© 2020. Published by The Company of Biologists Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

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in any medium provided that the original work is properly attributed.

Silencing of CCR4-NOT complex subunits affect heart structure and

function

Lisa Elmén1 PhD, Claudia B. Volpato2 PhD, Anaïs Kervadec1 PhD, Santiago Pineda1,# PhD,

Sreehari Kalvakuri1, PhD, Nakissa N. Alayari1+ MS, Luisa Foco2 PhD, Peter P. Pramstaller2

MD, Karen Ocorr1, PhD, Alessandra Rossini2 PhD, Anthony Cammarato3 PhD, Alexandre R.

Colas1 PhD, Andrew A. Hicks2* PhD, Rolf Bodmer1,* PhD

1 Development Aging and Regeneration Program, Sanford Burnham Prebys Medical

Discovery Institute, 10901 N Torrey Pines Rd, La Jolla, CA 92037, USA.

2 Institute for Biomedicine, Eurac Research, Affiliated Institute of the University of Lübeck,

Bolzano, Italy

3 Johns Hopkins University, Division of Cardiology, 720 Rutland Ave., Baltimore, MD 21205,

USA.

# Present address: University of California Los Angeles, Molecular, Cell, and Developmental

Biology, Terasaki Life Sciences Building, 610 Charles E. Young Dr. South, CA 90095, USA.

+ Present address: Illumina Inc., 5200 Illumina Way, San Diego CA 92122, USA.

* Corresponding authors: Rolf Bodmer, PhD, Development Aging and Regeneration

Program, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines

Road, La Jolla, CA 92037. Telephone: (858) 795-5295. E-mail: rolf@sbpdiscovery.org.

Andrew A. Hicks, PhD, Institute for Biomedicine, Eurac Research, Via Luigi Galvani 31,

Bolzano, 39100, Italy. Telephone +39 3347788721. E-mail: andrew.hicks@eurac.edu.

Summary statement

This work demonstrates the successful approach of combining GWAS studies with in vitro

human cell assays and a suitable in vivo model organism, and clearly connects CNOT1,

CNOT7 and overall, the CCR4-NOT complex, to heart morbidity.

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.044727Access the most recent version at First posted online on 29 May 2020 as 10.1242/dmm.044727

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.

Key words: CNOT1, GWAS, arrhythmia, long QT-syndrome, Drosophila heart, human iPSC,

cardiomyocytes.

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INTRODUCTION

Despite the medical advances that have been made over the last decades,

cardiovascular disease remains the most common cause of mortality worldwide (WHO, 2018).

Understanding the mechanisms of heart morbidity is crucial for finding new therapies, and

determining which genetic variants predispose individuals to heart disease is necessary to

provide better preventative care. The challenge of connecting human genetic variants with

disease can be met by combining Genome Wide Association Studies (GWAS) with patient

sequencing and validation using disease-in-a-dish and in vivo cardiac model systems.

Drosophila melanogaster benefits from well-conserved genes and permits functional

assessment of genes of interest, which when manipulated may not be well-tolerated by the

vertebrate heart.

The QT interval on an electrocardiogram is a measure that reflects myocardial

repolarization. Short-QT syndrome (Rudic et al., 2014) and Long-QT syndrome (Amin et al.,

2013) are caused by different underlying mechanisms but are both risk factors for atrial and

ventricular arrhythmias and sudden cardiac death (Rudic et al., 2014, Amin et al., 2013,

Vacanti et al., 2017). Genome-wide association in up to 100,000 individuals has successfully

identified at least 35 common variant QT interval loci that collectively explain ∼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 (CNOT1) of the CCR4-

NOT complex. CCR4-NOT is conserved throughout the eukaryotic kingdoms and is involved

in the sequential processes of gene expression. Its activities can be divided in functional

modules involved in transcription (Kruk et al., 2011) (CNOT2, CNOT3), mRNA-degradation

(Bhandari et al., 2014, Yi et al., 2018, Temme et al., 2010, Webster et al., 2018), deadenylation

(CNOT6/6L, CNOT7/8) and protein quality control via ubiquitination (CNOT4) (Halter et al.,

2014, Collart, 2016, Collart and Panasenko, 2017).

The CCR4-NOT complex has previously been implicated in heart disease; we have

demonstrated that silencing of genes UBC4 and not3 cause cardiac dilation and dysfunction

in Drosophila (Neely et al., 2010). In addition, CNOT3 heterozygous knockout mouse hearts

displayed reduced contractility and increased susceptibility to failure following aortic

constriction (Neely et al., 2010). CNOT3 has also been found to interact with Atg7, which

affects cardiomyocyte (CM) survival and QT intervals in mice (Yamaguchi et al., 2018). In the

present study we investigated the individual role of additional CCR4-NOT complex subunits,

starting with positively QT-interval-associating variants in the CNOT1 putative promoter

region, to determine whether and in what direction they functionally influence reporter gene

expression. We further explored the effects of RNAi-mediated knockdown of CNOT1 and

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complex subunit genes CNOT2, CNOT4, CNOT6/6L and CNOT7/8 in human induced

pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) (Cunningham et al., 2017, Yu et

al., 2018) on proliferation and electrophysiological properties, in particular action potential

duration (APD), (McKeithan et al., 2017), and in vivo, on cardiac structure and contractile

function using the Drosophila heart model (Ocorr et al., 2014). Overall, we find that silencing

CNOT1 and other CCR4-NOT components compromise cardiac development and function in

the two model systems, suggesting an important role of this complex in cardiac health and

disease.

RESULTS

Functional validation of CNOT1 promoter polymorphisms

Three of the strongest QT-interval associating variants (SNPs), in strong linkage

disequilibrium (LD) over CNOT1, are located in the putative promoter region of the gene (Fig.

