME isoform that associates with SC35-containing speckles and interacts with the splicing regulator PTBP1. Consistently, the functional inactivation of pCHARME influences the splicing of cardiac-specific pre-mRNAs and impacts their expression, which reflects a decline in cardiomyocyte differentiation and physiology. In line with a possible association with disease, large-scale analysis of the lncRNA expression across cardiomyopathy patients reveals increased levels of pCHARME in hypertrophic and dilated hearts. We also find that HSCHARME dosage can modulate the expression of a subset of disease-associated targets. Our findings provide mechanistic insights into the role of pCHARME in cardiac cells with potential implications for disease. Subject terms: Long non-coding RNAs, Stem-cell differentiation, Alternative splicing __________________________________________________________________ Cardiomyopathies are linked to dysregulated gene expression. Here, the authors identify the human long noncoding RNA HSCHARME as a disease-associated regulator of cardiomyocyte differentiation, acting through alternative splicing control. Introduction Heart diseases represent the primary cause of death globally^[42]1. As the cardiac muscle fails to renew cardiomyocytes (CM)^[43]2 regenerative medicine therapies are considered crucial for stimulating the repair of damaged hearts, thereby reducing the need for transplants. Nonetheless, the restoration of fully matured and functional cardiomyocytes is still challenging due to the partial knowledge of the factors regulating their endogenous development and maturation^[44]3. Over the years, considerable attention has been placed on coding genes^[45]4. More recently, the increasing discovery of long-noncoding RNAs (lncRNAs) has revealed unexplored modalities that are essential for CM to acquire their identity and functionality^[46]5,[47]6. LncRNAs represent a heterogeneous class of transcripts longer than 500 nucleotides, with limited protein-coding capability^[48]7. Since their discovery, they have been implicated in regulating every step of the gene life cycle^[49]8, as well as a wide range of processes, from physiological to pathological^[50]9. The remarkable temporal and tissue-specific expression of these molecules designates them as optimal regulators of physiological organ development. In the heart, several studies conducted in murine models have underscored the critical roles of lncRNAs in sustaining cardiac homeostasis, with their dysregulation frequently associated with the onset of disease^[51]6,[52]10–[53]14. Despite the presence of many orthologues, their direct investigations in human cells or tissues remain scarce. Nonetheless, several lncRNAs have been implicated in human CM processes, including proliferation and regeneration^[54]5,[55]15,[56]16. In the nucleus, lncRNAs fine-tune gene expression by interacting with DNA/RNA molecules, RNA binding proteins, or by shaping the genome three-dimensional organization^[57]17. Their role as RNA scaffolds is exemplified by their ability to form RNA-rich condensates and create high-local molecule concentrations at specific loci. This is the case of NEAT1 and MALAT1, which accumulate within the nucleus as RNA components of specific sub-nuclear compartments enriched in splicing factors, thereby regulating pre-mRNA splicing^[58]18,[59]19. In mice, we have previously identified pCharme, an architectural lncRNA coordinating the activation of pro-myogenic genes at specific nuclear condensates^[60]20,[61]21. In the heart, the genetic ablation of pCharme causes persistent expression of fetal-like gene programs delaying CM maturation, which ultimately leads to dilated cardiomyopathy^[62]22. This highlights an important role for the lncRNA in the pathophysiology of the heart and encourages further analyses of its possible contribution in humans. Built on this rationale, we used comparative genomics to further investigate conserved synteny at the previously identified pCharme locus^[63]20. We found that the human syntenic gene, HSCHARME (Human Syntenic CHARME), is expressed in the human heart, both in fetal and adult CM. By using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM), we show that the pCHARME transcript localizes close to SC35-containing speckles and directly binds to the Polypyrimidine tract-binding protein 1 (PTBP1) to influence the splicing of cardiac-specific pre-mRNAs involved in CM differentiation. Finally, we found that the endogenous pCHARME expression is significantly increased in dilated (DCM) and hypertrophic (HCM) human cardiomyopathies, correlating with dysregulation of disease-associated genes. Overall, these results identify pCHARME as a gene linked to cardiac disease, with potential implications for therapeutic approaches in human CM. Results Identification of conserved synteny of the pCharme locus in Homo