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

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 limited to, the subunits considered in this promoter region of the gene (Fig. 1B; redrawn from fig. S2 in study) (Fig. 1A). It has been shown in HeLa and HEK293T cells that Arking et al., 2014). All GWAS-associated variants over the whole short interfering RNA (siRNA) depletion of CNOT1 decreases the length of this gene are in strong LD with the putative promoter amount of CCR4-NOT subunits and reduces deadenylase activity of variants, and the Genotype-Tissue Expression (GTEx) data (https:// the complex; simultaneous siRNA silencing of the entire deadenylase doi.org/10.1089/bio.2015.0032) indicate strong tissue-specific module (CNOT6/6L, CNOT7/8) results in apoptosis similar to that of expression quantitative trait loci (eQTL) variants over the whole CNOT1 silencing alone (Ito et al., 2011). Therefore, we decided to use gene; this observation led us to postulate that the promoter variants hiPSC-CMs to investigate the effect of silencing CNOT6/6L and would demonstrate functionally different expression levels. CNOT7/8, genes encoding enzymes that affect translation efficiency, Sequence analysis of a fragment spanning all three of these by removing the mRNA poly(A) tail (Yi et al., 2018). This approach variants strongly associated with QT interval confirmed the wasalsousedtostudythegeneCNOT4/Not4, which encodes a RING presence of four SNPs (rs27097, rs37037, rs9941290 and E3-ligase important for assembly of the proteasome and proposed to rs863433). Three of these SNPs associate strongly with QT be involved in co-translational quality control (Halter et al., 2014). We interval, whereas rs37037 (which is not in strong LD with the also chose to examine CNOT2, as it associates with CNOT3, for which other three variants) is still associated but with a less significant we previously identified a role in cardiac function (Neely et al., 2010). P-value (Fig. S1C). We identified two human subjects homozygous First, to evaluate a potential role of the CCR4-NOT complex in for alleles at the four SNPs that fall within ∼3.2 kb of the 5′ region human cardiac physiology, we knocked down each of the CNOT of the CNOT1 coding sequence (Fig. 1C). The two haplotypes (one genes and evaluated their effect on hiPSC-CM proliferation. with risk alleles and one with alternate alleles) from the putative Individual knockdown of CNOT1, CNOT2, CNOT3, CNOT4 and CNOT1 promoter region were isolated and cloned in two forms into CNOT6 led to decreased 5-ethynyl-2′-deoxyuridine (EdU) a plasmid vector (pGL4.1) to drive the firefly luciferase gene incorporation in day 25 hiPSC-CMs and reduced CM number (Fig. 1D). The ‘minimal’ putative promoter region contained 657 bp compared with control (Fig. 2A-D), thereby suggesting a general role of the region around just one SNP (rs27097), located closest to the of the CCR4-NOT complex in the regulation of CM proliferation. start codon of the CNOT1 gene. The larger 3172 bp fragment A number of studies (Itzhaki et al., 2011; Matsa et al., 2011; Lahti contained all of the strongest QT interval-associating variants, et al., 2012) have shown that hiPSC-CMs from patients with long- potentially capturing the ‘complete’ promoter region of CNOT1 QT syndromes consistently show prolonged APD phenotypes, (with respect to the significantly associated variants in this region of suggesting that APD modulation in hiPSC-CMs represents a the gene). Both constructs were sequenced to confirm that the four reliable model system to evaluate the role of candidate genes for variant positions under study were the only ones differing between QT-interval modulation. Therefore, we asked whether the CCR4- these natural promoter regions and that the alleles were NOT complex could also have such a role and transfected day 25 homozygous. For both the minimal and complete constructs, one hiPSC-CMs with siRNAs directed against each member of the haplotype consisted of alleles that significantly associate with complex; APD parameters were determined using a fluorescence- increases in QT interval length (‘TGAG’ haplotype), whereas the based, single-cell and high-throughput voltage transient recording other haplotype consisted of the alternate alleles at these variants assay, based on that used in a previous study (McKeithan et al.,

(‘GAGT’ haplotype) (see also Fig. S1C) (Pfeufer et al., 2009). The 2017; Fig. 2E). Interestingly, we found that CNOT7, but not Disease Models & Mechanisms