1B), redrawn from Fig. S2 in Arking et al. (Arking et al., 2014)). All GWAS associated variants

over the whole length of this gene are in strong LD with the putative promoter variants, and

the GTEx data (https://doi.org/10.1089/bio.2015.0032) indicates strong tissue specific eQTL

variants over the whole gene, leading us to postulate that the promoter variants would

demonstrate functionally different expression levels. A fragment spanning all three of these

strongly QT-associated variants was confirmed by sequencing to contain four SNPs in total

(rs27097, rs37037, rs9941290 and rs863433, all associating strongly with QT interval, apart

from rs37037 which is not in strong linkage disequilibrium with the other three variants, but still

associated although with a less significant p value, Figure S1C). We identified two human

subjects homozygous for alleles at the four SNPs that fall within ~3.2 kb of the 5’ region of the

CNOT1 coding sequence (Fig. 1C). The two haplotypes (one with risk alleles and one with

alternate alleles) from the putative CNOT1 promoter region were isolated and cloned in two

forms into a plasmid vector (pGL4.1) to drive the firefly luciferase gene (Fig. 1D). The “minimal”

putative promoter region contained 657bp of the region around just 1 SNP (rs27097), located

closest to the start codon of the CNOT1 gene. The larger 3172bp fragment contained all of

the strongest QT-associating variants, potentially capturing the “complete” promoter region of

CNOT1 (with respect to the significantly associated variants in this region of the gene). Both

constructs were sequenced to confirm that the four variant positions under study were the only

ones differing between these natural promoter regions, and that the alleles were homozygous.

For both the “minimal” and “complete” constructs, one haplotype consisted of alleles that

significantly associate with increases in QT interval length (“TGAG” haplotype), while the other

haplotype consisted of the alternate alleles at these variants (“GAGT” haplotype) (see also

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Fig. S1C) (Pfeufer et al., 2009). The “minimal” promoter constructs consisted of alleles T and

G respectively at rs27097 (Fig. 1C).

The “complete” and “minimal” promoter-luciferase constructs were transfected into HL-

1 cells, a cardiac muscle cell line that contracts and retains phenotypic characteristics of adult

CM, along with renilla luciferase in order to normalize signals. Both “minimal” and “complete”

promoter constructs were able to drive increased luciferase expression in the HL-1 cells with

significant differences between the two haplotypes. When considering the SNP closest to the

gene alone (“minimal” construct), expression was effectively silenced in the HL-1 cell line for

the haplotype that was associating with longer QT intervals (Fig. 1E). These differences were

also observed in both HeLa and T293 cells (Fig. S1A, B). With the larger promoter fragment,

expression was re-established in all lines, but was significantly reduced for the haplotype

associated with a longer QT compared with the haplotype carrying the alternate alleles. From

these experiments we conclude that the variants which significantly associate with QT interval

in human GWAS, are indeed functionally able to alter expression of the CNOT1 gene in

cardiac tissue, with the most significant differences for the “complete” haplotype being seen

in the cardiac cell line, and that the direction of the effect is such that reduced CNOT1 possibly

contributes to QT interval prolongation.

CCR4-NOT complex genes regulate proliferation and action potential duration in human

iPSC-derived cardiomyocytes (hiPSC-CMs)

CCR4-NOT is a multisubunit complex with different functional modules (including, but

not limited to, the subunits in this study) (Fig. 1A). It has been shown in HeLa and HEK293T

cells that siRNA depletion of CNOT1 decreases the amounts CCR4-NOT subunits and

reduces the complex’ deadenylase activity, and that simultaneous siRNA silencing of the

entire deadenylase module (CNOT6/6L, CNOT7/8) result in apoptosis similar of that of CNOT1

silencing alone (Ito et al., 2011). Therefore, we next decided to use hiPSC-CMs to investigate

the effect of silencing CNOT6/6L and CNOT7/8, genes encoding enzymes that affect

translation efficiency by removing mRNA poly(A) tails (Yi et al., 2018), and CNOT4/not4,

encoding a RING E3-ligase, important for assembly of the proteasome and proposed to be

involved in co-translational quality control (Halter et al., 2014). We also chose to examine

CNOT2, as it associates with CNOT3, for which we previously had identified a role in cardiac

function (Neely et al., 2010).

First, to evaluate a potential role of CCR4-NOT complex on human cardiac physiology,

we knocked down each of the CNOT genes and evaluated their effect on human iPSC-derived

CM (hiPSC-CM) proliferation. Individual knockdown of CNOT1, CNOT2, CNOT3, CNOT4 and

CNOT6 led to decreased EdU incorporation in day 25 hiPSC-CMs and reduced CM number

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as compared to siControl (Fig. 2A-D), thereby suggesting a general role of the CCR4-NOT

complex in the regulation of proliferation in CMs.

A number of studies (Itzhaki et al., 2011, Matsa et al., 2011, Lahti et al., 2012) have

shown that hiPSC-CMs from patients with long QT syndromes consistently show prolonged

action potential duration (APD) phenotypes, thus suggesting that APD modulation in hiPSC-

CMs represent a reliable model system to evaluate the role of candidate genes for QT interval

modulation. Therefore, we asked whether the CCR4-NOT complex could also play such a role

and transfected day 25 hiPSC-CMs with siRNAs directed against each member of the complex

and APD parameters using a fluorescence-based single cell and high-throughput voltage

transient recording assay, based on (McKeithan et al., 2017) (Fig. 2E). Interestingly, we found

that CNOT7, but not CNOT1, knockdown led to a significant shortening (>20ms) of APD (Fig.

2F-I). Although it is possible that the level of CNOT1 knockdown was insufficient to cause a

change in APD, it did produce a proliferation deficit which indicates that the siRNAs were

transfected and active in hiPSC-CMs. This suggests a potential new role for CNOT7 and

deadenylation in the regulation of the QT interval in humans, an observation supported by the

suggestive association of a variant (rs183286310, p = 1.1x10-6) near the CNOT7 gene with

QT interval in ~5000 individuals within the CHRIS study (Pattaro et al., 2015).

Cardiac-specific in vivo knockdown of CNOT1/not1 and CNOT7/8/pop2 in Drosophila

results in dilated cardiomyopathy

Since experiments with hiPSC-CMs provided evidence that CNOT1 and CNOT7

regulate CM proliferation and APD respectively, we asked the question how the same

manipulations would affect the heart in vivo. The CNOT genes are conserved in Drosophila

and have the following orthologs to the human CNOT genes: CNOT2/not2, CNOT4/not4,

CNOT6 and CNOT6L/twin, CNOT7 and CNOT8/pop2 (Table 1).

