Conceptually similar to modifications of DNA, mRNAs undergo chemical modifications, which can affect their activity, localization, and stability. The most prevalent internal modification in mRNA is the methylation of adenosine at the N⁶-position (m⁶A). This returns mRNA to a role as a central hub of information within the cell, serving as an information carrier, modifier, and attenuator for many biological processes. Still, the precise role of internal mRNA modifications such as m⁶A in human and murine-dilated cardiac tissue remains unknown. Transcriptome-wide mapping of m⁶A in mRNA allowed us to catalog m⁶A targets in human and murine hearts. Increased m⁶A methylation was found in human cardiomyopathy. Knockdown and overexpression of the m⁶A writer enzyme Mettl3 affected cell size and cellular remodeling both in vitro and in vivo. Our data suggest that mRNA methylation is highly dynamic in cardiomyocytes undergoing stress and that changes in the mRNA methylome regulate translational efficiency by affecting transcript stability. Once elucidated, manipulations of methylation of specific m⁶A sites could be a powerful approach to prevent worsening of cardiac function.

We introduce Single-cell Nanopore Spatial Transcriptomics (scNaST), a software suite to facilitate the analysis of spatial gene expression from second- and third-generation sequencing, allowing to generate a full-length near-single-cell transcriptional landscape of the tissue microenvironment. Taking advantage of the Visium Spatial platform, we adapted a strategy recently developed to assign barcodes to long-read single-cell sequencing data for spatial capture technology. Here, we demonstrate our workflow using four short axis sections of the mouse heart following myocardial infarction. We constructed a de novo transcriptome using long-read data, and successfully assigned 19,794 transcript isoforms in total, including clinically-relevant, but yet uncharacterized modes of transcription, such as intron retention or antisense overlapping transcription. We showed a higher transcriptome complexity in the healthy regions, and identified intron retention as a mode of transcription associated with the infarct area. Our data revealed a clear regional isoform switching among differentially used transcripts for genes involved in cardiac muscle contraction and tissue morphogenesis. Molecular signatures involved in cardiac remodeling integrated with morphological context may support the development of new therapeutics towards the treatment of heart failure and the reduction of cardiac complications.

BACKGROUND: The myocardium adapts to ischemia/reperfusion (I/R) by changes in gene expression, determining the cardiac response to reperfusion. mRNA translation is a key component of gene expression. It is largely unknown how regulation of mRNA translation contributes to cardiac gene expression and inflammation in response to reperfusion and whether it can be targeted to mitigate I/R injury. METHODS: To examine translation and its impact on gene expression in response to I/R, we measured protein synthesis after reperfusion in vitro and in vivo. Underlying mechanisms of translational control were examined by pharmacological and genetic targeting of translation initiation in mice. Cell type–specific ribosome profiling was performed in mice that had been subjected to I/R to determine the impact of mRNA translation on the regulation of gene expression in cardiomyocytes. Translational regulation of inflammation was studied by quantification of immune cell infiltration, inflammatory gene expression, and cardiac function after short-term inhibition of translation initiation. RESULTS: Reperfusion induced a rapid recovery of translational activity that exceeds baseline levels in the infarct and border zone and is mediated by translation initiation through the mTORC1 (mechanistic target of rapamycin complex 1)–4EBP1 (eIF4E-binding protein 1)–eIF (eukaryotic initiation factor) 4F axis. Cardiomyocyte-specific ribosome profiling identified that I/R increased translation of mRNA networks associated with cardiac inflammation and cell infiltration. Short-term inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory cytokines such as Ccl2 (C-C motif chemokine ligand 2) of border zone cardiomyocytes, thereby attenuating Ly6C ^hi monocyte infiltration and myocardial inflammation. In addition, we identified a systemic immunosuppressive effect of eIF4F translation inhibitors on circulating monocytes, directly inhibiting monocyte infiltration. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin attenuated translation, reduced infarct size, and improved cardiac function after myocardial infarction. CONCLUSIONS: Global protein synthesis is inhibited during ischemia and shortly after reperfusion, followed by a recovery of protein synthesis that exceeds baseline levels in the border and infarct zones. Activation of mRNA translation after reperfusion is driven by mTORC1/eIF4F-mediated regulation of initiation and mediates an mRNA network that controls inflammation and monocyte infiltration to the myocardium. Transient inhibition of the mTORC1-/eIF4F axis inhibits translation and attenuates Ly6C ^hi monocyte infiltration by inhibiting a proinflammatory response at the site of injury and of circulating monocytes.

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We present Sci-ModoM, the first next-generation RNome database offering a holistic view of the epitranscriptomic landscape. Sci-ModoM has a simple yet powerful interface, underpinned by FAIR data principles, a standardized nomenclature, and interoperable formats, fostering the use of common standards within the epitranscriptomics community. Sci-ModoM provides quantitative measurements per site and dataset, enabling users to assess confidence levels based on score, coverage, and stoichiometry. Data in Sci-ModoM is directly traceable to its sources. Users can Search and Compare over six million modifications across 156 datasets, Browse or download datasets, and retrieve metadata. A comparison tool offers a novel and unique opportunity to compare modifications site-wise across datasets, with the ability to securely upload and compare user data against latest published research. Sci-ModoM empowers researchers, including non-experts, to access a broad spectrum of recent quantitative RNA modification data, thereby enhancing the utility and impact of latest discoveries, and opening new avenues in biological and medical research.