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

Abstract

Genome wide association studies (GWAS) have identified variants that associate with QT-interval length. Three of the strongest associating variants (SNPs) are located in the putative promotor region of CNOT1, a gene encoding the central subunit of CCR4-NOT, a multi-functional, 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 may 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-CNOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes, CNOT1/not1 and CNOT7/8/pop2, in vivo, 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. In sum, 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 critical role of the CCR4-NOT complex in heart development and function.