Using the Drosophila UAS–Gal4 system (Brand and Perrimon, 1993) we silenced

CNOT1/not1 with Hand-Gal4, a driver specific for myocardial and pericardial cells of the heart,

acting throughout development (Han and Olson, 2005). At one-week post-eclosion (young

adult flies), we dissected the animals to expose the heart for video recording as previously

described (Ocorr et al., 2007a, Ocorr et al., 2014, Fink et al., 2009) (Fig. 3A). The Hand-Gal4-

driven CNOT1/not1 knockdown hearts exhibited normal beating frequency (shown as heart

period, Fig. 3B). However, these hearts were fragile and exhibited diastolic and systolic

diameters that were significantly larger compared to controls (Fig. 3C, D), which resulted in a

substantial decrease in fractional shortening (Fig. 3E), thus exhibiting reduced capacity for the

heart to contract. Fluorescent staining of actin revealed an abnormal myofibrillar structure with

large gaps and disarray in CNOT1/not1 knockdown fly hearts, as compared to control hearts

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displaying typical tightly packed circumferential myofibrils (Fig. 3F, G). Knockdown with two

different CNOT1/not1 RNAi-lines (GD12571 and KK106587) resulted in similar phenotypes,

and knockdown with a third line, TRiP 28681 exhibited the same trend but was not statistically

significant from control fly hearts. The CNOT1/not1 RNAi-line VDRC GD12571 is shown in all

figures.

We had previously observed double beat EAD-associated arrhythmias in M-mode

traces from movies of CNOT3/not3 knockdown fly hearts (Neely et al., 2010), and therefore

an effort was made to record electrophysiology traces. However, this proved nearly impossible

due to the fragility of the CNOT1/not1 knockdown hearts. One successful recording did show

abnormal fibrillatory events, with increased event duration and number of peaks per burst (Fig.

S2A, B).

As the results of the hiPSC-CMs indicated the importance of the deadenylase CNOT7

for cardiac rhythm control, we next asked the question how knockdown of the Drosophila

ortholog CNOT7/8/pop2 would affect the fly heart. RNAi-mediated knockdown of

CNOT7/8/pop2, via the Hand-Gal4 driver line, resulted in pupal lethality at 25C. By lowering

the incubation temperature during development to 18C, and thereby reducing Gal4-

production, the flies did eclose. When one-week-old CNOT7/8/pop2 knockdown hearts were

functionally analyzed, they exhibited no change in heart period (Fig. 4A). However, significant

cardiac dilation, as measured by increased diastolic and systolic diameters (Fig. 4B, C), and

reduced contractility (Fig. 4D) were evident, consistent with the phenotype observed in

CNOT1/not1 knockdown fly hearts. Furthermore, fluorescent staining of CNOT7/8/pop2

knockdown hearts also revealed myofibrillar structure abnormalities (Fig. 4E, F), as seen in

CNOT1/not1 knockdown hearts (Fig. 3F, G). Electrophysiological recordings indicated that

cardiac-restricted silencing of CNOT7/pop2, as observed with the single CNOT1/not1

knockdown heart, triggered longer event durations and multiple peaks per burst (Fig. 4G-I)

compared to control hearts. While we cannot be certain that the single CNOT1/not1 recording

is representative, taken together with the CNOT7/8/pop2 electrophysiological recordings, we

find that this phenotype is consistent with a propensity for arrhythmia.

Hand-Gal4 driven RNAi-knockdown of CNOT2/not2, CNOT4/not4 and CNOT6/6L/twin

also caused cardiac dilation, as observed with the silencing of CNOT1/not1 and

CNOT7/8/pop2. Knockdown of CNOT2/not2, CNOT4/not4 and CNOT6/6L/twin induced a

significant increase in diastolic diameter (Fig. 5A), but only CNOT2/not2 and CNOT6/6L/twin

silencing also resulted in increased systolic diameters compared to controls (Fig. 5B). This

dilation did however not result in significantly diminished fractional shortening (Fig. 5C). The

results suggest that CNOT2/not2, CNOT4/not4 and CNOT6/6L/twin are needed for normal

heart dimensions in vivo, but their reduction did not significantly affect overall contractility.

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We repeated all RNAi-experiments with a second driver line, TinCΔ4-Gal4 (Lo and

Frasch, 2001), expressed in the myocardium during early development, during late pupal

stages of cardiac remodeling, and in the adult heart, but not during larval and early pupal

stages. Surprisingly, TinCΔ4-Gal4-mediated CNOT1/not1 knockdown did not cause a dilated

cardiac phenotype, except for a small increase in systolic heart diameter that resulted in

modestly decreased fractional shortening (Fig. S2C-E). TinCΔ4-Gal4 driven knockdown of

CNOT2/not2, CNOT4/not4 and CNOT6/6L /twin did not engender a cardiac phenotype

compared to controls, except for not4 knockdown, which resulted in substantially increased

diastolic and systolic diameters, but without an effect on fractional shortening (Fig. S3A-C).

Knockdown of CNOT1/not1 and CNOT7/8/pop2 during Drosophila development (larval

stages)

The finding that knockdown of CNOT1/not1 and CNOT7/8/pop2 with the pupal/adult

TinCΔ4-Gal4 myocardial driver (not expressed in larval hearts), did not recapitulate the results

obtained with the continuously expressed Hand-Gal4 heart driver, raised the question of

whether developmental expression in larvae/ early pupae was critical for normal adult heart

function. We therefore tested the hypothesis that the discrepancies observed between the

drivers were due to temporal expression differences. To test our hypothesis, we used the

driver NP1029-Gal4 that conferred larval/ early pupal-specific gene silencing (Monier et al.,

2005). CNOT1/not1 knockdown using NP1019-Gal4 was partially larval lethal, and completely

pupal lethal at 25C, while CNOT7/8/pop2-knockdown flies did eclose. Analysis of one-week-

old fly hearts upon larval/ early pupal CNOT7/8/pop2 knockdown revealed significant dilation

and reduction in fractional shortening and normal heart period (Fig. 6A-D), similar to the results

obtained with the Hand-Gal4 driver. Although we did not test for a cardiac phenotype at early

pupal stages, it is unlikely to be manifest similarly at adult stages, because most of the larval

heart will undergo histolysis and more anteriorly located portions of the larval aorta will

metamorphose during later pupal stages and become the adult heart (see Monier et al. 2005).