sapiens To explore the phylogenetic conservation of the murine pCharme^[64]20,[65]21 and precisely map the chromosomal location of the gene in the human genome assembly, we performed a cross-species analysis through the UCSC genome browser “LiftOver” tool (Fig. [66]1A). Conversion from the murine (mm10; chr7:44,473,538-44,486,138) into the human coordinates revealed the existence of a syntenic locus (hg38; chr19:50,486,482-50,500,386), herein named HSCHARME (gene aliases lncFAM^[67]23 and MYREM^[68]24), whose mapping located a 94-nucleotide extension at the beginning of the gene, as compared to the previous annotation^[69]20. Fig. 1. Identification of conserved synteny of the HSCharme locus in Homo sapiens. [70]Fig. 1 [71]Open in a new tab A UCSC visualization of the chromosome position and the genomic coordinates of Charme gene in mice and humans (HSCHARME). Default tracks for Vertebrate Multiz Alignment & Conservation and Histone modifications (H3K9ac, green; H3K4me3, blue; H3K27me3, orange) are also shown, together with the RNA-seq reads taken from GEO and FANTOM5 CAGE datasets. B HSCHARME expression across fetal tissues based on CAGE TSS usage data (Phase1 and 2 datasets, ROI coordinates: hg38 chr19:50,476,155-50,510,700) from ZENBU FANTOM5 Human hg19 promoterome and across cardiac cells subtypes from Descartes scRNA-seq atlas^[72]29. Bars represent Relative Logaritmic Expression (RLE) of Tag Per Million values of TSS usage or UMI/cell values, respectively. C HSCHARME expression in adult tissues from scRNA-seq data of Tabula Sapiens^[73]30 organized by full details or by tissue and cell type. Bars represent the Parts Per Million values of HSCHARME TSS usage per sample. D Integrated UMAP plot of single-cell transcriptomic profiles from hiPSC-CM (2857 cells from Day 14, 2321 cells from Day 45, as in ref. ^[74]31), describing cell identity assignment to CM, Early CM, Neural, Neural progenitors, Endothelial, or Unknown (cells that could not be assigned to any specific identity) subpopulations. HSCHARME expression is shown at single-cell resolution over the same UMAP representation. E UCSC visualization of ReMap ChIP-seq track across HSCHARME promoter. The analysis was limited to human CM biotypes and spans the 2.5 kb region upstream of the HSCHARME locus. The genomic coordinates, the HSCHARME transcriptional start site ( + 1, black arrow) and the TBX5 binding sites (black squares) predicted with JASPAR 2022 (relative profile score threshold=80%) are shown. F RNA-seq quantification of HSCHARME expression in n = 2 wild type (WT), n = 3 TBX5 knockout (KO) and n = 2 TBX5 overexpressing (OE) hiPSC-derived CM ([75]GSE81585^[76]31). Data are presented as log(TPM + 1) to avoid negative values, where TPM = Transcripts Per Million mapped reads. Dots indicate individual replicates; KO bars represent mean ± SEM of 3 biological replicates. As shown in Fig. [77]1A, we found an overall high-level sequence conservation of the ~26 kb-long region, especially in Rhesus, Mouse, X. Tropicalis and Zebrafish, as well as the synteny of the EMC10 and JOSD2 nearby coding genes. More specifically, sequence conservation analysis between the mouse and human genes (intronic and exonic regions) highlighted a degree of 45.1% of sequence identity (Supplementary Fig. [78]1A), which positions HSCHARME between the range of well-known lncRNAs, such as NEAT1 and XIST^[79]25,[80]26 (Supplementary Fig. [81]1B). Consistent with the transcriptional activity of the locus, Transcriptional Start Site (TSS) mapping by CAGE (Cap Analysis of Gene Expression) confirmed the presence of a sharp peak on the negative strand of the HSCHARME gene, attributable to the existence of a transcript produced in antisense direction (Fig. [82]1A). We then searched available datasets for epigenetic and gene expression signatures across the human gene focusing on cardiac samples. We found that embryonal whole-heart samples show deposition of histone H3 acetyl-lysine 9 (H3K9ac; [83]GSM706849^[84]27), trimethylation of histone H3 lysine 4 (H3K4me3; [85]GSM772735^[86]27) and the absence of the repressive trimethylation of histone H3 lysine 27 (H3K27me3; [87]GSM621450^[88]27) marks, correlating with the transcriptional activation of the locus in fetal hearts (90-120 days), as further confirmed by the gene expression RNA-seq outputs ([89]GSM1059494; [90]GSM1059495) (Fig. [91]1A). CAGE-sequencing data from the FANTOM5 human promoterome catalog^[92]28 ([93]https://fantom.gsc.riken.jp/zenbu), further confirmed the specific expression of HSCHARME in both fetal hearts (84-217 days) and skeletal muscle cells (Fig. [94]1B). In line with our previous finding in mice^[95]22, scRNA-seq data from Descartes^[96]29 and Tabula Sapiens^[97]30 atlases revealed the highest expression of HSCHARME in fetal (72–129 days) cardiomyocytes (Fig. [98]1B). Restriction of lncRNA expression to cardiomyocytes