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Fig. 1. The CCR4-NOT complex and functional validation of CNOT1 promoter polymorphisms. (A) Cartoon of the CCR4-NOT complex with the subunits investigated in this study. (B) GWAS identifies CNOT1 SNPs associated with human QT syndrome. The four putative promoter SNPs are highlighted in the red box. Redrawn from fig. S2 in Arking et al. (2014). This image is not published under the terms of the CC-BY license of this article. For permission to reuse, please see Arking et al. (2014). (C) Constructs generated for the CNOT1 complete promoter region and the minimal promoter region, cloned from two subjects carrying haplotype ‘GAGT’ and haplotype ‘TGAG’. Numbers above each SNP denote how close the variant is to the open reading frame (e.g. 1 is the closest). (D) Schematic of experimental procedure. (E) Box plots summarizing the intensity of the ratio between luciferase and renilla signal in HL1 cells. Boxes, interquartile range; central line, median; whiskers, upper and lower adjacent values as defined by Tukey (1977); dots, outside values. Two-sided P-values were computed using the Wilcoxon rank-sum test (***P≤0.001). For complete promoter, the number of independent experiments n=3; for each experiment, the number of independent biological replicates per haplotype n=3. Number of observations for each haplotype n=9, total n=18. For minimal promoter, the number of independent experiments n=5; for each experiment, the number of independent biological replicates per haplotype n=3, except one experiment (n=4). Number of observations for each haplotype n=16, total n=32. CNOT1, knockdown led to a significant shortening (>20 ms) of near the CNOT7 gene with QT interval in ∼5000 individuals within APD (Fig. 2F-I). Although it is possible that the level of CNOT1 the CHRIS study (Pattaro et al., 2015). knockdown was insufficient to cause a change in APD, it did produce a proliferation deficit, indicating that the siRNAs were Cardiac-specific in vivo knockdown of CNOT1/Not1 and transfected and active in hiPSC-CMs. This finding suggests a CNOT7/8/Pop2 in Drosophila results in dilated potential new role for CNOT7 and deadenylation in the regulation of cardiomyopathy the QT interval in humans, an observation supported by the As experiments with hiPSC-CMs provided evidence that CNOT1 and −6 suggestive association of a variant (rs183286310; P=1.1×10 ) CNOT7 regulate CM proliferation and APD, respectively, we Disease Models & Mechanisms

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Fig. 2. Genes encoding the CCR4-NOT complex regulate proliferation and action potential in hiPSC-CMs. (A) Schematic of the proliferation assay in hiPSC-CMs. (B) Representative immunofluorescence images for EdU and ACTN1 in control (siCtrl), siCNOT1 and siCNOT3 conditions. Scale bars: 25 µm. (C,D) Histograms showing the normalized percentage of EdU-positive hiPSC-CMs and normalized number of hiPSC-CMs. Student’s t-test was used to calculate P-values. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. (E) Schematic overview of the single-cell, high-throughput voltage assay. RFU, relative fluorescence units. (F) Two-dimensional graph for APD75 and Kolmogorov-Smirnov distance (KS-D) representing screen results for CCR4-NOT component knockdown. (G) Population distribution of APD75 measurements for siCNOT7- versus siCtrl-transfected hiPSC-CMs. (H) Median action potential traces for siCNOT7- and siCtrl-transfected hiPSC-CMs. (I) Table summarizing average and s.d. values for APD50, APD75 and APD90 for siCNOT7- and siCtrl-transfected hiPSC-CMs. considered how the same manipulations would affect the heart in vivo. Using the Drosophila UAS-Gal4 system (Brand and Perrimon, The CNOT genes are conserved in Drosophila and have the following 1993) we silenced CNOT1/Not1 with Hand-Gal4, a driver specific orthologs to the human CNOT genes: CNOT2/Not2, CNOT4/Not4, for myocardial and pericardial cells of the heart that acts throughout

CNOT6 and CNOT6L/twin, CNOT7 and CNOT8/Pop2 (Table 1). development (Han and Olson, 2005). At 1 week post-eclosion Disease Models & Mechanisms

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Table 1. CCR4-NOT genes investigated in this study myofibrils (Fig. 3F,G). Knockdown with two different CNOT1/ Human Drosophila Not1 RNAi lines (GD12571 and KK106587) resulted in similar phenotypes, whereas knockdown with a third line (TRiP 28681) CNOT1 Not1 resulted in the same trend, but was not statistically significant from CNOT2 Not2/Rga CNOT3 Not3 control fly hearts. The CNOT1/Not1 RNAi line VDRC GD12571 is CNOT4 Not4/Cnot4 shown in all figures. CNOT6 and CNOT6L twin We had previously observed double-beat early afterdepolarization CNOT7 and CNOT8 Pop2 (EAD)-associated arrhythmias in M-mode traces from movies of CNOT3/Not3 knockdown fly hearts (Neely et al., 2010), thus an effort was made to record electrophysiological traces. This proved (young adult flies), we dissected the animals to expose the heart for nearly impossible, however, owing to the fragility of the CNOT1/ video recording as previously described (Ocorr et al., 2007a, 2014; Not1 knockdown hearts. One successful recording did show Fink et al., 2009) (Fig. 3A). The Hand-Gal4-driven CNOT1/Not1 abnormal fibrillatory events, with increased event duration and knockdown hearts exhibited normal beating frequency (shown as number of peaks per burst (Fig. S2A,B). heart period, Fig. 3B). However, these hearts were fragile and As the results of the hiPSC-CMs indicated the importance of the exhibited diastolic and systolic diameters that were significantly deadenylase CNOT7 for cardiac rhythm control, we asked the larger than controls (Fig. 3C,D). These changes resulted in a question how knockdown of the Drosophila ortholog CNOT7/8/ substantial decrease in fractional shortening (Fig. 3E) and a reduced Pop2 would affect the fly heart. RNAi-mediated knockdown of capacity for the heart to contract. Fluorescent staining of actin CNOT7/8/Pop2, through the Hand-Gal4 driver line, resulted in revealed an abnormal myofibrillar structure with large gaps and pupal lethality at 25°C. By lowering the incubation temperature disarray in CNOT1/Not1 knockdown fly hearts compared with during development to 18°C, and thereby reducing Gal4 control hearts, displaying typical tightly packed circumferential production, the flies did eclose. When 1-week-old CNOT7/8/Pop2