Thus, the observed adult heart phenotype is expected to be established during later pupal

stages but requiring CNOT7/8/pop2 function already during larval/early pupal stages, and

perhaps even in the embryo.

To test the latter, we further explored the developmental requirements by knocking

down CNOT7/8/pop2 in the cardiac mesoderm during early embryonal stages using the driver

line Tin-D-Gal4 (Reim and Frasch, 2005). This did not have any significant effects, except for

a prolonged heart period in CNOT7/8/pop2-knockdown flies compared to control (Fig. 6E-H).

The converse experiment was also performed, which was to knockdown of CNOT7/8/pop2 in

adult flies with the Hand-Gal4 Gene Switch system (Monnier et al., 2012) that activates RNAi-

mediated silencing in the myocardial and pericardial cells only when induced with RU-486.

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Adult flies were placed in food vials containing RU-486 at eclosion and were analyzed at one

week of age. Knockdown of CNOT7/8/pop2 in adult flies had no statistically significant effect

on any of the cardiac parameters measured (Fig. 6I-L).

Taken together, our data strongly suggest that the CCR4-NOT complex, in particular

CNOT1/not1 and CNOT7/8/pop2, along with CNOT3 (Neely et al., 2010, Yamaguchi et al.,

2018), are required during larval and/or early pupal stages of development, and thus during

the initial stages of cardiac remodeling from the larval to adult heart.

DISCUSSION

GWAS studies have successfully identified many genetic loci associated with multiple

disorders, including cardiovascular disease (Buniello et al., 2019). However, how to use

GWAS results, for recognizing the specific targets of these associations and for understanding

the biology of disease, is a major challenge in order to make progress towards diagnostics

improvement and personalized therapy. Here, we started with QT interval associating variants

in the CNOT1 gene, an integral component of the CCR4-NOT complex, and expanded on this

observation to include an investigation of additional complex subunits, with the combined

approach of using hiPSC-CMs and Drosophila, enabling a human and whole organ

assessment of cardiac physiology.

We examined the functionality of human QT associating variants in the CNOT1

promoter region and determined that the alleles of variants that significantly associate with

increases in QT interval are capable of lowering reporter gene expression, which may reflect

reduced transcription of CNOT1 as well. We do note, however, that the human SNPs tested

in vitro are located in the CNOT1 promoter region, and that mutation of the actual gene may

have different consequences in humans as compared with altered gene expression in cardiac

tissue alone. Since CNOT1 is essential for the function of the CCR4-NOT complex (Ito et al.,

2011), we expanded our study to include other subunits, to assess the range of potential

functional differences. We found that knockdown of not only CNOT1, but also CNOT2,

CNOT3, CNOT6, CNOT6L and CNOT7 decreased proliferation of hiPSC-CMs, and that

knockdown of CNOT7 also caused significant APD shortening. Knockdown of CNOT8 did not

change CM proliferation, which may be due to compensation by CNOT7 (Fig. 2).

Consistent with our observations in hiSPCs, in vivo findings also show that

CNOT1/not1 KD flies exhibit dilated hearts with reduced contractile ability, and severe

structural defects, similar to the myofibrillar reduction and cardiomyocyte death observed in

CNOT1 and CNOT3 muscle-specific knockout mice (Yamaguchi et al., 2018). Importantly,

silencing of CNOT7/8/pop2 resulted in cardiac damage similar to CNOT1/not1 knockdown,

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and electrophysiological recordings demonstrated extended event duration and multiple peaks

per burst, which is indicative of a propensity for arrhythmias. Muscle tissue defects and

electrical activity have been linked in mouse and humans (Chinchilla and Franco, 2006).

Moreover, mutations in seizure, the Drosophila homolog of the human K+-channel hERG, both

important in cardiac repolarization, not only cause bradycardia and arrhythmia, but also

structural defects, such as myofibrillar disorganization (Ocorr et al., 2017). Silencing of

CNOT2/not2, CNOT4/not4 and CNOT6/6L/twin subunits led overall to similar, albeit weaker

phenotypes, limited to increased diastolic and systolic diameters. Importantly, the role of

CNOT subunits in the action potential (AP) repolarization phase was demonstrated not only

in the Drosophila heart model (Ocorr et al., 2017), but also in humans (Tse et al., 2017).

Notably, in our hands, dilated cardiomyopathy resulted as the main phenotype

produced by Hand-Gal4 driven knockdown of CNOT/not1 in the Drosophila heart.

Unfortunately, transthoracic echocardiography (TTE), the first-line imaging test in the

assessment of ventricular dilation (Mathew et al., 2017), was not performed in the individuals

from which we isolated the natural variants of the CNOT1 promoter and therefore we could

not evaluate structural alterations in those individuals. However, genetic based forms of long-

QT (LQT) have been associated with the development of dilated cardiomyopathy (DCM). An

overlap between DCM and LQT3 formed due to abnormalities of SCN5a gene, have been

described in multiple reports (Kwon et al., 2012, Shi et al., 2008) but there is also evidence for

the association between LQT1 and idiopathic DCM (Allen et al., 2016). In addition, it has been

reported that patients with both severe or mild forms of cardiomyopathies such as DCM or

hypertrophic cardiomyopathy (HCM), can show QT prolongation (Johnson et al., 2011, Jouven

et al., 2002, Ryerson and Giuffre, 2006). Of note, the role of the causative mutation in the

overlap between channelopathies and cardiomyopathies has not been fully understood and

the role of possible new players acting as phenotype modifiers, like the CCR4-NOT complex,

is yet to be determined.

When using a cardiac driver for gene knockdown that excluded the larval and early

pupal stages of fly heart development (TinCd4-Gal4), we failed to observe a strong

requirement for CNOT1/not1 and CNOT7/8/pop2. In contrast, when using a driver that was

restricted specifically to larval and early pupal stages (NP1029-Gal4), CNOT1/not1-

knockdown was lethal, but CNOT7/8/pop2 silencing at this stage of development resulted in

dilation and reduced contractility similar to knockdown exerted throughout life, whereas an

embryonic or adult-only driver had no effect. These findings suggest that CCR4-NOT function

is critical during cardiac remodeling from the larval to the adult heart. It is, however, also

possible that knockdown in adult flies would have an effect under stress conditions.