persists in adult hearts, with no evident difference between atria and ventricles (Fig. [99]1C). Further examination of scRNA-seq data derived from hiPSC differentiated into cardiac cells^[100]31 definitively confirmed the highly specific expression of HSCHARME within the CM cluster (Fig. [101]1D, and Supplementary Fig. [102]1C–E and Supplementary Data [103]1, see Methods for details). Searching for possible regulators of HSCHARME expression we mined ReMap ChIP-seq atlas and found the binding of notable transcription factors in the 2.5 Kb region from the TSS (Fig. [104]1E), with only TBX5 (T-box transcription factor) showing clear and specific expression in CM (Supplementary Fig. [105]1F). Consistent with the functional implication of TBX5 in HSCHARME expression, computational analyses of available transcriptomic datasets from WT, TBX5-KO and TBX5-OE hiPSC-derived CM^[106]31, demonstrate a marked decrease of the lncRNA upon the loss-of-function of TBX5 (Fig. [107]1F). Along with our previous findings in mice^[108]22, these results underscore a conserved role for TBX5 in positively regulating the expression of HSCHARME in the human heart. Since the overexpression of TBX5 alone does not influence the levels of the lncRNA, we argue that it may establish its basal transcriptional expression, while other factors could be involved in the transcriptional regulation of the locus in cardiac cells. Overall, these data provide a high-resolution map of HSCHARME expression in the human heart and detail the cell-type specific restriction of the lncRNA to CM. The similarities of HSCHARME with its murine counterpart, which has already been shown to play an important role in cardiac remodeling^[109]20–[110]22, encouraged further investigations into the relevance of the human transcript in disease. HSCHARME characterization in hiPSC-derived human cardiomyocytes Considering the specific expression of HSCHARME in CM, we used hiPSCs as a model to study cardiomyogenic differentiation. The applied protocol^[111]32 exploits the induction of the WNT/β-catenin pathway (Day 0, CHIR addition), to guide cells towards mesoderm followed by its subsequent inhibition (Day 3, IWR-1 addition) to promote a CM fate (Fig. [112]2A). In line with the acquisition of the CM identity, RT-qPCR analyses performed at specific time points of hiPSC differentiation show the expected dynamic wave of cardiac gene expression^[113]32 (Fig. [114]2B), alongside the onset of spontaneous beating of cellular foci, starting at days 8-10 (Supplementary Movie [115]1). The expression of HSCHARME at day 10 and its progressive increase (Fig. [116]2B) suggest its potential association with human CM commitment and differentiation. To directly test this hypothesis, we used a CRISPR/Cas9-based gene editing technology to generate isogenic HSCHARME knockout (KO) hiPSCs. Specifically, sgRNAs were designed to produce a genomic deletion ( ~ 4.8 Kb sized) overlapping the HSCHARME TSS, which also includes the TBX5 binding sites (delta promoter = ∆P) (Fig. [117]2C). Mutant hiPSCs-CM were checked to confirm the correct genomic editing, the abrogation of HSCHARME expression (Supplementary Fig. [118]2A) and the absence of possible off-targets (Supplementary Fig. [119]2B, Supplementary File [120]1 and Supplementary Data [121]2). Following these verifications, we proceeded with the transcriptome analysis of WT versus isogenic ∆P-CM. Fig. 2. HSCHARME characterization in hiPSC-derived human CM. [122]Fig. 2 [123]Open in a new tab A Schematic representation of the applied CM differentiation protocol from hiPSCs. B RT-qPCR amplification of readout mRNAs and HSCHARME from hiPSCs-derived CM at specific time points (D0-D20). Data were normalized to ATP5O mRNA and represent relative expression means (2^^-ΔCt) ± SEM of 3 biological replicates. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero represented by the control sample (highest expression time point). Significant p-values are indicated. C Schematic representation of the genome editing strategy design followed to obtain ∆P hiPSCs using CRISPR/Cas9 technology. HR template = Homologous Recombination template. D Schematic representation of HSCHARME isoforms as reconstructed by genome assembly. Arrows represent the position of primers for RT-qPCR. E RT-qPCR amplification of pCHARME and mCHARME from hiPSC-derived CM at specific time points (D0-D20). Data were normalized to ATP5O mRNA and represent relative expression means (2^^-ΔCt) ± SEM of 3 biological experiments. