Straightjacket/α2δ3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1

  1. Emilie Auxerre-Plantié
  2. Masayuki Nakamori
  3. Yoan Renaud
  4. Aline Huguet
  5. Caroline Choquet
  6. Cristiana Dondi
  7. Lucile Miquerol
  8. Masanori P Takahashi
  9. Geneviève Gourdon
  10. Guillaume Junion
  11. Teresa Jagla
  12. Monika Zmojdzian  Is a corresponding author
  13. Krzysztof Jagla  Is a corresponding author
  1. GReD, CNRS UMR6293, INSERM U1103, University of Clermont Auvergne, France
  2. Osaka University Graduate School of Medicine, Japan
  3. Imagine Institute, Inserm UMR1163, France
  4. Centre de Recherche en Myologie, Inserm UMRS974, Sorbonne Universités, Institut de Myologie, France
  5. Aix-Marseille Univ, CNRS UMR 7288, IBDM, France
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 3 supplements
Cardiac-specific knockdown of MBNL1 ortholog and overexpression of CELF1 counterpart in Drosophila lead to asynchronous heartbeats.

(A) The adult Drosophila heart expressing Hand-Gal4-driven GFP (Hand>GFP). Note that this Gal4 line drives expression exclusively in the heart. Arrows indicate cardiomyocytes (CM) and pericardial …

Figure 1—figure supplement 1
Bru-3 and Mbl are expressed in the adult fly heart and their transcript and protein levels are affected in Hand>Bru-3 and Hand>MblRNAi flies.

(A–B) RT-qPCR analyses of Bru-3 overexpression and Mbl attenuation in the adult hearts from Hand>Bru-3 (A) and Hand>MblRNAi (B) flies. Three biological replicates (n = 10–12 hearts per replicate) …

Figure 1—figure supplement 2
Effect of simultaneous cardiac attenuation of Mbl and overexpression of Bru-3 on conduction defects.

Barplot graph showing percentage of flies with conduction defects. Note a moderate additive effect of simultaneous attenuation of Mbl and overexpression of Bru-3. Number of fly hearts tested (n) is …

Figure 1—video 1
An example of asynchronous heartbeat phenotype observed in Hand>MblRNAi context.

High-speed 20 s time-lapse movie.

Cardiac-specific transcriptional profiling using TU-tagging method.

(A) Pipeline of heart-targeted transcriptional profiling using TU-tagging. Note that Gal4- inducible UPRT transgene (UAS-UPRT) has been combined with UAS-MblRNAi and with UAS-Bru-3 for the purpose …

Figure 3 with 2 supplements
Heart-specific transcriptional profiling of DM1 flies identifies deregulation of genes controlling cellular calcium level.

(A) Venn diagrams of genes deregulated in Hand>UPRT;MblRNAi and in Hand>UPRT;Bru-3 contexts (FC > 1.7) followed by heatmap of commonly deregulated genes. (B) Genemania interaction network of …

Figure 3—source data 1

A list of Drosophila genes and their Human orthologs deregulated in both Hand>Bru-3 and Hand>MblRNAi contexts.

Orthology scores were obtained from DIOPT tool (DRSC Integrative Ortholog Prediction Tool) (Hu et al., 2011) which predicts orthologs, based on sequence homology, phylogenetic trees, and functional similarity (www.flyrnai.org/cgi-bin/DRSC_orthologs.pl).

https://cdn.elifesciences.org/articles/51114/elife-51114-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Attenuation of inaD, Syn1 and Rgk2 all lead to an increase of diastolic (DD) and systolic (SD) heart diameters.

For each candidate gene effects of attenuation were tested using two different UAS-RNAi lines crossed with Hand-Gal4 driver. More than 20 flies for each genotype were analysed by SOHA. Statistical …

Figure 3—figure supplement 2
The expression levels of CG42617 and CG16868, two additional α2δ protein-coding genes are not affected in the heart of DM1 fly models.

Normalized RNAseq IGV tracks in control (Hand>lacZ) and in pathogenic DM1 contexts (Hand>MblRNAi and Hand>Bru-3) are shown aligned with genomic exon/intron organization of CG42617 (A) and CG16868 (B).

Calcium waves in asynchronous Drosophila heart.

In wild type hearts, calcium waves underlie cardiac contractions so that calcium peaks (arrows) align with the onset of contractions (arrowheads) in both A3 and A4 segments. In contrast, in …

Figure 5 with 2 supplements
Increased cardiac expression of Stj contributes to the conduction defects observed in DM1 fly models.

(A) Stj protein is detected at a low level in cardiomyocytes (CM) but at a higher level in the ventral longitudinal muscles (VLMs) that underlie adult heart tube. Stj in VLMs and in heart …

Figure 5—source data 1

Corrected total cell fluorescence (CTCF) of Stj signal in circular fibers of cardiomyocytes.

https://cdn.elifesciences.org/articles/51114/elife-51114-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Stj protein levels in different genetic contexts visualized by immunostaining.

