RTTN Mutations: Centrosome Dysfunction in Infantile Dilated Cardiomyopathy
Executive Summary
This research paper identifies a novel genetic cause for infantile dilated cardiomyopathy (iDCM): mutations in the RTTN gene, which encodes the centrosomal protein Rotatin. The study demonstrates that RTTN mutations disrupt centrosome reduction, a critical process during cardiomyocyte maturation, leading to impaired microtubule network formation, sarcomere and mitochondrial defects, and ultimately, cardiac dysfunction. The research team used a combination of patient-derived iPSC models, CRISPR/Cas9 gene editing, in vivo models (zebrafish and Drosophila), and single-cell RNA sequencing (scRNA-seq) to elucidate the underlying mechanisms and identify a potential therapeutic target.
Background and Problem
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iDCM is a rare but severe condition leading to significant morbidity and mortality in infants. While genetic factors are known to contribute to iDCM in a significant percentage of cases (27-54%), the specific genes and mechanisms are not fully understood. Previously identified mutations are typically in genes relating to sarcomere structure, nuclear envelope, and cytoskeleton. Current management often necessitates heart transplantation, highlighting the urgent need for targeted therapies based on a deeper understanding of the disease pathogenesis. The centrosome, a microtubule organizing center (MTOC), undergoes a significant reorganization during cardiomyocyte maturation called "centrosome reduction." The role of this process in cardiac cell biology and its potential link to cardiac disease has been largely unknown.
Key Findings and Ideas:
RTTN Mutations as a Novel Cause of iDCM: Whole exome sequencing of an infant with iDCM, combined with CRISPR/Cas9-mediated gene knockout and correction in iPSC-derived cardiomyocytes, identified RTTN as the causal gene. The patient had compound heterozygous mutations in RTTN: an in-frame deletion inherited from the mother and a missense mutation inherited from the father. "Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient’s condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy."
Recapitulation of iDCM Phenotype in iPSC Model: iPSC-derived cardiomyocytes from the patient (iDCM-CMs) recapitulated the cardiac defects observed in the explanted heart, including sarcomere disorganization, mitochondrial abnormalities, and reduced contractility. "Compared with cardiomyocytes from healthy donors, the iDCM-derived cardiomyocytes (iDCM-CMs) exhibited profound sarcomere defects (Figure 1B), recapitulating the cardiac defects seen in the explanted heart (Figure 1A)." Evolutionarily Conserved Role of RTTN in Cardiac Development: Genetic knockdown of RTTN in zebrafish and ana3 (the RTTN homolog) mutant flies resulted in cardiac structural and functional defects, confirming an evolutionarily conserved role for RTTN in heart development. "Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function."
RTTN Mutation Leads to Dilated Cardiomyopathy in Multiple In Vivo Systems" Impaired Cardiomyocyte Maturation: Single-cell RNA sequencing (scRNA-seq) revealed impaired maturation of iDCM-CMs, with downregulation of genes involved in sarcomere organization, cardiac muscle contraction, and mitochondrial function, and upregulation of genes related to glycolysis and apoptosis. "Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits."
Defective Centrosome Reduction and Microtubule Network Disruption: The study found that RTTN mutations disrupt the normal reorganization of the centrosome during cardiomyocyte maturation (centrosome reduction). In iDCM-CMs, the centrosome remained localized at the centriole, contrasting with the expected perinuclear reorganization. This led to global microtubule network defects. "We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects." "RTTN-mediated centrosome reduction is upstream of other canonical maturation events"
Small Molecule Rescue: Treatment with a small molecule (C1935) that promotes centrosome reduction restored centrosome reorganization, improved sarcomere structure, and enhanced contractility in iDCM-CMs. "In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes."
Implications
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Novel Disease Mechanism: The study identifies a previously unrecognized role for centrosome dysfunction in iDCM, expanding our understanding of the disease's pathogenesis. It is the first to demonstrate a case of human disease caused by a defect in centrosome reduction.
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Therapeutic Potential: The identification of a small molecule that can rescue the iDCM phenotype suggests a potential therapeutic strategy for centrosome-related iDCM.
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Future Research Directions: The authors propose that future studies aimed at identifying variants in other centrosome components may uncover additional contributors to human cardiac disease. "Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease." "Because centrosome dynamics play fundamental roles in cardiomyocyte cell cycle exit, structure, and function, a better understanding of this tissue-specific, developmentally programmed process will be highly relevant to cardiac regenerative therapy efforts."