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero represented by the control sample (highest expression time point). Significant p-values are indicated. F Quantification of the subcellular distribution of pCHARME and mCHARME from D10 CM. The histogram shows the RT-qPCR quantification of the relative % of RNA abundance in cytoplasmic versus nuclear compartments and represents mean ± SEM of 3 biological experiments. GAPDH and pre-GAPDH RNAs were used, respectively, as cytoplasmic and nuclear controls. G Representative RNA-FISH staining for pCHARME (red) combined with TNNT2 immunofluorescence (green) in WT and ∆P D20 CM. The images include the maximun 2D projection of full-size confocal caption, digital magnification of highlighted region (white squares) and pCHARME signal distribution inside the nuclei (dotted lines). A plot displaying the Average Fluorescence Intensity (AFI) of pCHARME and DAPI signals of a single focal plane is also shown. Overlapping lines indicate colocalization along the horizontal distance of the selection (white square). The images are representative of 3 biological replicates with similar results. De novo assembly of reads from the WT condition suggested that two HSCHARME full-length isoforms are produced in CM: the partially spliced transcript (precursor) pCHARME stably retaining the first intron and the fully spliced transcript (mature) mCHARME (Fig. [124]2D and Supplementary Fig. [125]2C). Both isoforms were induced throughout CM differentiation, with the pCHARME transcript being consistently more abundant than mCHARME and enriched to nuclei (Fig. [126]2E-F). Subcellular fractionation of CMs followed by RT-qPCR analyses revealed a cytoplasmic enrichment for mCHARME. The presence of open reading frames within mCHARME, possibly translated into micro-peptides^[127]33–[128]36, was excluded by the Coding Potential Calculator tool (CPC2, [129]http://cpc2.gao-lab.org/index.php). In line with the biochemical fractionation, high-resolution RNA-fluorescence in situ hybridization (RNA-FISH) experiments using probes against pCHARME confirmed its nuclear localization (Fig. [130]2G). No fluorescence was detected in ∆P-CM, which proves the specificity of pCHARME signals and the efficiency of the lncRNA KO. The latter was further confirmed by i) RT-qPCR analysis of WT and ∆P-CM with pCHARME and mCHARME specific primers (Fig. [131]3A), ii) TPM quantification of the transcriptomic reads and ii) the RNA-seq genomic plots (Fig. [132]3B and Supplementary Fig. [133]2D). Fig. 3. Genome-wide profiling of WT and ∆P hiPSC-derived CM. [134]Fig. 3 [135]Open in a new tab A RT-qPCR amplification of pCHARME and mCHARME from WT vs ∆P D10 and D20 CM. Data were normalized to ATP5O mRNA and represent relative expression means (2^^-ΔCt) ± SEM of 5 biological replicates. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero. Significant p-values are indicated. A schematic of the genome editing strategy is shown on the left. B RNA-seq quantification of pCHARME and mCHARME expression in WT vs ∆P D10 and D20 CM. Data are expressed as Log(TPM + 1) and represent means ± SEM of 3 biological replicates. C Volcano plots showing differentially expressed genes (DEGs) in WT vs ∆P CM at D10 and D20. Statistical analysis was performed with DESeq2 using a negative binomial distribution. P-values were adjusted for multiple testing using the Benjamini–Hochberg method. Significantly up-regulated (FDR < 0.05; log[2]FC > 1) and down-regulated (FDR < 0.05; log[2]FC < −1) genes are represented in red and blue, respectively. D RT-qPCR quantification of down-regulated and up-regulated DEGs in WT vs ∆P (black) and WT2 vs PA (gray) D20 CM. Data were normalized to ATP5O mRNA and represent relative expression means (2^^-ΔCt) ± SEM of 5 biological experiments. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero represented by the control sample (WT for ∆P-CM and WT2 for PA-CM). Significant p-values are indicated. E Gene ontology (GO) enrichment analysis on down-regulated and up-regulated DEGs in WT vs ∆P D20 CM. The analysis was performed with EnrichR using default parameters (Fisher’s exact test with Benjamini–Hochberg correction). Bars indicate +/–log10 adjusted p-value (log10Adjp and –log10Adjp) of the top enriched biological processes. F GSEA plot showing the enrichment of “cardiac muscle contraction”, “cardiac cell development” and “sarcomere organization” processes resulting down-regulated in WT vs ∆P D20 CM. As previously observed for the murine orthologue^[136]22, the staining of pCHARME in human CM exhibits a discrete nuclear pattern. Nonetheless, here we noticed that the fluorescent signals encompass multiple foci, which suggests trans-regulatory roles for the lncRNA. In this direction, differential expression analysis performed to compare the WT and ∆P transcriptomic datasets, revealed an impact of HSCHARME on the expression of 175 and 3637 genes at 10 (D10) and 20 (D20) differentiation days, respectively (−1 1; FDR < 0.05) (Fig. [137]3C, and Supplementary Fig. [138]3A and Supplementary Data [139]3). Of them, a total of 1465 differential expressed genes (DEGs) were up-regulated (n = 68 at D10; n = 1397 at D20), whereas 2347 were down-regulated (n = 107 at D10; n = 2240 at D20) in ∆P-CM. Importantly, none