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Silencing of CCR4-NOT Complex Subunits Affects Heart Structure and Function Lisa Elmén1, Claudia B

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Silencing of CCR4-NOT Complex Subunits Affects Heart Structure and Function Lisa Elmén1, Claudia B © 2020. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2020) 13, dmm044727. doi:10.1242/dmm.044727 RESEARCH ARTICLE Silencing of CCR4-NOT complex subunits affects heart structure and function Lisa Elmén1, Claudia B. Volpato2, Anaïs Kervadec1, Santiago Pineda1,*, Sreehari Kalvakuri1, Nakissa N. Alayari1,‡, Luisa Foco2, Peter P. Pramstaller2, Karen Ocorr1, Alessandra Rossini2, Anthony Cammarato3, Alexandre R. Colas1, Andrew A. Hicks2,§ and Rolf Bodmer1,§ ABSTRACT establish a crucial role of the CCR4-NOT complex in heart development The identification of genetic variants that predispose individuals to and function. cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies This article has an associated First Person interview with the first author have identified variants that associate with QT-interval length (a of the paper. measure of myocardial repolarization). Three of the strongest KEY WORDS: CNOT1, GWAS, Arrhythmia, Long-QT syndrome, associating variants (single-nucleotide polymorphisms) are located Drosophila heart, hiPSC, Cardiomyocytes 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 INTRODUCTION turnover. We isolated the minimum fragment of the CNOT1 Despite medical advances over the past few decades, cardiovascular promoter containing all three variants from individuals homozygous disease remains the most common cause of mortality worldwide for the QT risk alleles and demonstrated that the haplotype [https://www.who.int/news-room/fact-sheets/detail/cardiovascular- associating with longer QT interval caused reduced reporter diseases-(cvds)]. Understanding the mechanisms of heart morbidity expression in a cardiac cell line, suggesting that reduced CNOT1 is crucial for finding new therapies, and determining which genetic expression might contribute to abnormal QT intervals. Systematic variants predispose individuals to heart disease is necessary to siRNA-mediated knockdown of CCR4-NOT components in human provide better preventative care. The challenge of connecting human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) genetic variants with disease can be met by combining genome-wide revealed that silencing CNOT1 and other CCR4-NOT genes reduced association studies (GWAS) with patient sequencing and validation their proliferative capacity. Silencing CNOT7 also shortened action using disease-in-a-dish and in vivo cardiac model systems. potential duration. Furthermore, the cardiac-specific knockdown of Drosophila melanogaster benefits from well-conserved genes and Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and permits functional assessment of genes of interest, which when CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, manipulated might not be well tolerated by the vertebrate heart. reduced contractility or a propensity for arrhythmia. Silencing The QT interval on an electrocardiogram is a measure that reflects CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac myocardial repolarization. Short-QT syndrome (Rudic et al., 2014) chamber size and contractility. Developmental studies suggested that and long-QT syndrome (Amin et al., 2013) are caused by different CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac underlying mechanisms, but are both risk factors for atrial and remodeling from larval to adult stages. To summarize, we have ventricular arrhythmias and sudden cardiac death (Rudic et al., demonstrated how disease-associated genes identified by GWAS can 2014; Amin et al., 2013; Vacanti et al., 2017). Genome-wide be investigated by combining human cardiomyocyte cell-based and association in up to 100,000 individuals has successfully identified in vivo at least 35 common variant QT-interval loci that collectively explain whole-organism heart models. Our results also suggest a ∼ potential link of CNOT1 and CNOT7/8 to QT alterations and further 8-10% of QT variation (Arking et al., 2014). Some of the strongest QT-associating variants identified center around the CNOT1 gene, which encodes the central scaffolding subunit CCR4-NOT 1Development Aging and Regeneration Program, Sanford Burnham Prebys transcription complex subunit 1 (CNOT1) of the CCR4-NOT Medical Discovery Institute, 10901 N Torrey Pines Rd, La Jolla, CA 92037, USA. complex. CCR4-NOT is conserved throughout the eukaryotic 2Institute for Biomedicine, Eurac Research, Affiliated Institute of the University of Lübeck, Via Galvani 31, 39100 Bolzano, Italy. 