Periodic Reporting for period 1 - MAACS (N6 - methyladenosine RNA modification in acute coronary syndrome)
Periodo di rendicontazione: 2023-03-01 al 2025-02-28
Cardiovascular diseases (CVD) remain the leading cause of death worldwide, with acute coronary syndromes (ACS)—such as heart attacks—posing a particularly severe threat due to their sudden onset and risk of post-infarction heart failure. Despite advances in medical diagnostics and treatment, many patients remain at high risk of long-term complications and death. There is a pressing need for personalised diagnostic tools that can identify high-risk individuals early and guide timely clinical intervention.
The MAACS project (m⁶A RNA Modifications in Acute Coronary Syndromes) was designed to address this gap by exploring a new frontier in biomedical research: epitranscriptomics, the study of chemical modifications on RNA molecules that can influence gene expression and cellular function. The project specifically focused on a modification called N6-methyladenosine (m⁶A), believed to play a critical role in inflammation and heart tissue remodelling after an infarction.
Hosted by the Luxembourg Institute of Health (LIH), and supported by partners at McGill University (Canada), the project aimed to discover new RNA-based biomarkers in blood cells that could predict the likelihood of heart failure following ACS. The research also sought to understand how these molecular changes influence immune system responses, potentially uncovering new therapeutic targets.
By combining clinical research with cutting-edge RNA sequencing and computational biology, MAACS contributes to the EU’s health policy goals of promoting personalized medicine, reducing preventable mortality, and supporting excellence in cardiovascular research.
The MAACS project (m⁶A RNA Modifications in Acute Coronary Syndromes) was designed to address this gap by exploring a new frontier in biomedical research: epitranscriptomics, the study of chemical modifications on RNA molecules that can influence gene expression and cellular function. The project specifically focused on a modification called N6-methyladenosine (m⁶A), believed to play a critical role in inflammation and heart tissue remodelling after an infarction.
Hosted by the Luxembourg Institute of Health (LIH), and supported by partners at McGill University (Canada), the project aimed to discover new RNA-based biomarkers in blood cells that could predict the likelihood of heart failure following ACS. The research also sought to understand how these molecular changes influence immune system responses, potentially uncovering new therapeutic targets.
By combining clinical research with cutting-edge RNA sequencing and computational biology, MAACS contributes to the EU’s health policy goals of promoting personalized medicine, reducing preventable mortality, and supporting excellence in cardiovascular research.
The MAACS project began with the recruitment of two carefully selected patient groups: 200 individuals who had experienced an acute myocardial infarction (MI), a severe form of acute coronary syndrome (ACS), and 150 individuals with stable angina (SA). Blood samples were collected from MI patients within 24 to 48 hours after the heart attack—a critical time window for capturing immune system activity that may influence the risk of complications such as heart failure. Importantly, MI patients were monitored for 12 months to track changes in heart function and identify those who showed either improvement or decline over time.
From these samples, we isolated peripheral blood mononuclear cells (PBMCs)—a type of immune cell known to interact with damaged heart tissue. High-quality RNA was extracted from these cells and used in multiple streams of investigation:
• Quantification of RNA modifications, specifically N6-methyladenosine (m⁶A), using highly sensitive liquid chromatography-mass spectrometry (LC/MS) at LIH Metabolomic platofrm.
• Direct RNA sequencing using Oxford Nanopore Technologies (ONT) at LIH in collaboration with McGill University, enabling the detection of RNA modifications at the single-nucleotide level.
• Bioinformatic analysis to uncover gene activity patterns and RNA modification profiles linked to adverse cardiac outcomes.
This integrated approach revealed striking differences between MI and SA patients. We identified a set of genes that were more or less active, especially those related to immune cells such as monocytes and neutrophils—key players in inflammation and healing. At the same time, hundreds of RNA molecules showed altered m⁶A modification, though these changes were not specific to a particular immune cell type. Additionally, we found that several different types of RNA modifications measured by LC/MS were associated with ACS, suggesting broader regulatory disruptions in RNA biology during acute cardiac events.
Using advanced computational tools, we analyzed the data from multiple angles. One key analysis revealed that a group of genes most responsible for variability between patients was highly linked to immune system responses. A separate group, composed only of RNA modification sites, was found to be strongly associated with MI, suggesting that RNA modifications may provide an independent layer of regulation beyond gene activity alone. These modifications were linked to processes such as RNA metabolism, transport, and cellular signaling—indicating potential new targets for understanding and treating complications following a heart attack.
Together, these findings represent a major step forward in understanding how the immune system and RNA biology interact after heart attacks, and lay the groundwork for future diagnostic and therapeutic strategies.
From these samples, we isolated peripheral blood mononuclear cells (PBMCs)—a type of immune cell known to interact with damaged heart tissue. High-quality RNA was extracted from these cells and used in multiple streams of investigation:
• Quantification of RNA modifications, specifically N6-methyladenosine (m⁶A), using highly sensitive liquid chromatography-mass spectrometry (LC/MS) at LIH Metabolomic platofrm.
