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Isoform changes of action potential regulators in the ventricles of arrhythmogenic phospholamban-R14del humanized
Malgorzata E Rogalska1, Elizabeth Vafiadaki2, Zoi Erpapazoglou3
1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona 08003, Spain.
Insights
The phospholamban (PLN) R14del mutation causes arrhythmogenic cardiomyopathy (ACM) through altered mRNA splicing, affecting cardiac cell action potentials and calcium handling, increasing arrhythmia risk.
Area of Science:
- Cardiovascular Biology
- Molecular Genetics
- Cardiac Electrophysiology
Background:
- Arrhythmogenic cardiomyopathy (ACM) is a severe heart condition linked to sudden cardiac death.
- The phospholamban (PLN) R14del mutation is a known genetic cause of ACM.
- The precise molecular mechanisms underlying PLN-R14del ACM pathogenesis are not fully understood.
Purpose of the Study:
- To investigate transcriptome-wide mRNA splicing alterations in a humanized PLN-R14del mouse model and human iPSC-CMs.
- To identify the specific molecular pathways and regulatory factors involved in PLN-R14del ACM.
- To elucidate the role of aberrant splicing in cardiac dysfunction and arrhythmogenesis.
Main Methods:
- Utilized a humanized PLN-R14del mouse model and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).
- Performed transcriptome-wide mRNA sequencing to identify alternative splicing (AS) events.
- Conducted bioinformatic and enrichment analyses to identify affected genes and regulatory factors.
Main Results:
- Identified over 200 significant AS events, with distinct profiles in right and left ventricles of PLN-R14del hearts.
- Found that the 'cardiac cell action potential' biological process was significantly affected, particularly in the right ventricle.
- Observed altered splicing of key calcium-regulating genes (Trpm4, Camk2d) and identified Srrm4 and Nova1 as potential upstream regulators.
Conclusions:
- Aberrant mRNA splicing is a key molecular mechanism in PLN-R14del ACM pathogenesis.
- Altered splicing impacts calcium homeostasis and cardiac action potential, contributing to arrhythmogenesis.
- PLN-R14del mutation-induced splicing changes highlight novel therapeutic targets for ACM.
Abstract:
Arrhythmogenic cardiomyopathy (ACM) is characterized by life-threatening ventricular arrhythmias and sudden cardiac death and affects hundreds of thousands of patients worldwide. The deletion of Arginine 14 (p.R14del) in the phospholamban (PLN) gene has been implicated in the pathogenesis of ACM. PLN is a key regulator of sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Despite global gene and protein expression studies, the molecular mechanisms of PLN-R14del ACM pathogenesis remain unclear. Using a humanized PLN-R14del mouse model and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), we investigated the transcriptome-wide mRNA splicing changes associated with the R14del mutation. We identified >200 significant alternative splicing (AS) events and distinct AS profiles were observed in the right (RV) and left (LV) ventricles in PLN-R14del compared to WT mouse hearts. Enrichment analysis of the AS events showed that the most affected biological process was associated with "cardiac cell action potential", specifically in the RV. We found that splicing of 2 key genes, Trpm4 and Camk2d, which encode proteins regulating calcium homeostasis in the heart, were altered in PLN-R14del mouse hearts and human iPSC-CMs. Bioinformatical analysis pointed to the tissue-specific splicing factors Srrm4 and Nova1 as likely upstream regulators of the observed splicing changes in the PLN-R14del cardiomyocytes. Our findings suggest that aberrant splicing may affect Ca2+-homeostasis in the heart, contributing to the increased risk of arrythmogenesis in PLN-R14del ACM.