Fig. 3. Cardiac-specific in vivo knockdown of CNOT1/Not1 in Drosophila. (A) Schematic of heart analysis procedure in Drosophila. Dissection in artificial hemolymph exposes the beating fly heart for video recording. Automated quantification generates M-modes used to measure heart period, heart size and contractility. (B-E) RNAi-mediated knockdown of CNOT1/Not1 (VDRC, GD12571) using the cardiomyocyte and pericardial cell-specific driver Hand-Gal4; n=40 female flies per genotype. Reduced CNOT1/Not1 expression increased diastolic and systolic diameters and reduced fractional shortening. Student’s t-test was used to calculate two-sided P-values. ****P≤0.0001. Boxes, interquartile range; central line, median; plus, mean; whiskers, upper and lower adjacent values as defined by Tukey (1977); dots, outside values. (F,G) Immunofluorescence staining with phalloidin visualized F-actin of Drosophila hearts. A representative Not1 knockdown heart shows myofibrillar disarray and gaps in muscle tissue compared with the tightly packed circumferential myofibrils seen in the control heart. Scale bar: 200 µm. Disease Models & Mechanisms

5 RESEARCH ARTICLE Disease Models & Mechanisms (2020) 13, dmm044727. doi:10.1242/dmm.044727 knockdown hearts were functionally analyzed, they exhibited no only CNOT2/Not2 and CNOT6/6L/twin silencing also resulted in change in heart period (Fig. 4A). However, significant cardiac increased systolic diameters compared with controls (Fig. 5B). This dilation, as measured by increased diastolic and systolic diameters dilation did not result in significantly diminished fractional (Fig. 4B,C), and reduced contractility (Fig. 4D) were evident, shortening, however (Fig. 5C). The results suggest that CNOT2/ consistent with the phenotype observed in CNOT1/Not1 Not2, CNOT4/Not4 and CNOT6/6L/twin are needed for normal knockdown fly hearts. Furthermore, fluorescent staining of heart dimensions in vivo, but their reduction did not significantly CNOT7/8/Pop2 knockdown hearts revealed myofibrillar structural affect overall contractility. abnormalities (Fig. 4E,F), as seen in CNOT1/Not1 knockdown We repeated all RNAi experiments with a second driver line, hearts (Fig. 3F,G). Electrophysiological recordings indicated that TinCΔ4-Gal4 (Lo and Frasch, 2001), which is expressed in the cardiac-restricted silencing of CNOT7/Pop2, as observed with the myocardium during early development, during late pupal stages single CNOT1/Not1 knockdown heart, triggered longer event of cardiac remodeling and in the adult heart, but not during larval durations and multiple peaks per burst (Fig. 4G-I) compared with and early pupal stages. Surprisingly, TinCΔ4-Gal4-mediated control hearts. Although we cannot be certain that the single CNOT1/Not1 knockdown did not cause a dilated cardiac CNOT1/Not1 recording is representative, taken together with the phenotype, except for a small increase in systolic heart diameter CNOT7/8/Pop2 electrophysiological recordings, we find that this that resulted in modestly decreased fractional shortening (Fig. phenotype is consistent with a propensity for arrhythmia. S2C-E). TinCΔ4-Gal4-driven knockdown of CNOT2/Not2, Hand-Gal4-driven RNAi knockdown of CNOT2/Not2, CNOT4/ CNOT4/Not4 and CNOT6/6L/twin did not engender a cardiac Not4 and CNOT6/6L/twin also caused cardiac dilation, as observed phenotype compared with controls, although CNO4/Not4 with the silencing of CNOT1/Not1 and CNOT7/8/Pop2. knockdown resulted in substantially increased diastolic and Knockdown of CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin systolic diameters, but with no effect on fractional shortening induced a significant increase in diastolic diameter (Fig. 5A), but (Fig. S3).

Fig. 4. Cardiac-specific in vivo knockdown of CNOT7/8/Pop2 in Drosophila. (A-D) RNAi-mediated knockdown of CNOT7/8/Pop2 (TRiP HM05235) using the cardiomyocyte and pericardial cell specific driver Hand-Gal4 (n=32 female flies per genotype). Reduced Pop2 expression increased diastolic and systolic diameters and reduced fractional shortening. Student’s t- test was used to calculate two-sided P-values. *P≤0.05, ***P≤0.001, ****P≤0.0001. Boxes, interquartile range; central line, median; plus, mean; whiskers, upper and lower adjacent values as defined by Tukey (1977); dots, outside values. (E,F) Immunofluorescence staining with phalloidin visualizes F-actin of Drosophila hearts. The CNOT7/8/Pop2 knockdown heart shows dilation and gaps in muscle tissue compared with the tightly packed circumferential myofibrils in control heart. Scale bar: 100 µm. (G) Table summarizing electrophysiological measurements of CNOT7/8/Pop2 knockdown fly hearts. (H,I) Representative 10 s M-modes show greater peaks per burst and longer event duration of CNOT7/8/Pop2 knockdown (Hand-Gal4>CNOT7/8/Pop2) fly hearts compared with control (w1118×CNOT7/8/ Pop2). Disease Models & Mechanisms