• Direct RNA sequencing using Oxford Nanopore Technologies (ONT) at LIH in collaboration with McGill University, enabling the detection of RNA modifications at the single-nucleotide level.
• Bioinformatic analysis to uncover gene activity patterns and RNA modification profiles linked to adverse cardiac outcomes.
This integrated approach revealed striking differences between MI and SA patients. We identified a set of genes that were more or less active, especially those related to immune cells such as monocytes and neutrophils—key players in inflammation and healing. At the same time, hundreds of RNA molecules showed altered m⁶A modification, though these changes were not specific to a particular immune cell type. Additionally, we found that several different types of RNA modifications measured by LC/MS were associated with ACS, suggesting broader regulatory disruptions in RNA biology during acute cardiac events.
Using advanced computational tools, we analyzed the data from multiple angles. One key analysis revealed that a group of genes most responsible for variability between patients was highly linked to immune system responses. A separate group, composed only of RNA modification sites, was found to be strongly associated with MI, suggesting that RNA modifications may provide an independent layer of regulation beyond gene activity alone. These modifications were linked to processes such as RNA metabolism, transport, and cellular signaling—indicating potential new targets for understanding and treating complications following a heart attack.
Together, these findings represent a major step forward in understanding how the immune system and RNA biology interact after heart attacks, and lay the groundwork for future diagnostic and therapeutic strategies.
The MAACS project has produced a unique and pioneering body of work that significantly advances both cardiovascular and epitranscriptomic research. To our knowledge, this is the first study to use direct Oxford Nanopore Technologies (ONT) RNA sequencing to explore the epitranscriptomic landscape of blood immune cells in patients following myocardial infarction (MI). This innovative approach enabled us to generate the first dataset of its kind, offering unprecedented resolution of RNA modifications at the single-nucleotide level in a clinically relevant context.
Our work led to the identification of specific m⁶A-modified sites on individual genes associated with MI, revealing an entirely new layer of biological regulation. Notably, we demonstrated that the molecular pathways regulated by m⁶A RNA modifications differ significantly from those identified through gene expression analysis alone. While differentially expressed genes highlighted classical immune pathways, m⁶A modifications revealed distinct mechanisms related to RNA metabolism, transport, and signaling, suggesting that RNA modifications contribute independently to disease progression.
Additionally, using LC/MS technology, we were the first to identify several types of chemically modified RNA nucleotides—beyond m⁶A—that are significantly associated with ACS. These findings open new avenues for the development of RNA modification-based biomarkers for patient stratification and prognosis.
In summary, MAACS:
• Identified novel m⁶A RNA biomarkers in immune cells that differentiate patients with preserved versus reduced heart function after ACS.
• Generated the first ONT-based epitranscriptomic dataset in post-MI immune cells.
• Showed that m⁶A regulates immune responses through different biological pathways than traditional gene expression changes.
• Discovered multiple RNA modifications linked to ACS, advancing the field beyond m⁶A alone.
Key needs moving forward include:
• Continued validation in larger, independent cohorts to confirm the diagnostic and prognostic value of identified RNA modifications;
• Advanced deconvolution analysis to pinpoint the specific immune cell subtypes contributing to adverse outcomes in ACS, leveraging multiomics approaches;
• Expansion of international partnerships to support clinical translation, regulatory alignment, and future integration into personalized cardiovascular care.
Our work led to the identification of specific m⁶A-modified sites on individual genes associated with MI, revealing an entirely new layer of biological regulation. Notably, we demonstrated that the molecular pathways regulated by m⁶A RNA modifications differ significantly from those identified through gene expression analysis alone. While differentially expressed genes highlighted classical immune pathways, m⁶A modifications revealed distinct mechanisms related to RNA metabolism, transport, and signaling, suggesting that RNA modifications contribute independently to disease progression.
Additionally, using LC/MS technology, we were the first to identify several types of chemically modified RNA nucleotides—beyond m⁶A—that are significantly associated with ACS. These findings open new avenues for the development of RNA modification-based biomarkers for patient stratification and prognosis.
In summary, MAACS:
• Identified novel m⁶A RNA biomarkers in immune cells that differentiate patients with preserved versus reduced heart function after ACS.
• Generated the first ONT-based epitranscriptomic dataset in post-MI immune cells.
• Showed that m⁶A regulates immune responses through different biological pathways than traditional gene expression changes.
• Discovered multiple RNA modifications linked to ACS, advancing the field beyond m⁶A alone.
Key needs moving forward include:
• Continued validation in larger, independent cohorts to confirm the diagnostic and prognostic value of identified RNA modifications;
• Advanced deconvolution analysis to pinpoint the specific immune cell subtypes contributing to adverse outcomes in ACS, leveraging multiomics approaches;
• Expansion of international partnerships to support clinical translation, regulatory alignment, and future integration into personalized cardiovascular care.