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Considering the effects on hiPSC-CM proliferation and the developmental defects

observed by silencing CNOT1/not1 and CNOT7/8/pop2 in Drosophila, we speculate that the

CCR4-NOT encoding genetic variants identified by GWAS in adult humans are those resulting

in less severe consequences, as the lack of proper mRNA regulation might be lethal at certain

stages of embryogenesis. Depletion of CNOT1 and the entire deadenylase module

(CNOT6/6L, CNOT7/8), respectively, has been demonstrated to promote ER-stress and

apoptosis in vitro (Ito et al., 2011). In turn, it has also been shown that activation of the

unfolded protein response impairs cardiac ion channel biogenesis leading to a prolongation of

the action potential duration (APD) (Liu et al., 2018). Taken together, these findings suggest

that disruption of CCR4-NOT complex function affect both structural (i.e. decreased CM

proliferation, myofibrillar structure abnormalities) and electrophysiological (i.e. shortened APD,

decreased contractility in flies) components of the heart. Whether knockdown of a specific

subunit produces one or both of those phenotypes may be influenced by silencing efficiency,

e.g. less ER-stress may lead to electrical remodeling, while more result in apoptosis. In

addition, specific RNA binding proteins that connect and guide CCR4-NOT to target specific

mRNAs are likely to have an influence. Collectively, our results show a prominent role of the

deadenylase module (CNOT7/8/pop2) both in vitro and in vivo.

This work demonstrates how the combinatorial use of GWAS studies and cardiac

model systems, enabled to connect CNOT1, CNOT7 and overall, the CCR4-NOT complex

function to cellular and whole heart phenotypes in the context human heart disease. However,

in this context, direct CCR4-NOT complex targets that influence heart rhythm and physiology

remain to be identified. Finally, strategies to modulate the expression of key components of

the CCR4-NOT complex, or stabilize its function, might be promising avenues for regulating

QT interval and preventing pro-arrhythmogenic substrates, especially targeted to those

individuals at increased risk due to their genetic background.

MATERIALS AND METHODS

Ethics statement

The DNA for promoter isolation was drawn from individual participants in the MICROS

study in South Tyrol (Pattaro et al., 2007). MICROS was approved by the Ethics Committee

of the Autonomous Province of Bolzano (Südtiroler Sanitätsbetrieb/Azienda Sanitaria dell’Alto

Adige). Each participant gave written informed consent.

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Generation of hiPSC-CMs

hiPSC were dissociated with 0.5 mM EDTA in PBS without CaCl2 and MgCl2 (Corning)

for 7min at RT, resuspended in mTeSR-1 media (StemCell Technologies) with 2 µM

Thiazovivin (StemCell Technologies) and 3 x 105 cells/well were plated in a Matrigel-coated

12-well plate. 24 hours after passage, cells were fed daily with mTeSR-1 media (without

Thiazovivin) for 3-5 days until ≥ 90% confluence. hiPSC-CMs were differentiated as previously

described (Burridge et al., 2015). Day 0, WNT signaling was activated by adding 6 µM

CHIR99021 (Selleck Chemicals) in S12 medium(Pei et al., 2017) for 48 hours. Day 2, cells

were treated with 2 µM Wnt-C59 (Selleck Chemicals) in S12 to inhibit WNT. Day 4, S12

medium was fully changed. Day 5, cells were dissociated with TrypLE Express (Gibco) for 4

minutes and blocked with RPMI (Gibco) +10% FBS (Omega Scientific). Cells were

resuspended in S12 supplemented with 4mg/L Recombinant Human Insulin (Gibco) (S12+

media) and 2µM Thiazovivin and 9 x 105 cells/well were plated in a Matrigel-coated 12-well

plate. S12+ media was changed Day 8 and replaced Day 10 by RPMI (Gibco) media + 213

µg/µL L-ascorbic acid (Sigma), 500 mg/L BSA-FV (Gibco), 0.5 mM L-carnitine (Sigma) and 8

g/L AlbuMAX Lipid-Rich BSA (Gibco) (CM medium). Under these conditions, hiPSC-CMs start

to beat around day 9-10. Day 15, cells were purified with lactate media, consisting of RPMI

without glucose, 213 µg/µL L-ascorbic acid, 500 mg/L BSA-FV and 8 mM Sodium-DL-Lactate

(Sigma) (Burridge et al., 2015, Tohyama et al., 2016), for 4-5 days and was replaced by CM

media until day 25.

Proliferation assay in hiPSC-CMs

At day 25 of differentiation, hiPSC-CMs were dissociated with TrypLE Select 10X

(Gibco), 12 min and neutralized with RPMI+10% FBS. Cells were resuspended in RPMI with

2% KOSR (Gibco) and 2% B27 50X with vitamin A (Life Technologies) supplemented with 2

µM Thiazovivin and plated at a density of 5000 cells/well in a Matrigel-coated 384-well plate.

hiPSC-CMs were transfected with siRNA (Dharmacon) targeting siCNOT1: L-015369-01,

siCNOT2: L020313-02, siCNOT3: L-020319-00, siCNOT4: L-020323-00, siNOT6: L-019101-

00, siNOT6L: L-016411-00, siCNOT7: L-012897-00 and siCNOT8: L-018791-00, using

lipofectamine RNAiMax (ThermoFisher). Each siRNA was tested in quadruplicate. 48 hours

post-transfection, cells were labelled with 10µM EdU (ThermoFisher). After 24h of EdU

incubation, cells were fixed with 4% paraformaldehyde for 30 min. EdU was detected

according to protocol and cells were stained with cardiac specific marker ACTN2 (Sigma,

dilution 1/800) and DAPI. Cells were imaged with ImageXpress Micro XLS microscope

(Molecular Devices) and custom algorithms were used to quantify EdU+ hiPSC-CMs.