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Fig. 5. RNAi-mediated knockdown of CNOT2/Not2 (VDRC GD20826), CNOT4/Not4 (TRiP JF03203) and CNOT6/6L/twin (VDRC GD13365) using the cardiomyocyte and pericardial cell specific driver Hand-Gal4. (A) Reduced expression of CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin significantly increased diastolic diameter. (B) Reduced expression of CNOT2/ Not2 and CNOT6/6L/twin significantly increased systolic diameter. (C) Fractional shortening was not changed by reduced expression of either CNOT2/Not2, CNOT4/Not4 or CNOT6/6L/twin. n=15 female flies per genotype. One- way ANOVA with Tukey’s multiple comparisons test was used to calculate two-sided P-values. *P≤0.05, **P≤0.01, ****P≤0.0001. Boxes, interquartile range; central line, median; plus, mean; whiskers, upper and lower adjacent values as defined by Tukey (1977); dots/arrowheads, outside values.

Knockdown of CNOT1/Not1 and CNOT7/8/Pop2 during with CNOT3 (Neely et al., 2010; Yamaguchi et al., 2018), are Drosophila development (larval stages) required during the larval and/or early pupal stages of development, The finding that knockdown of CNOT1/Not1 and CNOT7/8/Pop2 and thus during the initial stages of cardiac remodeling from the with the pupal/adult TinCΔ4-Gal4 myocardial driver (not expressed larval to adult heart. in larval hearts) did not recapitulate the results obtained with the continuously expressed Hand-Gal4 heart driver raised the question DISCUSSION as to whether developmental expression in larvae/early pupae was GWAS studies have successfully identified many genetic loci crucial for normal adult heart function. We therefore tested the associated with multiple disorders, including cardiovascular disease hypothesis that the discrepancies observed between the drivers were (Buniello et al., 2019). Nevertheless, how to use GWAS results to due to temporal expression differences. To test our hypothesis, we recognize the specific targets of these associations and for used the driver NP1029-Gal4 that conferred larval/early pupal- understanding the biology of disease remains a major challenge. specific gene silencing (Monier et al., 2005). CNOT1/Not1 Progress in this area will enable the future improvement of knockdown using NP1019-Gal4 was partially larval lethal and diagnostics and personalized therapy. Here, we started with QT completely pupal lethal at 25°C, whereas CNOT7/8/Pop2 interval-associating variants in the CNOT1 gene, an integral knockdown flies did eclose. Analysis of 1-week-old fly hearts component of the CCR4-NOT complex, and expanded on this upon larval/early pupal CNOT7/8/Pop2 knockdown revealed observation to include an investigation of additional complex significant dilation and reduction in fractional shortening and subunits. A combined approach using hiPSC-CMs and Drosophila normal heart period (Fig. 6A-D), similar to the results obtained with enabled a human and whole-organ assessment of cardiac the Hand-Gal4 driver. Although we did not test for a cardiac physiology. phenotype at early pupal stages, it is unlikely to be manifest We examined the functionality of human variants associated with similarly at adult stages, because most of the larval heart will QT interval in the CNOT1 promoter region and determined that the undergo histolysis and more anteriorly located portions of the larval alleles of variants that significantly associate with increases in QT aorta will metamorphose during later pupal stages to become the interval are capable of lowering reporter gene expression, which adult heart (see Monier et al., 2005). Thus, the observed adult heart might also reflect reduced transcription of CNOT1. We do note, phenotype is expected to be established during later pupal stages, however, that the human SNPs tested in vitro are located in the although CNOT7/8/Pop2 function is already required during larval/ CNOT1 promoter region, and that mutation of the actual gene might early pupal stages and perhaps even in the embryo. have different consequences in humans as compared with altered To test this idea, we further explored the developmental gene expression in cardiac tissue alone. As CNOT1 is essential for requirements by knocking down CNOT7/8/Pop2 in the cardiac the function of the CCR4-NOT complex (Ito et al., 2011), we mesoderm during early embryonal stages using the driver line Tin- expanded our study to include other subunits to assess the range of D-Gal4 (Reim and Frasch, 2005). This knockdown did not have any potential functional differences. We found that knockdown of not significant effects, except for a prolonged heart period in CNOT7/8/ only CNOT1, but also CNOT2, CNOT3, CNOT6, CNOT6L and Pop2 knockdown flies compared with control (Fig. 6E-H). The CNOT7, decreased proliferation of hiPSC-CMs, and that converse experiment was also performed, which was to knockdown knockdown of CNOT7 also caused significant APD shortening. CNOT7/8/Pop2 in adult flies with the Hand-Gal4 Gene Switch Knockdown of CNOT8 did not affect CM proliferation, which system (Monnier et al., 2012), which activates RNAi-mediated might be due to compensation by CNOT7 (Fig. 2). silencing in the myocardial and pericardial cells only when induced Consistent with our observations in hiSPCs, in vivo findings with RU-486. Adult flies were placed in food vials containing RU- show that CNOT1/Not1 knockdown flies exhibit dilated hearts with 486 at eclosion and were analyzed at 1 week of age. Knockdown of reduced contractile ability and severe structural defects, similar to CNOT7/8/Pop2 in adult flies had no statistically significant effect the myofibrillar reduction and cardiomyocyte death observed in on any of the cardiac parameters measured (Fig. 6I-L). CNOT1 and CNOT3 muscle-specific knockout mice (Yamaguchi Taken together, our data strongly suggest that the CCR4-NOT et al., 2018). Importantly, silencing of CNOT7/8/Pop2 resulted in complex, in particular CNOT1/Not1 and CNOT7/8/Pop2, along cardiac damage similar to CNOT1/Not1 knockdown, and Disease Models & Mechanisms