of the putative off-target genes was found among DEGs (Supplementary Fig. [140]3B), which conclusively excludes their involvement in the transcriptomic alterations. Moreover, we did not find alteration in the expression of pCHARME neighboring loci ( ± 260 Kb), which conclusively excludes a role for the lncRNA on nearby gene expression in cardiac cells (Supplementary Fig. [141]3C). To strengthen these results, we produced an independent knockout cell line (PA-hiPSC), with a different HSCHARME mutation and genetic background (WTSIi004-A; referred to as WT2). Specifically, the PA-hiPSC were generated through the insertion of a strong termination cassette inside the HSCHARME locus using a strategy previously setup in mice^[142]20 (Supplementary Fig. [143]3D). Upon checking the correct abrogation of pCHARME and mCHARME (Supplementary Fig. [144]3E), the absence of off-target mutations was confirmed by DNA sequencing (Supplementary Fig. [145]3F, and Supplementary File [146]1 and Supplementary Data [147]2). To note, no off-target gene was found in common between the two genotypes, which excludes effects arising from unwanted Cas9 background activities. Validation by RT-qPCR analyses performed on the top-most down-regulated DEGs from ΔP-CM showed their significant decrease in both the mutant cell lines (Fig. [148]3D and Supplementary Fig. [149]3G). As both CRISPR/Cas9 strategies demonstrate high efficiency in the pCHARME knockout ( ~ 99%), the minor alterations in gene expression observed within the PA context can be attributed to the intrinsic features of the iPSC-WTSIi004-A background. Gene Ontology (GO) term enrichment analysis performed on transcriptomic datasets revealed that at early stages of CM differentiation (D10), down-regulated DEGs are associated with developmental pathways, such as limb development (Supplementary Fig. [150]3H and Supplementary Data [151]3). At later stages (D20), enriched GO categories relate to CM structure and function, including myofibril assembly, heart contraction and cardiac muscle cell development gene classes (Fig. [152]3E and Supplementary Data [153]3). Gene set enrichment analysis (GSEA) confirmed the significant downregulation of genes belonging to these categories (Fig. [154]3F), with key examples that include NKX2-5, crucial for CM development^[155]37, along with structural and functional components such as MYH6^[156]38, MYO18B^[157]39, and CACNA1C^[158]40. Conversely, upregulated genes enriched GO categories relates to axonogenesis and nervous system development (Fig. [159]3E and Supplementary Data [160]3), may reflect the predisposition of the embryonic stem cells to differentiate toward default neuronal fate in the absence of additional stimuli^[161]41. We argued that HSCHARME depletion, by inhibiting the signals activating CM specification, might promote the upregulation of alternative pathways leading to a default neuronal state. To assess the evolutionarily conservation of pCHARME function between human and mouse, significant DEGs from WT and Charme KO post-natal hearts ([162]GSE200878^[163]22) were analyzed, identifying 847 human homologs (Supplementary Fig. [164]3I). Of them, 208 genes (24.55% of the murine DEGs) were i. commonly deregulated upon pCHARME ablation in cardiac muscle (i.e. MYO18B, CACNA1C, and NPPB), ii. enriched in pathways related to muscle homeostasis (e.g., glycolytic process, heart development), muscle function (e.g., contraction, fatty acid metabolism), and iii. linked to cardiac disorders such as familial atrial fibrillation and hypertrophic cardiomyopathy. Collectively, the transcriptomic output, the evolutionary conservation, and the functional interpretation of our datasets support the role of HSCHARME as a positive regulator of genes whose expression is physiologically relevant for the differentiation of human CM. Functional implication of HSCHARME in human cardiomyogenesis To functionally characterize WT and gene-edited hiPSC-CM, we tested their capacity to contract. By tracking the contraction dynamics through MUSCLEMOTION (Supplementary Fig. [165]4A) we found a significant decrease in the beat rate of mutant ∆P-CM as well as the alteration of other parameters, such as time to peak, duration and peak to peak time (Fig. [166]4A). These changes are consistent with a general decrease of the spontaneous beating frequency in ∆P-CM compared to WT cells, with individual contractions becoming longer upon the pCHARME transcript loss. Importantly, the inspection of cells over differentiation evidenced a significant delay in the onset of beating of mutant CM (∆P and PA) compared to their WT counterparts (11 versus 8 days on average, Fig. [167]4B and Supplementary Fig. [168]4B). Other phenotypical traits associated with cellular morphology^[169]42 were influenced by pCHARME loss-of-function, with mutant CM appearing smaller and rounder than