3Johns Hopkins University, Division kingdom and is involved in the sequential processes of gene of Cardiology, 720 Rutland Ave., Baltimore, MD 21205, USA. expression. Its activities can be divided into functional modules *Present address: University of California Los Angeles, Molecular, Cell, and Developmental Biology, Terasaki Life Sciences Building, 610 Charles E. Young involved in transcription (Kruk et al., 2011) (CNOT2, CNOT3), Dr. South, CA 90095, USA. ‡Present address: Illumina Inc., 5200 Illumina Way, mRNA degradation (Bhandari et al., 2014; Yi et al., 2018; Temme San Diego CA 92122, USA. et al., 2010; Webster et al., 2018), deadenylation (CNOT6/6L, §Authors for correspondence ([email protected]; [email protected]) CNOT7/8) and protein quality control through ubiquitination (CNOT4) (Halter et al., 2014; Collart, 2016; Collart and A.A.H., 0000-0001-6320-0411; R.B., 0000-0001-9087-1210 Panasenko, 2017). This is an Open Access article distributed under the terms of the Creative Commons Attribution The CCR4-NOT complex has previously been implicated in heart License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, disease; we have demonstrated that silencing of genes Ubc4 and distribution and reproduction in any medium provided that the original work is properly attributed. Not3 cause cardiac dilation and dysfunction in Drosophila (Neely Handling Editor: Ross Cagan et al., 2010). In addition, CNOT3 heterozygous knockout mouse Received 28 February 2020; Accepted 19 May 2020 hearts displayed reduced contractility and increased susceptibility to Disease Models & Mechanisms 1 RESEARCH ARTICLE Disease Models & Mechanisms (2020) 13, dmm044727. doi:10.1242/dmm.044727 failure following aortic constriction (Neely et al., 2010). CNOT3 minimal promoter constructs consisted of alleles T and G, has also been found to interact with Atg7, which affects respectively, at rs27097 (Fig. 1C). cardiomyocyte (CM) survival and QT intervals in mice The complete and minimal promoter-luciferase constructs were (Yamaguchi et al., 2018). In the present study, we investigated the transfected into HL-1 cells, a cardiac muscle cell line that contracts individual role of additional CCR4-NOT complex subunits, starting and retains phenotypic characteristics of adult CM, along with with variants in the CNOT1 putative promoter region that positively renilla luciferase in order to normalize signals. Both minimal and associate with QT intervals, to determine whether and in what complete promoter constructs were able to drive increased luciferase direction these variants functionally influence reporter gene expression in the HL-1 cells with significant differences between expression. We further explored the effects of RNA interference the two haplotypes. When considering the SNP closest to the gene (RNAi)-mediated knockdown of CNOT1 and complex subunit alone (minimal construct), expression was effectively silenced in the genes CNOT2, CNOT4, CNOT6/6L and CNOT7/8 in human HL-1 cell line for the haplotype that was associating with longer QT induced pluripotent stem cell-derived cardiomyocytes (hiPSC- intervals (Fig. 1E). These differences were also observed in both CMs) (Cunningham et al., 2017; Yu et al., 2018); the effects of HeLa and T293 cells (Fig. S1A,B). With the larger promoter knockdown on proliferation and electrophysiological properties – in fragment, expression was re-established in all lines, but was particular, action potential duration (APD) (McKeithan et al., 2017) significantly reduced for the haplotype associated with a longer QT – were investigated. In addition, the effects of gene knockdown on interval compared with the haplotype carrying the alternate alleles. cardiac structure and contractile function were studied in vivo using From these experiments, we conclude that the variants that the Drosophila heart model (Ocorr et al., 2014). Overall, we find significantly associate with QT interval in human GWAS are that silencing CNOT1 and other CCR4-NOT components indeed functionally able to alter expression of the CNOT1 gene in compromises cardiac development and function in the two model cardiac tissue, with the most significant differences for the complete systems, suggesting an important role of this complex in cardiac haplotype being seen in the cardiac cell line. The direction of the health and disease. effect is such that reduced CNOT1 possibly contributes to QT-interval prolongation. RESULTS Functional validation of CNOT1 promoter polymorphisms Genes encoding the CCR4-NOT complex regulate Three of the strongest QT interval-associating variants [single- proliferation and APD in hiPSC-CMs nucleotide polymorphisms (SNPs)], in strong linkage CCR4-NOT is a multisubunit complex with different functional disequilibrium (LD) over CNOT1, are located in the putative modules (including, but not
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