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Fig. 6. RNAi-mediated knockdown of CNOT7/8/Pop2 (TRiP HM05235) during developmental stages and in adult fly hearts. (A-D) RNAi-mediated knockdown of CNOT7/8/Pop2 using the larval stage heart- specific driver NP1029-Gal4 (n=19 female flies per genotype). Reduced CNOT7/8/Pop2 expression increased diastolic and systolic diameters and reduced fractional shortening. (E-H) RNAi-mediated knockdown of CNOT7/ 8/Pop2 using embryonal stage heart-specific driver TinD-Gal4 extended heart period, but had no effect on heart diameters and contractility (n=19 female flies per genotype). Student’s t-test was used to calculate two- sided P-values in A-D and E-H. *P≤0.05, **P≤0.01, ****P≤0.0001. (I-L) RNAi-mediated knockdown of CNOT7/8/Pop2 using the RU- 486 inducible Hand-Gal4 Gene Switch driver. Reduced expression of CNOT7/8/Pop2 in adult fly hearts had no effect on heart diameters or contractility (n=12 female flies per condition and genotype). Two-way ANOVA was used to calculate P-values. Boxes, interquartile range; central line, median; plus, mean; whiskers, upper and lower adjacent values as defined by Tukey (1977); dots, outside values.

electrophysiological recordings demonstrated extended event Notably, in our hands, dilated cardiomyopathy (DCM) resulted as duration and multiple peaks per burst, which is indicative of a the main phenotype produced by Hand-Gal4-driven knockdown of propensity for arrhythmias. Muscle tissue defects and electrical CNOT/Not1 in the Drosophila heart. Unfortunately, transthoracic activity have been linked in mouse and humans (Chinchilla and echocardiography, the first-line imaging test in the assessment of Franco, 2006). Moreover, mutations in seizure [the Drosophila ventricular dilation (Mathew et al., 2017), was not performed in the homolog of the human K+ channel gene hERG (also known as individuals from whom we isolated the natural variants of the KCNH2), which is important in cardiac repolarization] not only CNOT1 promoter; thus, we could not evaluate structural alterations cause bradycardia and arrhythmia, but also structural defects such as in those individuals. However, genetic-based forms of long-QT myofibrillar disorganization (Ocorr et al., 2017). Silencing of have been associated with the development of DCM. An overlap CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin subunits led between DCM and long-QT3 resulting from abnormalities of the overall to similar, albeit weaker, phenotypes limited to increased sodium channel gene SCN5A have been described in multiple diastolic and systolic diameters. Importantly, the role of CNOT reports (Kwon et al., 2012; Shi et al., 2008), but there is also subunits in the action potential repolarization phase was evidence for an association between long-QT1 and idiopathic DCM demonstrated not only in the Drosophila heart model (Ocorr (Allen et al., 2016). In addition, it has been reported that patients et al., 2017), but also in humans (Tse et al., 2017). with both severe or mild forms of cardiomyopathies, such as DCM Disease Models & Mechanisms