Stj protein expression (red) in circular fibers of cardiomyocytes in wild-type (wt) (A) and in different genetic contexts (B-E) tested. Circular fibers are visualized with phalloidin staining …

Figure 5—figure supplement 2
stj-RC transcript isoform carrying long 3’UTR and alternatively spliced exon 15 is up regulated in Hand>MblRNAi and in Hand>Bru-3 hearts.

(A) RT-qPCR probes for stj-RC specific exon 15 and for total stj transcripts are indicated on the scheme. (B) RT-qPCR analysis of stj-RC and total stj transcript levels in control (Hand>lacZ) fly …

Figure 6 with 1 supplement
Increased cardiac expression of Straightjacket human ortholog α2δ3 is associated with conduction defects in DM1 patients.

(A) In mouse, α2δ3 protein is detected at a high level in atrial cardiac cells and at a low level in ventricles. LV denotes left ventricle. Scale bars, 100 mm. (B) RT-qPCR analysis of mouse α2δ3 …

Figure 6—figure supplement 1
RT-qPCR analysis showing that Cav1.2 transcripts are elevated in human cardiac samples from DM1 patients with conduction defects.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene
(D. melanogaster)
stjFLYB: FBgn0261041
Gene
(D. melanogaster)
mblFLYB: FBgn0265487
Gene
(D. melanogaster)
bru3FLYB: FBgn0264001
Genetic reagent
(D. melanogaster)
UAS-stjFlyORF Zurich ORFeome ProjectFlyORF ID: F001252
Genetic reagent
(D. melanogaster)
stj TRIPBloomington Drosophila Stock CenterRRID:BDSC25807RNAi line
Genetic reagent
(D. melanogaster)
Mbl RNAiVDRC Vienna Drosophila Resource CenterVDRC: GD 28732RNAi line
Genetic reagent
(D. melanogaster)
P{XP}bru3d09837Exelixis at Harvard Medical SchoolHarvard: d09837UAS carrying P element insertion line
Genetic reagent
(D. melanogaster)
UAS-GFPBloomington Drosophila Stock CenterRRID:BDSC_32201
10 Genetic reagent
(D. melanogaster)
UAS-HA-UPRTBloomington Drosophila Stock CenterRRID:BDSC_27604
11 Genetic reagent
(D. melanogaster)
UAS-lacZBloomington Drosophila Stock CenterRRID:BDSC_1776
12 Genetic reagent
(D. melanogaster)
UAS-GCaMP3Bloomington Drosophila Stock CenterRRID:BDSC_32116
13 Genetic reagent
(D. melanogaster)
Ca-alpha1D TRIPBloomington Drosophila Stock CenterRRID:BDSC_25830RNAi line
14 Genetic reagent
(D. melanogaster)
Rgk2 TRIPBloomington Drosophila Stock CenterRRID:BDSC_65932RNAi line
15 Genetic reagent
(D. melanogaster)
Rgk2 RNAiVDRC Vienna Drosophila Resource CenterVDRC: GD44611RNAi line
16 Genetic reagent
(D. melanogaster)
Syn1 RNAiVDRC Vienna Drosophila Resource CenterVDRC: GD27893RNAi line
17 Genetic reagent
(D. melanogaster)
Syn1 RNAiBloomington Drosophila Stock CenterRRID: BDSC_27504RNAi line
18 Genetic reagent
(D. melanogaster)
inaD RNAiVDRC Vienna Drosophila Resource CenterVDRC: GD26211 (unavailable)RNAi line
19 Genetic reagent
(D. melanogaster)
inaD RNAiVDRC Vienna Drosophila Resource CenterVDRC: 330227RNAi line
20 Genetic reagent
(D. melanogaster)
Hand-GAL4Laurent Perrin, TAGC,
Marseille, France
GAL4 driver line
21 Genetic reagent
(D. melanogaster)
TinCΔ4-GAL4Rolf Bodmer, SBPMD Institute, San Diego, USGAL4 driver line
22 Antibodyanti-StjHugo Bellen, Baylor College of Medicine, Huston, USAguinea pig polyclonalUse 1:500 (IHC)
23 Antibodyanti-α2δ3Greg Neely, University of Sidney, Australiarabbit polyclonalUse 1:200 (IHC)
24 Antibodyanti-α2δ3GeneTexGTX16618 - rabbit polyclonalUse 1:500 (WB)
25 Antibodyanti-Bru3Millegen, Toulouse, Francerabbit polyclonalUse 1:1000 (IHC)
26 Antibodyanti-MblDarren Monckton, Glasgow, UKsheep polyclonalUse 1:200 (IHC)
27 Antibodyanti-GFPAbcamab5450 – goat polyclonalUse 1:500 (IHC)
28 AntibodyAnti-GAPDHSigma AldrichSAB2108266 – rabbit polyclonalUse 1:3000 (WB)

Additional files

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