the isogenic WT (Fig. [170]4C and Supplementary Fig. [171]4C), indicating a possible delay in CM differentiation. Flow cytometry analyses of PDGFRA^+/CD56^+ cardiac progenitors (Fig. [172]4D and Supplementary Fig. [173]4D) and PDGFRA^+/CD82^+ cardiac-committed progenitors (Fig. [174]4E and Supplementary Fig. [175]4D) show that pCHARME loss leads to a 43% reduction in cardiac progenitors and 20% reduction in the cardiac committed progenitors. Given that CD82 is a key marker of CM fate specification^[176]43, these data align with an early role for the lncRNA in CM specification, which is also coherent with the emergence of default alternative fates observed by transcriptomic analyses. Along the same direction, flow cytometry analysis performed at later stages (D20) shows an 80% reduction of differentiated CM, as inferred by the quantification of TNNT2^[177]44 positive (+) cells (Fig. [178]4F and Supplementary Fig. [179]4E-F). These findings perfectly match with cellular deconvolution analysis used to interpret our hiPSC-derived CM in terms of cell-type composition. Indeed, we found that pCHARME ablation causes a reduction of approximately 75% of CM (D20) (Fig. [180]4G and Supplementary Data [181]1), with most of the downregulated DEGs enriched in this population (KL-GSEA, Supplementary Fig. [182]4G). However, as the impact of pCHARME ablation in CM was higher than in precursor cells, we asked whether the lncRNA may also play a more intrinsic role in differentiated cells. To this end, we used antisense LNA-GapmeRs to deplete pCHARME directly in CM (D20). Our findings indicate that 80% reduction of pCHARME reduces, although to a lesser extent than in the KO cells, the expression of genes induced with differentiation (i.e TNNT2, MYH7 and CACNA1C) (Fig. [183]4H). These findings indicate that pCHARME plays a dual regulatory role in physiology, both for the acquisition and the maintenance of human CM identity. Fig. 4. Functional implication of HSCHARME in human cardiomyogenesis. [184]Fig. 4 [185]Open in a new tab A Quantitative analysis of beat rate (beats per minute=BPM) and contraction parameters in WT and ∆P D20 CM. Representative images are shown above each graph. Data represent means ± SEM of 5 biological replicates. Statistical test: paired two-tailed Student’s t-test for normally distributed data; Mann–Whitney test for non-normally distributed data (see Source Data). Significant p-values are indicated. B Onset (days) of spontaneous contraction of WT and ∆P CM. Data represent means ± SEM of 5 biological replicates. Statistical test: paired two-tailed Student’s t-test. Significant p-values are indicated. C Representative images of WT and ∆P D20 CM used for morphological analyses (white dashed outlines) following TNNT2 (green) and DAPI (blue) staining. Scale bar = 50 µm. Quantitative measurement of morphological features of CM is shown on the right. Data represent means ± SEM of 5 biological replicates. Statistical test: paired two-tailed Student’s t-test. Significant p-values are indicated. D Representative flow-cytometry density plot of WT and ΔP D10 CM stained for cardiac progenitor markers (PDGFRA/CD56). Percentages refer to PDGFRA^+/CD56^+ gated cells relative to secondary antibody-only controls. Data represent mean ± SEM of 3 biological replicates. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero. Significant p-values are indicated. E Representative flow-cytometry density plot of WT and ΔP D10 CM stained for cardiac-committed progenitor markers (PDGFRA/CD82). Percentages refer to PDGFRA^+/CD82^+ gated cells relative to secondary antibody-only controls. Data represent mean ± SEM of 3 biological replicates. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero. F Flow cytometry quantification of cardiac troponin-T (TNNT2) positive cells in WT and ∆P D20 CM. Data represent mean ± SEM of 3 biological replicates. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero. Significant p-values are indicated. G Pie-chart showing estimated proportion of CM (red) and other cell types (white, Supplementary Data [186]1) based on deconvolution analysis of WT and ∆P D20 CM. H RT-qPCR quantification of pCHARME and CACNA1C, TNNT2, MYH6, CACNB1 and TUBB2B mRNA expression in D20 CM transfected with control (GAP-SCR) or pCHARME-targeting (GAP-pCHARME) antisense LNA-GapmeRs. Data were normalized to ATP5O mRNA and represent relative expression means (2^^-ΔCt) ± SEM of 3 biological experiments. Statistical test: one-sample, two-tailed Student’s t-test on LogFC values against the null hypothesis of zero represented by the control sample (GAP-SCR). Significant p-values are indicated. pCHARME regulates the alternative splicing of cardiac-expressed pre-mRNAs The high-local distribution of pCHARME in the nucleus led us to investigate whether specific features are associated with these lncRNA-enriched domains. We first explored