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Voltage assay in hiPSC-CMs

Voltage assay was performed as described in McKeithan et al., 2017. Day 25 of

differentiation hiPSC-CMs were dissociated with TrypLE Select 10X for up to 12 min and

neutralized with RPMI+10% FBS. Cells were resuspended in RPMI with 2% KOSR (Gibco),

2% B27 50X with vitamin A (Life Technologies) and supplemented with 2µM Thiazovivin and

plated at 6.000 cells/well in a Matrigel-coated 384-well plate. hiPSC-CMs were transfected

with CCR4-NOT-NOT siRNAs as described above. Three days post-transfection, cells were

washed with pre-warmed Tyrode’s solution (Sigma) by removing 50 µL of media and adding

50 µL, five times. After the fifth wash, 50µL of 2x dye solution: voltage sensitive dye Vf2.1 Cl

(Fluovolt, 1:4000, ThermoFisher) diluted in Tyrode’s solution with 1µL of 10% Pluronic F127

(in water, ThermoFisher) and 20µg/mL Hoechst 33258 (in water, ThermoFisher) was added

to each well. The plate was returned to 37°C 5% CO2 incubator for 45 min. After incubation,

cells were washed four times with pre-warmed Tyrode’s. hiPSC-CMs were imaged with

ImageXpress Micro XLS microscope at 100 Hz for 5sec, with excitation wavelength at

485/20nm and emission filter 525/30nm. A single image of Hoechst was acquired before the

time series. Fluorescence over time quantification and trace analysis were automatically

quantified using custom software packages developed by Molecular Devices and Colas lab.

Three independent experiments were performed, each condition in quadruplicate.

Cell culture

HL-1 mouse atrial cardiomyocytes (Claycomb et al., 1998) were kindly donated by

William Claycomb (Louisiana State University, New Orleans) and cultured in Claycomb

medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS), 4 mM L-

glutamine, 100 U/mL Penicillin, 100 mg/mL Streptomycin, 0.3 mM Ascorbic Acid and 10 mM

Norepinephrine as previously described (Meraviglia et al., 2015).

Hela and 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM),

GlutaMAX supplement (Thermo Fisher Scientific), supplemented with 10% Fetal bovine serum

(FBS) (Sigma) and 1% penicillin-streptomycin (Thermo Fisher Scientific). All cells were

maintained at 37°C in a saturated humidity atmosphere containing 5% CO2.

Luciferase assay

HL1, HeLa and T293 cells were seeded at 30,000, 60,000, and 150,000 cells/well,

respectively, into 24-well plates (Corning). 24 hours after seeding, 10ng of the reporter plasmid

pG4.74[hRluc/TK], was co-transfected with 10ng of either pGL4.10 vector (complete or

minimal for both haplotypes) or negative control vector (pG4.13 [luc2/SV40]). Transfection

was performed with the Lipofectamine plus reagent (Invitrogen), according to manufacturer’s

protocol. 48 hours post-transfection, cells were washed with PBS and lysed with 100 μl of

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Passive Lysis Buffer (Promega) for 15 min at RT. Cell lysates were immediately used to

measure luciferase activity, using the Dual Luciferase Reporter Assay System kit (Promega).

Each lysate (20 μl) was incubated with 100 μl of Luciferase Assay Reagent II (LAR II). Firefly

luminescence was measured for 10 seconds using a luminometer (Victor X3-2030, Perkin

Elmer). After 2 seconds, 100μl of Dual-Glo Stop & Glo Reagent was added to each well.

Subsequently, renilla luminescence was measured for 10 seconds using the same

luminometer. Luciferase activity was calculated based on the ratio of the activities of firefly

and renilla luciferases. At least three independent experiments were performed in triplicate.

Fly stocks

All transgenic RNAi-fly lines were purchased from Vienna Drosophila RNAi Center

(VDRC) and from Bloomington Drosophila Stock Center at Indiana University (Transgenic

RNAi Project at Harvard Medical School, TRiP). VDRC IDs: not1 GD12571 and KK106587,

not2 GD20826, pop2 GD28396, twin GD13365. TRiP/ BDSC IDs: not1 28681, not4 JF03203,

pop2 HM05235, twin HMS00493. Control flies with corresponding genetic background: VDRC

w1118 (GD RNAi library), TRiP-fly line with attP2 docking site. Cardiac-specific drivers were

kind gifts from the following investigators: Manfred Frasch: TinCΔ4 12a-Gal4 (Lo and Frasch,

2001) and tin-D-Gal4 (Reim and Frasch, 2005), Eric Olsen: Hand-Gal4 (Han and Olson, 2005),

Laurent Perrin: NP1029-Gal4 (Monier et al., 2005) and Hand-Gal4 Gene Switch (Monnier et

al., 2012).

Fly medium

Ingredients: cornmeal (7.0%), malt (5.2%), molasses (5.2%), soy flour (1.7%), agar

(0.4%), autolyzed yeast (2.1%). All ingredients were mixed with water and cooked for 15

minutes without boiling. Preservatives, Tegosept in EtOH (1.8%) and propionic acid (2.1%),

were added once the batter cooled <65C. All percentages refer to final concentration. Dry

ingredients: weight/ total volume batter, liquid ingredients: volume/ total volume batter.

Fly crosses

Driver-line virgins were crossed to RNAi-males and corresponding isogenic control

males. Flies were raised on standard fly food and kept in 25C or 18C. Female progeny was

collected and aged to 1 week in 25C at which point they were imaged and analyzed.

Hand-Gal4 Gene-Switch knockdown

Eclosed female progeny was collected and placed in vials with fly food containing

100µg/ml RU-486, (40µl of 25mg/ml stock RU-486 dissolved in EtOH was added to 10ml fly

food). Control food contained an equal amount of EtOH. Flies were aged to 1 week in 25C at

which point they were dissected and analyzed.

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Fly heart dissection

Flies were anesthetized with FlyNap and dissected in artificial hemolymph according to a

previously described protocol (Vogler and Ocorr, 2009). The procedure includes removing the

fly head, intestines and some fat, resulting in a semi-intact preparation that visualizes the

beating heart. Artificial hemolymph was re-oxygenated for 20 minutes post dissection allowing

the hearts to stabilize before video recording.