8 RESEARCH ARTICLE Disease Models & Mechanisms (2020) 13, dmm044727. doi:10.1242/dmm.044727 or hypertrophic cardiomyopathy, can show QT prolongation Generation of hiPSC-CMs (Johnson et al., 2011; Jouven et al., 2002; Ryerson and Giuffre, hiPSC-CMs were dissociated with 0.5 mM EDTA in PBS without CaCl2 2006). Of note, the role of the causative mutation in the overlap and MgCl2 (Corning) for 7 min at room temperature (RT), resuspended in mTeSR-1 medium (StemCell Technologies) with 2 µM thiazovivin between channelopathies and cardiomyopathies is not fully 5 understood, and the role of possible new players acting as (StemCell Technologies) and 3×10 cells/well were plated in a Matrigel- coated 12-well plate. At 24 h after passage, cells were fed daily with phenotype modifiers, like the CCR4-NOT complex, has yet to be mTeSR-1 medium (without thiazovivin) for 3-5 days until ≥90% determined. confluence. hiPSC-CMs were differentiated as previously described When using a cardiac driver for gene knockdown that excluded (Burridge et al., 2015). On day 0, WNT signaling was activated by the larval and early pupal stages of fly heart development (TinCd4- adding 6 µM CHIR99021 (Selleck Chemicals) in S12 medium (Pei et al., Gal4), we failed to observe a strong requirement for CNOT1/Not1 2017) for 48 h. On day 2, cells were treated with 2 µM Wnt-C59 (Selleck and CNOT7/8/Pop2. In contrast, when using a driver that was Chemicals) in S12 to inhibit WNT. On day 4, S12 medium was fully restricted specifically to larval and early pupal stages (NP1029- changed. On day 5, cells were dissociated with TrypLE Express (Gibco) for Gal4), CNOT1/Not1 knockdown was lethal; CNOT7/8/Pop2 4 min and blocked with RPMI (Gibco)+10% fetal bovine serum (FBS; silencing at this stage of development resulted in dilation and Omega Scientific). Cells were resuspended in S12 supplemented with 4 mg/ reduced contractility, similar to knockdown exerted throughout life. l recombinant human insulin (Gibco) (S12+ medium) and 2 µM thiazovivin and 9×105 cells/well were plated in a Matrigel-coated 12-well plate. S12+ An embryonic or adult-only driver had no effect on cardiac outcome medium was changed on day 8 and replaced on day 10 by RPMI for either gene. These findings suggest that CCR4-NOT function is (Gibco)+213 µg/µl L-ascorbic acid (Sigma-Aldrich), 500 mg/l BSA-FV crucial during cardiac remodeling from the larval to the adult heart. (Gibco), 0.5 mM L-carnitine (Sigma-Aldrich) and 8 g/l AlbuMAX lipid- It is, however, also possible that knockdown in adult flies would rich BSA (Gibco) (CM medium). Under these conditions, hiPSC-CMs start have an effect under stress conditions. to beat around day 9-10. On day 15, cells were purified with lactate medium, Considering the effects on hiPSC-CM proliferation and the consisting of RPMI without glucose, 213 µg/µl L-ascorbic acid, 500 mg/l developmental defects observed by silencing CNOT1/Not1 and BSA-FV and 8 mM sodium-DL-lactate (Sigma-Aldrich) (Burridge et al., CNOT7/8/Pop2 in Drosophila, we speculate that the CCR4-NOT- 2015; Tohyama et al., 2016), for 4-5 days and was replaced by CM medium encoding genetic variants identified by GWAS in adult humans are until day 25. those that result in less severe consequences, as the lack of proper Proliferation assay in hiPSC-CMs mRNA regulation might be lethal at certain stages of embryogenesis. At day 25 of differentiation, hiPSC-CMs were dissociated with TrypLE Depletion of CNOT1 and the entire deadenylase module (CNOT6/6L, Select 10× (Gibco) for 12 min and neutralized with RPMI+10% FBS. Cells CNOT7/8), has been demonstrated to promote endoplasmic reticulum were resuspended in RPMI with 2% KnockOut Serum Replacement (ER) stress and apoptosis in vitro (Ito et al., 2011). In turn, it has also (KOSR; Gibco) and 2% B27 50× with vitamin A (Life Technologies) been shown that activation of the unfolded protein response impairs supplemented with 2 µM thiazovivin and plated at a density of 5000 cells/ cardiac ion channel biogenesis, leading to a prolongation of the APD well in a Matrigel-coated 384-well