the potential for pCHARME as a splicing regulator, given the similar sarcomere and contraction defects that splicing factor loss often causes in the heart^[187]45. In this direction, we combined RNA-FISH targeting pCHARME with SC35 immunofluorescence (IF) to study the possible association of the lncRNA with SC35-containing nuclear speckles^[188]46,[189]47. Intriguingly, in-depth quantification of three-dimensional signal distribution revealed that pCHARME stains in close contact with SC35 domains with 40% of pCHARME colocalising with SC35 domains and 60% of SC35 domains containing pCHARME (Fig. [190]5A, B). Fig. 5. pCHARME regulates the alternative splicing of cardiac-expressed pre-mRNAs. [191]Fig. 5 [192]Open in a new tab A Representative maximum 2D projection of confocal caption for RNA-FISH pCHARME (red) combined with SC35 (green) and DAPI (blue) IF in WT D20 CM. White lines indicate pCHARME-positive nuclei; digital magnifications of nuclei marked by asterisks are shown below. The images are representative of 3 biological replicates with similar results. B Graphic visualization of colocalized SC35 domains (boundary highlighted) overlaid with pCHARME. The images are representative of 3 biological replicates with similar results. Colocalization indexes between pCHARME and SC35 domains signals are indicated by Mander’s Overlap Coefficients (MOC). C Pie Chart depicting the portion of significantly altered splicing events (FDR < 0.05) detected by rMATS comparing WT vs ∆P D20 RNA-seq samples. Statistical analysis and multiple test correction were performed using the default framework of rMATS-turbo. The volcano plot depicts significant ES events (FDR < 0.05; Inclusion level variations stronger than 10%) in ∆P D20 CM. X-axis represent exon inclusion ratio while y-axis represent –log10 of p-value. A schematic representation of the investigated event is shown above. D Bar plot shows the proportion of exon skipping events identified in murine pCharme KO hearts and regulated by pCHARME in human CM. Events are stratified by the regulatory outcome: inclusion (Incl.) and exclusion (Excl.). Density plots represent distribution of conserved events expected by chance (gray). The observed conservation is marked by vertical dashed lines, red for inclusion and blue for exclusion. E Schematic representation of pre-mRNA regions analysed in the RBP binding enrichment analysis is shown alongside a heatmap displaying the log[2](Odds Ratio) of eCLIP binding site (enrichment or depletion) in pCHARME-regulated SE events, compared to unaffected control exons. Statistical significance was determined using Fisher’s exact test, and p-values were corrected for multiple testing using the Benjamini-Hochberg false method. Enrichment or depletion are indicated by red or blue colors, respectively, while white marks Not Significant (NS) region (FDR > 0.05). F Heatmap displaying the PTBP1 binding affinity for pCHARME RNA sequence. Bar plot in the top panel displays the total number of pCHARME-specific 7-mers within each defined segment of the transcript. On the right, the sequence logo generated from the pCHARME-specific k-mer signature is shown. Heatmap below shows the row-scaled (Z-score) PTBP1 binding affinity scores for pCHARME segments, based on data from two eCLIP experiments (HepG2 and K562) and one RNAcompete experiment (M227_0.6). G Representative confocal images of RNA-FISH for pCHARME (red) combined with IF for PTBP1 (white) and DAPI (blue) in WT D20 CM. Volume-rendered detail (blue square) of pCHARME (red) and PTBP1 (gray) signals is shown; asterisks indicate areas where pCHARME and PTBP1 signals overlap. The images are representative of 3 biological replicates with similar results. Colocalization indexes between pCHARME and PTBP1 signals are indicated by MOC. H Analysis of PTBP1-CLIP assay in WT D20 CM. Western blot detects PTBP1 protein, using GAPDH as a loading control. Input (Inp) samples represent 10% of the total protein extracts. RT-qPCR quantifies pCHARME (at both 5′ and 3′ intron-1 ends) and mCHARME recovery in PTBP1 IP and IgG samples. GAPDH RNA serves as a negative control. Dots represent percentage of input and indicate individual replicates. I PTBP1 protein recovery in WT and ∆P D20 CM was assessed by chemiluminescence quantification with ImageJ. RT-qPCR analyses quantify pCHARME and CACNB1 RNA recovery in PTBP1 IP and IgG samples. Dots represent percentage of input and indicate individual replicates. The observed RNA-protein proximity led us to test a possible involvement of pCHARME in splicing regulation. By large-scale analysis of splicing patterns from our RNA-seq datasets, we identified a total of 9615 expressed isoforms in both WT and ∆P cardiomyocytes (see Methods). We found that the abundance of a consistent fraction of these isoforms (16.3%; corresponding to 1877 transcript isoforms) was significantly altered