High speed digital video imaging and analysis

All fly hearts were filmed with an EM-CDD Hamamatsu digital camera, using a Leica

DMFLSA microscope equipped with a 10x dipping lens. 30-second recordings of each fly heart

were made with a camera speed of 120-140 frames per second (Ocorr et al., 2007b). M-modes

describing fly heart contractions were created by Semi-automated Optical Heartbeat Analysis

(Fink et al., 2009), SOHA.

Fluorescent staining and imaging

According to a previously described protocol (Alayari et al., 2009), flies were dissected

in artificial hemolymph and hearts relaxed with 10mM EGTA before fixation with formaldehyde.

Flies were stained with Phalloidin, Alexa488 to visualize F-actin. Apotome images were taken

with a Zeiss Axio Imager.Z1 microscope at 10x and 25x. Images were processed with Adobe

Photoshop.

Statistical Analysis

To determine any statistical significance between experimental and control groups in

hiPSC-CMs and Drosophila experiments, we calculated two-sided p-values with Student’s t-

test, One-way- and Two-way-ANOVA with Tukey’s multiple comparisons test, using GraphPad

Prism software (2016). 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 (StataCorp.

2013. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP). Population

distribution of siCtrl and siCNOT7-transfected hiPSC-CMs was generated with GraphPad

Prism using nonlinear regression. Unpaired nonparametric Kolmogorov-Smirnov test was

used to compare each treated condition to controls using APD75 of every measured cell.

Electrophysiology of Adult Hearts

Semi-intact heart preparations were incubated in artificial hemolymph containing 10µM

blebbistatin (Sigma Aldrich), left in the dark with oxygenation until the hearts stopped. Fresh

saline without blebbistatin was added and electrical potentials were recorded from the conical

chamber using glass electrodes (20-50 MΩ) filled with 3M KCl. Data were acquired using an

Axon-700B amplifier, signals were digitized using the DIGIDATA 1322A and data were

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captured and analyzed using PClamp 9.0 and Clampfit 10.0 software from Molecular Devices.

Data was quantified from representative 30s recordings where the resting membrane potential

had remained stable.

Competing interests - No competing interests declared.

Funding

This study was supported by grants from NIH R01-HL054732 to RB and R01-

HL124091 to AC, the Wanek Foundation to RB and AC and by the Department of Innovation,

Research and Universities of the Autonomous Province of Bolzano-South Tyrol (Italy) to CV,

LF, AR and AH.

Author contributions

Lisa Elmén performed the Drosophila experiments and data analysis together with

Anthony Cammarato, Rolf Bodmer and Nakissa Alayari. Claudia Volpato, Andrew Hicks and

Luisa Foco analysed the genetics data, and created the resources to perform the cell culture

experiments, which were analyzed with Alessandra Rossini. Santiago Pineda performed the

Drosophila electrophysiology experiments and analyzed the recorded data with Karen Ocorr

and Rolf Bodmer. Anaïs Kervadec performed all hiPSC-experiments and analyzed the data

with Alexandre Colas. Lisa Elmén, Andrew Hicks and Rolf Bodmer wrote and edited the

manuscript with contributions from Alessandra Rossini, Alexandre Colas and Anthony

Cammarato.

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Tables

Table 1. CCR4-NOT genes in this study

Human Drosophila

CNOT1 not1

CNOT2 not2/rga

CNOT3 not3

CNOT4 not4

CNOT6 and CNOT6L twin

CNOT7 and CNOT8 pop2

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Figures

Figure 1. 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 by red box. 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

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using the Wilcoxon rank-sum test. For complete promoter, n independent experiments=3; for

each experiment, n of independent biological replicates per haplotype=3. Total n of

observations for each haplotype=9, total n=18. For minimal promoter: n independent

experiments=5; for each experiment, n of independent biological replicates per haplotype=3,

except one experiment (n=4). Total n of observations for each haplotype=16, total n=32.

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Figure 2. A) Schematic of proliferation assay in hiPSC-CMs. B) Representative

immunofluorescence images for EdU and ACTN1 in siCtrl, siCNOT1 and siCNOT3 conditions.

Scale bar, 25 µm. C-D) Histogram showing normalized % of EdU positive hiPSC-CMs and

normalized number of hiPSC-CMs. T-test was used to calculate p-values. E) Schematic

overview of single cell and high throughput voltage assay. F) Two-dimensional graph for

APD75 and Kolgomorov Smirnov distance (KS-D) representing screen results for CCR4-

CNOT components knockdown. G) Population distribution of APD75 measurements for

siCNOT7 vs siControl-transfected hiPSC-CMs. H) Median action potential traces for siCNOT7

and siControl transfected hiPSC-CMs. I) Table summarizing average and standard deviation

values for APD50, APD75 and APD90 for siCNOT7 and siControl transfected hiPSC-CMs.

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Figure 3. 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 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. 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. Representative not1 knockdown heart show myofibrillar disarray

and gaps in muscle tissue compared to tightly packed circumferential myofibrils in control

heart. The scale bar is 200µm.

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Figure 4. A-D) RNAi-mediated knockdown of CNOT7/8/pop2 (TRiP HM05235) using

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. Boxes: interquartile

range, central line: median; plus: mean; whiskers: upper and lower adjacent values as defined

by Tukey (1977); dots: outside values. E-F) Immunofluorescent staining with phalloidin

visualizes f-actin of Drosophila hearts. CNOT7/8/pop2 knockdown heart show dilation and

gaps in muscle tissue compared to tightly packed circumferential myofibrils in control heart.

The scale bar is 100µm. G) Table summarizing electrophysiology measurements of

CNOT7/8/pop2-knockdown fly hearts. H-I) Representative 10s m-modes show greater peaks

per burst and longer event duration of CNOT7/8/pop2-knockdown (Hand-

Gal4>CNOT7/8/pop2) fly hearts compared to control (w1118 x CNOT7/8/pop2).

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Figure 5. RNAi-mediated knockdown of CNOT2/not2 (VDRC GD20826), CNOT4/not4 (TRiP

JF03203) and CNOT6/6L/twin (VDRC GD13365) using cardiomyocyte and pericardial cell

specific driver Hand-Gal4. n=15 female flies per genotype. 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. One-way ANOVA with Tukey’s multiple

comparisons test was used to calculate two-sided p-values. Boxes: interquartile range, central

line: median; plus: mean; whiskers: upper and lower adjacent values as defined by Tukey

(1977); dots: outside values.

