Silencing of CCR4-NOT complex subunits affects heart structure and function

Abstract

The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of <i>CNOT1</i>, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the <i>CNOT1</i> promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced <i>CNOT1</i> expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing <i>CNOT1</i> and other CCR4-NOT genes reduced their proliferative capacity. Silencing <i>CNOT7</i> also shortened action potential duration. Furthermore, the cardiac-specific knockdown of <i>Drosophila</i> orthologs of CCR4-NOT genes <i>in vivo</i> (<i>CNOT1/N</i><i>ot1</i> and <i>CNOT7/8/Pop2</i>) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing <i>CNOT2/Not2</i>, <i>CNOT4/</i><i>N</i><i>ot4</i> and <i>CNOT6/6L/twin</i> also affected cardiac chamber size and contractility. Developmental studies suggested that <i>CNOT1/Not1</i> and <i>CNOT7/8/Pop2</i> are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism <i>in vivo</i> heart models. Our results also suggest a potential link of <i>CNOT1</i> and <i>CNOT7/8</i> to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function.This article has an associated First Person interview with the first author of the paper.