upon pCHARME ablation and functionally linked to cardiomyogenic ontologies related to cardiac muscle cell development and heart contraction (Supplementary Fig. [193]5A). On this preliminary evidence, we used rMATS^[194]48 for alternative splicing (AS) analysis of our RNA-seq datasets, including exon-skipping (ES), intron retention (IR), alternative 3’ splice site (A3SS), alternative 5’ splice site (A5SS), and mutually excluded exons (MXE) and events. Also in this case, we observed a strong impact of HSCHARME ablation, with a total of 6.552 aberrant pre-mRNA splicing events (FDR < 0.05, Absolute Inclusion level> 0.1) (Fig. [195]5C, and Supplementary Fig. [196]5B and Supplementary Data [197]3). Splicing alterations were found in each of the analysed classes, with the ES events accounting the 70.1% of all the significant alterations (Fig. [198]5C). Among the most interesting examples we found CACNB1, a gene implicated in voltage-dependent calcium release^[199]49. In KO CM (∆P and PA), we found that the skipping of exon 7 of CACNB1 causes a shift from the EX6-EX7-EX8 isoform into the shorter EX6-EX8 one (Supplementary Fig. [200]5C–D). Another example is MYL6, a gene encoding for a hexameric ATPase cellular motor protein expressed in muscle and non-muscle tissues^[201]50. MYL6 gene produces two mRNA variants which differ in exon 6^[202]51 (Supplementary Fig. [203]5E). We found that HSCHARME KO (∆P and PA) leads to the overabundance of the longest EX5-EX6-EX7 isoform (Supplementary Fig. [204]5E–F), which is described in the literature as the major non-muscle transcript. Therefore, the HSCHARME-mediated regulation of MYL6 splicing facilitates the production of a structural myosin variant optimized for CM contraction. To evaluate whether pCHARME regulation of alternative splicing is also evolutionarily conserved between human and mouse CM, we performed AS analysis of RNA-seq datasets from WT and Charme KO post-natal hearts ([205]GSE200878^[206]22). We found that, in vivo, the lack of Charme results in 327 exon exclusion and 265 exon inclusion events (Supplementary Fig. [207]5G and Supplementary Data [208]3). We mapped the coordinates of these differentially spliced murine exons to the human genome and searched for overlaps with pCHARME-regulated exons. Focusing on concordant events, we observed conservation for 10.2% of inclusion events and 8.3% of exclusion events (Fig. [209]5D). Notably, these proportions were significantly higher than expected by chance (Fig. [210]5D), underscoring the conserved regulatory role for the lncRNA. Among the conserved splicing targets, we identified key cardiac genes, including TNNT2^[211]44, MFF^[212]52, SYNC^[213]53, SORBS2^[214]54, CREM^[215]55 and RBFOX2^[216]56 which are known to play crucial roles in heart function (Supplementary Data [217]3). Overall, these results indicate pCHARME as an evolutionarily conserved regulator of CM splicing. pCHARME modulates alternative splicing by direct interaction with PTBP1 To gain mechanistic insights into the splicing alterations observed in the absence of pCHARME, we leveraged data from 223 eCLIP experiments covering 150 RNA-binding proteins (RBP) across cell lines (K562 and HepG2)^[218]57 that do not encode the lncRNA. By focusing on ES, which represent the most altered and conserved pCHARME-regulated splicing events, and comparing them to a control set of similarly expressed but unregulated junctions (Supplementary Fig. [219]5H), we identified the splicing repressor PTBP1 as significantly enriched at the 5′ junctions of the excluded exons (Fisher’s exact test, FDR < 0.05, log2[Odds Ratio] > 0) (Fig. [220]5E and Supplementary Data [221]4). Among them we found several known PTBP1 splicing targets, such as TPM1, ACTN1, FHOD3, and TNNT2^[222]58, expressed in CM and displaying exon exclusion in pCHARME KO (Supplementary Data [223]4). Overall, these data prioritize PTBP1 as a regulator of the pCHARME-dependent ES events and suggest a possible interplay between the two in CM. To deepen this evidence with a focus into the physical engagement of pCHARME, we analyzed its nucleotide composition (7-mers) compared to the set of the 1987 lncRNAs expressed in CM (Supplementary Fig. [224]5I). We found that pCHARME possesses a unique UC-rich sequence signature, primarily driven by an extended simple repeat (75-2955 nt) in the 5’ region of intron 1 (Supplementary Data [225]4). RNA secondary structure predictions using the RNAfold software^[226]59 suggested these UC-rich stretches as unstructured and likely accessible to RBP (Supplementary Fig. [227]5J). On this prediction, we employed four independent approaches to identify RBP with high affinity for the UC-rich sequence, and specifically (i) RBP affinity scores derived from crosslinking-induced truncations from eCLIP datasets, (ii) in vitro RNAcompete experiments assessing RBP binding preferences for short