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Figure 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 larval stage heart specific driver NP1029-Gal4. n=19 females 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. I-L) RNAi-mediated knockdown of CNOT7/8/pop2 using the RU-

486 inducible Hand-Gal4 Gene Switch driver. Reduced expression of CNOT7/8/pop2in adult

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fly hearts had no effect on heart diameters or contractility. n=12 females 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.

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Complete CNOT1 promoter

luci

fera

ce/ r

enill

a ra

tio 2.0

1.5

1.0

0.5

0

GAGTGAGT

p=0.0575

HeLa T293

p=0.0464

Figure S1

A Minimal CNOT1 promoterB

1.0

0.8

0.6

0.4

0

GAGTGAGT

p=0.0003 p=0.0004

0.2

luci

fera

ce/ r

enill

a ra

tioHeLa T293

C

Marker Name

rs27097

rs37037

rs9941290

rs863433

Allele1 Allele2

T

A

A

T

Effect (ms) StdErr (ms) P-value

G

G

G

G

Direction

1.6218

-0.6233

1.617

-1.6166

3.62E-12

0.1997

2.71E-12

0.2313

0.2313

2.71E-12

0.2333

0.001805

++++++

-+----

++++++

------

G and T

G and T

G and T

G and T

Figure S1. Box plots summarizing the intensity of the ratio between luciferase and renilla signal in HeLa and T293 cells. A) Complete CNOT1 promoter construct. B) Minimal CNOT1 promoter construct. C) This table lists the four SNPs in strong linkage disequilibrium within the putative promoter region of the CNOT1 gene, with association p-values derived from the QTSCD consortium data18. This region was reported originally as the GINS3-NDRG4-SETD6-CNOT1 region as no resolution as to the functional gene in this region was possible without further experimental validation, provided herein. Allele 1 represents the effect allele identified in the GWAS meta-analysis and sequencing of this region from individuals homozygous for these variants revealed haplotypes consistent with effect direction (e.g. T at rs27097, G at rs37037, A at rs9941290 and G at rs863433) which were predicted from these results, and subsequently demonstrated experimentally, to lengthen QT interval, with the opposite haplotype predicted to shorten QT interval. The Effect column shows the observed effect in milliseconds (ms) on QT interval, and the Direction column shows the consistent direction of this effect in the six individual cohorts contributing to the QTSCD consortium.

Disease Models & Mechanisms: doi:10.1242/dmm.044727: Supplementary information

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w1118 x CNOT1/ not1

Time (s) 10 5

20

0

-20

-40

-60

IN 0

(mV

)

0

20

40

60

80

100

120

M

Diastolic Diameter

0

20

40

60

80

M

Systolic Diameter*

0.0

0.2

0.4

0.6

%

Fractional Shortening **

Hand-Gal4 > CNOT1/ not1

Time (s)

20

0

-20

-40

-60

IN 0

(mV

)

10 5

C D E

A B

Figure S2

w11

18 x C

NOT1/not1

TinC∆4

> CNOT1/n

ot1

Figure S2. A-B) Electrophysiological recordings of CNOT1/not1 (VDRC, GD12571) knockdown Drosophila hearts. One single electrophysiology trace obtained from fragile Hand-Gal4 driven CNOT1/not1-knockdown fly hearts show extended events and multiple peaks per burst com-pared to control fly. C-E) TinCΔ4-Gal4 driven CNOT1/not1-knockdown increased systolic diameter and reduced fractional shortening. n=40 female flies per genotype. Student’s t-test was used to calculate two-sided p-values. Boxes: interquartile range,central line: median; plus: mean; whiskers: upper and lower adjacent values as defined by Tukey (1977); dots: outside values.

w11

18 x C

NOT1/not1

TinC∆4

> CNOT1/n

ot1

w11

18 x C

NOT1/not1

TinC∆4

> CNOT1/n

ot1

Disease Models & Mechanisms: doi:10.1242/dmm.044727: Supplementary information

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0

20

40

60

80

100

120

M

Diastolic Diameter ****

0

20

40

60

80

M

Systolic Diameter

***

0.0

0.1

0.2

0.3

0.4

0.5

Fractional ShorteningA B C

Figure S3

TinCΔ

4> C

NOT7/8/ p

op2

TinCΔ

4> C

NOT4/ no

t4

TinCΔ

4> C

NOT2/ no

t2

TinCΔ

4> C

NOT6/6L/

twin

w11

18 x T

inCΔ4

TinCΔ

4> C

NOT7/8/ p

op2

TinCΔ

4> C

NOT4/ no

t4

TinCΔ

4> C

NOT2/ no

t2

TinCΔ

4> C

NOT6/6L/

twin

w11

18 x T

inCΔ4

TinCΔ

4> C

NOT7/8/ p

op2

TinCΔ

4> C

NOT4/ no

t4

TinCΔ

4> C

NOT2/ no

t2

TinCΔ

4> C

NOT6/6L/

twin

w11

18 x T

inCΔ4

%

Figure S3. A-C) RNAi-mediated knockdown of CNOT7/8/pop2 (TRiP HM05235), CNOT2/not2 (VDRC GD20826), CNOT4/not4 (TRiP JF03203) and CNOT6/6L/twin (VDRC GD13365) using cardiomyocyte specific driver TinC∆4-Gal4 (n=15, n=20, n=19, n=20 of female flies per respective genotype). Reduced expression of CNOT7/8/pop2, CNOT2/not2 and CNOT6/6L/twin had no effect on diastolic diameter, systolic diameter or fractional shortening. Reduced expression of CNOT4/not4 increased both systolic and diastolic diameters but had no effect on contractility. One-way ANOVA with Tukey’s multiple comparisons test was used to calculate two-sided p-values. Boxes: interquartile range, central line: median; plus: mean; whiskers: upper and lower adjacent values as defined by Tukey (1977); dots: outside values.

Disease Models & Mechanisms: doi:10.1242/dmm.044727: Supplementary information

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