plate. hiPSC-CMs were transfected with (Liu et al., 2018). Taken together, these findings suggest that siRNA (Dharmacon) targeting siCNOT1 (L-015369-01), siCNOT2 disruption of CCR4-NOT complex function affects both structural (L020313-02), siCNOT3 (L-020319-00), siCNOT4 (L-020323-00), (i.e. decreased CM proliferation, myofibrillar structural abnormalities) siNOT6 (L-019101-00), siNOT6L (L-016411-00), siCNOT7 (L-012897- and electrophysiological (i.e. shortened APD, decreased contractility 00) and siCNOT8 (L-018791-00), using lipofectamine RNAiMax (Thermo Fisher Scientific). Each siRNA was tested in quadruplicate. Cells were in flies) components of the heart. Whether knockdown of a specific labeled with 10 µM EdU (Thermo Fisher Scientific) 48 h post-transfection. subunit produces one or both of these phenotypes might be influenced After 24 h of EdU incubation, cells were fixed with 4% paraformaldehyde by silencing efficiency; for example, less ER stress might lead to for 30 min. EdU was detected according to protocol and cells were stained electrical remodeling, whereas more might result in apoptosis. In with the cardiac-specific marker ACTN2 (Sigma-Aldrich, dilution 1/800) addition, specific RNA-binding proteins that connect and guide and DAPI. Cells were imaged with an ImageXpress Micro XLS microscope CCR4-NOT to target-specific mRNAs are likely to have an influence. (Molecular Devices) and custom algorithms were used to quantify Collectively, our results show a prominent role of the deadenylase EdU-labeled hiPSC-CMs. module (CNOT7/8/Pop2) both in vitro and in vivo. The combined use of GWAS studies and cardiac model systems Voltage assay in hiPSC-CMs in this study has enabled us to connect CNOT1, CNOT7 and, overall, Voltage assay was performed as described in McKeithan et al. (2017). At CCR4-NOT complex function to cellular and whole-heart day 25 of differentiation, hiPSC-CMs were dissociated with TrypLE Select phenotypes in the context of human heart disease. In this context, 10× for up to 12 min and neutralized with RPMI+10% FBS. Cells were resuspended in RPMI with 2% KOSR (Gibco), 2% B27 50× with vitamin A however, direct CCR4-NOT complex targets that influence heart (Life Technologies) and supplemented with 2 µM thiazovivin and plated at rhythm and physiology remain to be identified. Finally, strategies to 6000 cells/well in a Matrigel-coated 384-well plate. hiPSC-CMs were modulate the expression of key components of the CCR4-NOT transfected with CCR4-NOT-NOT siRNAs as described above. Three days complex, or to stabilize its function, might be promising avenues for post-transfection, cells were washed five times with pre-warmed Tyrode’s regulating QT interval and preventing pro-arrhythmogenic solution (Sigma-Aldrich) by removing 50 µl of medium and adding 50 µl. substrates, especially targeted to those individuals at increased After the fifth wash, 50 µl of 2× dye solution [voltage-sensitive dye Vf2.1 Cl risk owing to their genetic background. (Fluovolt, 1:4000, Thermo Fisher Scientific) diluted in Tyrode’s solution with 1 µl of 10% pluronic F127 (in water, Thermo Fisher Scientific) and 20 µg/ml Hoechst 33258 (in water, Thermo Fisher Scientific)] was added to MATERIALS AND METHODS each well. The plate was returned to an incubator at 37°C 5% CO2 for Ethics statement 45 min. After incubation, cells were washed four times with pre-warmed The DNA for promoter isolation was drawn from individual participants in Tyrode’s solution. hiPSC-CMs were imaged with an ImageXpress Micro the MICROS study in South Tyrol (Pattaro et al., 2007). MICROS was XLS microscope at 100 Hz for 5 s, with excitation wavelength at 485/20 nm approved by the Ethics Committee of the Autonomous Province of Bolzano and emission filter 525/30 nm. A single image of Hoechst was acquired (Südtiroler Sanitätsbetrieb/Azienda Sanitaria dell’Alto Adige). Each before the time series. Fluorescence over time quantification and trace participant gave written informed consent. analysis were automatically quantified using custom software packages Disease Models & Mechanisms

