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 CNOT1, 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 CNOT1 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 CNOT1 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 CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 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 in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 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.

In vivoidentification and validation of novel potential predictors for human cardiovascular diseases

“…Across the hGWAS set of 40 genes, comparative heart rate analysis showed statistically significant heart rate phenotypes in a total of 16 genes ( Fig 3B). The five positive controls, known to play key roles in heart functions such as cardiac contraction (TTN and NACA) and heart rate regulation (CASQ2, Beyond known cardiac genes, we revealed new genes linked to various biological functions (CCDC141, GIGYF1, HOMEZ, MYRF, SMG6, CMYA5, CNOT1, SLC17A3, TRAPPC12, SSPO and PADI4) which up to now, had little to no experimental evidence in cardiac function (Rathjens et al 2020;Benson et al 2017;Yamaguchi et al 2018;Elmen et al 2020).…”

The Regulatory Properties of the Ccr4–Not Complex

“…Some of the strongest QT-associating variants identified, centre around the CNOT1 gene. Using reporter assays it was shown that reduced CNOT1 expression contributes to abnormal QT-intervals [402]. Cardiac specific depletion of Not1 and Pop2 in Drosophila was either lethal or resulted in dilated cardiomyopathy, reduced contractility, or a propensity for arrhythmia [186,402].…”

“…Using reporter assays it was shown that reduced CNOT1 expression contributes to abnormal QT-intervals [402]. Cardiac specific depletion of Not1 and Pop2 in Drosophila was either lethal or resulted in dilated cardiomyopathy, reduced contractility, or a propensity for arrhythmia [186,402]. Similarly, the loss of Not2, Not3, Not4 and twin affected cardiac chamber size and contractility [186,402].…”

“…Cardiac specific depletion of Not1 and Pop2 in Drosophila was either lethal or resulted in dilated cardiomyopathy, reduced contractility, or a propensity for arrhythmia [186,402]. Similarly, the loss of Not2, Not3, Not4 and twin affected cardiac chamber size and contractility [186,402]. The loss of NOT3 in heterozygous knock out mice resulted in impairment of cardiac contractility and susceptibility to heart failure although these could be reversed with HDAC inhibitors [186].…”

First person – Lisa Elmén

“… ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Lisa Elmén is first author on ‘ Silencing of CCR4-NOT complex subunits affects heart structure and function ’, published in DMM. Lisa conducted the research described in this article while a lab manager/research associate in Rolf Bodmer's lab at Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.…”