9 RESEARCH ARTICLE Disease Models & Mechanisms (2020) 13, dmm044727. doi:10.1242/dmm.044727 developed by Molecular Devices and the Colas Laboratory. Three in ethanol was added to 10 ml fly food). Control food contained an equal independent experiments were performed, each condition in quadruplicate. amount of ethanol. Flies were aged to 1 week at 25°C, at which point they were dissected and analyzed. Cell culture HL-1 mouse atrial cardiomyocytes (Claycomb et al., 1998) were kindly Fly heart dissection donated by William Claycomb (Louisiana State University, New Orleans, Flies were anesthetized with FlyNap and dissected in artificial hemolymph LA, USA) and cultured in Claycomb medium (Sigma-Aldrich) according to a previously described protocol (Vogler and Ocorr, 2009). supplemented with 10% FBS, 4 mM L-glutamine, 100 U/ml penicillin, The procedure includes removing the fly head, intestines and some fat, 100 mg/ml streptomycin, 0.3 mM ascorbic acid and 10 mM norepinephrine, resulting in a semi-intact preparation that visualizes the beating heart. as previously described (Meraviglia et al., 2015). Artificial hemolymph was re-oxygenated for 20 min post-dissection HeLa and 293T cells were cultured in Dulbecco’s modified Eagle medium allowing the hearts to stabilize before video recording. (DMEM), GlutaMAX supplement (Thermo Fisher Scientific), supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Thermo High-speed digital video imaging and analysis Fisher Scientific). All cells were maintained at 37°C in a saturated humidity All fly hearts were filmed with an EM-CDD Hamamatsu digital camera, atmosphere containing 5% CO2. using a Leica DMFLSA microscope equipped with a 10× dipping lens. Recordings of each fly heart were made for 30 s with a camera speed of Luciferase assay 120-140 frames per second (Ocorr et al., 2007b). M-modes describing fly HL1, HeLa and T293 cells were seeded at 30,000, 60,000 and 150,000 cells/ heart contractions were created by semi-automated optical heartbeat analysis well, respectively, into 24-well plates (Corning). A total of 24 h after seeding, (SOHA; Fink et al., 2009). 10 ng of the reporter plasmid pG4.74[hRluc/TK] was co-transfected with 10 ng of either pGL4.10 vector (complete or minimal for both haplotypes) or Fluorescence staining and imaging negative control vector (pG4.13 [luc2/SV40]). Transfection was performed According to a previously described protocol (Alayari et al., 2009), flies were with the lipofectamine plus reagent (Invitrogen), according to the dissected in artificial hemolymph and hearts relaxed with 10 mM EGTA manufacturer’s protocol. Cells were washed with PBS 48 h post- before fixation with formaldehyde. Flies were stained with phalloidin, Alexa transfection and lysed with 100 μl of passive lysis buffer (Promega) for Fluor 488 to visualize F-actin. Apotome images were taken with a Zeiss Axio 15 min at RT. Cell lysates were immediately used to measure luciferase Imager.Z1 microscope at 10× and 25× magnification. Images were processed activity, using the Dual Luciferase Reporter Assay System Kit (Promega). with Adobe Photoshop. Each lysate (20 μl) was incubated with 100 μl of luciferase assay reagent II (LAR II). Firefly luminescence was measured for 10 s using a luminometer Statistical analysis μ (Victor X3-2030, Perkin Elmer). After 2 s, 100 l of Dual-Glo Stop and Glo To determine any statistical significance between experimental and control Reagent was added to each well. Subsequently, renilla luminescence was groups in hiPSC-CMs and Drosophila experiments, we calculated two- measured for 10 s using the same luminometer. Luciferase activity was sided P-values with Student’s t-test, or one-way or two-way ANOVA with calculated based on the ratio of the activities of firefly and renilla luciferases. Tukey’s multiple comparisons test, using GraphPad Prism software (2016). At least three independent experiments were performed in triplicate. We analyzed CNOT1 expression data from HL-1, HeLa and T293 cell lines with a two-sided non-parametric Wilcoxon rank-sum test, using Stata 13 Fly stocks (StataCorp. 2013; Stata Statistical Software Release 13; StataCorp, College All transgenic RNAi fly lines were purchased from Vienna Drosophila Station, TX, USA). Population distribution of control and siCNOT7- RNAi Center (VDRC) and from Bloomington Drosophila Stock Center transfected hiPSC-CMs was generated with GraphPad Prism using (BDSC) at Indiana University (Transgenic RNAi Project at Harvard nonlinear regression. Unpaired nonparametric Kolmogorov–Smirnov test Medical School, TRiP). VDRC IDs: Not1 (GD12571 and KK106587), Not2 was used to compare each treated condition with controls using APD75 of (GD20826), Pop2 (GD28396) and twin (GD13365). TRiP/BDSC IDs: Not1 every measured cell. (28681), Not4 (JF03203), Pop2 (HM05235) and twin (HMS00493). Control 1118 flies with corresponding genetic background: VDRC w (GD RNAi Electrophysiology of adult hearts library) and TRiP-fly line with attP2 docking site. Cardiac-specific drivers Semi-intact heart preparations were incubated in artificial hemolymph Δ were kind gifts from the following investigators: Manfred Frasch, TinC 4 containing 10 µM blebbistatin (Sigma-Aldrich) and left in the dark with 12a-Gal4 (Lo and Frasch, 2001) and tin-D-Gal4 (Reim and Frasch, 2005); oxygenation until the hearts stopped. Fresh saline without blebbistatin was Eric Olsen, Hand-Gal4 (Han and Olson, 2005); Laurent Perrin, NP1029-Gal4 added and electrical potentials were recorded from the conical chamber using (Monier et al., 2005) and Hand-Gal4 Gene Switch (Monnier et al., 2012). glass electrodes (20-50 MΩ) filled with 3 M KCl. Data were acquired using an Axon-700B amplifier, signals were digitized using the DIGIDATA 1322A Fly medium and data were captured and analyzed using PClamp 9.0 and Clampfit 10.0 Ingredients of fly medium comprised cornmeal (7.0%), malt (5.2%), software from Molecular Devices. Data were quantified from representative molasses (5.2%), soy flour (1.7%), agar (0.4%) and autolyzed yeast (2.1%). 30 s recordings where the resting membrane potential had remained stable. All ingredients were mixed with water and cooked for 15 min without boiling. Preservatives, Tegosept in ethanol (1.8%) and propionic acid Competing interests (2.1%) were added once the batter cooled to <65°C. All percentages refer to The authors declare no competing or financial interests. the final concentration. Dry ingredients, weight/total volume batter; liquid ingredients, volume/total volume batter. Author contributions Methodology: K.O.; Software: S.K., K.O.; Formal analysis: L.E., C.B.V., A.K., S.P., A.C.; Investigation: L.E., C.B.V., A.K., S.P., S.K., N.N.A., L.F., P.P.P., A.R., A.C., Fly crosses A.A.H.; Writing - original draft: L.E., C.B.V., A.K., A.R., A.C., A.R.C., A.A.H., R.B.; Driver-line virgins were crossed to RNAi males and corresponding isogenic Writing - review & editing: L.E., A.C., A.R.C., A.A.H., R.B.; Visualization: L.E., C.B.V., control males. Flies were raised on standard fly food and kept at 25°C or A.K., S.P., N.N.A., L.F., A.R., A.A.H.; Project administration: R.B.; Funding 18°C. Female progeny was collected and aged to 1 week at 25°C, at which acquisition: A.A.H., R.B. point they were imaged and analyzed. Funding This study was supported by the National Institutes of Health (R01-HL054732 to Hand-Gal4 gene-switch knockdown R.B.; R01-HL124091 to A.C.; R01-HL132241 to K.O.), the Wanek Foundation (R.B. Eclosed female progeny were collected and placed in vials with fly food and A.C.) and the Department of Innovation, Research and Universities of the containing 100 µg/ml RU-486 (40 µl of 25 mg/ml stock RU-486 dissolved Autonomous Province of Bolzano-South Tyrol (Italy) (C.B.V., L.F., A.R. and A.A.H.). Disease Models & Mechanisms

10 RESEARCH ARTICLE Disease Models & Mechanisms (2020) 13, dmm044727. doi:10.1242/dmm.044727

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