Deregulations of miR‐1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1

Figures

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  Figure 1. Heart‐targeted dmiR‐1 attenuation causes DCM in Drosophila A–C′(A) Control (UAS‐dmiR‐1 sponge) and (B) mutant (Hand > dmiR‐1 sponge) adult hearts from 1‐week‐old flies labeled for F‐actin (red). (A′, B′) Cross‐sections of cardiac tubes 3D‐reconstructed using Imaris software. White line shows the enlargement of lumen cardiac tube in Hand > dmiR‐1 sponge (B′) in comparison with control (UAS‐dmiR‐1 sponge) (A′). M‐mode records from 1‐week‐old control (UAS‐ dmiR‐1 sponge) (C) and Hand > dmiR‐1 sponge (C′) flies showing increased diastolic (green) and systolic diameters (red) in the dmiR‐1 sponge context (C′).D–FBox plots showing diastolic (D) and systolic (E) diameters and cardiac contractility analyses (percent fractional shortening) (F) performed using SOHA approach for controls (Hand‐Gal4 and UAS‐dmiR‐1 sponge) and Hand > sponge dmiR‐1 contexts at ages 1 and 5 weeks. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: Scale bar = 20 μm. P‐value < 0.05 is considered statistically significant. (*) P‐value = 0.033, (**) P‐value = 0.021, (***) P‐value = 0.0002. The…

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  Figure 2. DM1 flies develop DCM phenotype with a reduced dmiR‐1 level in cardiac cells A–FBox plots showing cardiac tube size (diastolic (A, D) and systolic (B, E) diameters) and contractility (percent fractional shortening (C, F)) analyses performed using SOHA approach for controls (Hand‐Gal4, UAS‐Bru3, and UAS‐mblRNAi) and DM1 contexts (Hand > Bru3 and Hand > mblRNAi), at ages 1 and 5 weeks. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile.G, G′Representative spot views generated using Imaris from in situ hybridization with miRCURY LNA probe for dmiR‐1 and used for quantification of dmiR‐1 levels. Spot views of dmiR‐1 in hearts of 1‐week‐old control (UAS‐mblRNAi) (G) and DM1 flies (Hand > mblRNAi) (G′) are shown. Each spot represents a pool of dmiR‐1 transcripts detected in the same area. The zoom region in G corresponds to area in which FISH signal was quantified.H, IScatter plot graph showing the signal intensity quantified in cardioblasts of 1‐ and 5‐week‐old flies for controls (UAS‐Bru3, UAS‐mblRNAi) and DM1 contexts (Hand > Bru3, Hand > mblRNAi). n = 9 hearts. Bars correspond to…

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  Figure EV1. Flies showing DCM present myofibrillar disarray in the heart tubes A–D′Z stack projection from hearts of controls (UAS‐mblRNAi, UAS‐Bru3, UAS‐dmiR‐1 sponge and UAS‐Mp) (A–D) and mutants (Hand > mblRNAi, Hand > Bru3, Hand > dmiR‐1 sponge and Hand > Mp) (A′–D′) aged of 5 weeks. The arrows point to irregularity in myofibrillar density and arrangement in the heart tubes. Scale bar = 20 μm.E–GBox plots showing cardiac size analyses (diastolic (E) and systolic (F) diameters) and percent fractional shortening (G) performed by SOHA approach for controls (Hand‐Gal4 and UAS‐960CTG) and mutant (Hand > 960CTG) at 1 and 5 weeks of age. Aged Hand > 960CTG flies present cardiac dilation characterized by a significant increase in diastolic and systolic diameters in comparison with control Hand > Gal4 (E, F). n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: P‐value < 0.05 considered statistically significant. (**) P‐value = 0.021, (***) P‐value = 0.0002. The nonparametric Mann–Whitney test was performed to compare control samples and samples of interest.

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  Figure EV2. Supplemental analyses of dmiR‐1 A–CScatter plot showing cardiac size analyses (diastolic (A) and systolic (B) diameters) and percent fractional shortening (C) performed by SOHA approach for control wt 1118 and heterozygous mutant context (dmiR‐1 KO −/+) at 1 and 5 weeks of age. Aged Hand > dmiR‐1 KO −/+ flies present cardiac dilation characterized by a significant increase in diastolic and systolic diameters in comparison with control. n = 20 hearts. Bars correspond to mean ± SD.D, ERTq–PCR analysis for pre‐dmiR‐1 transcript in adult heart of 1 and 5 weeks of age for control (Hand > LacZ) and DM1 contexts (Hand > mblRNAi, Hand > Bru3). n = 4 biological replicates.FAlignment of Drosophila and human miR‐1 sequences.GPotential binding site of human miR‐1 in 3′UTR of COL15A1 (COL15A1–201 transcript position 4428 to 4447). Data information: P‐value < 0.05 considered statistically significant. (*) P‐value = 0.033, (****) P‐value < 0.0001. The nonparametric Mann–Whitney test was performed to compare control samples and samples of interest.

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  Figure EV3. DM1 flies show reduced levels of dmiR‐1 in cardiac cells A–H′FISH for scramble (5′ 3′ DIG) (A′) and U6 (5′DIG) (B′) detected in w 1118 cardiac tube. FISH with miRCURY LNA probe for dmiR‐1 detection in controls (UAS‐dmiR‐1, UAS‐mblRNAi, UAS‐Bru3) (C′, E′, G′) and mutants (Hand > dmiR‐1, Hand > mblRNAi, Hand > Bru3) (D′, F′, H′) cardiac tubes labeled with dmiR‐1 probes (red) and actin (green) at 1 week of age. Representative spot views generated using Imaris from in situ hybridization with miRCURY LNA probe for dmiR‐1 and used for quantification of dmiR‐1 levels.I, I′Spot views of dmiR‐1 in hearts of 1‐week‐old control (UAS‐mblRNAi) (I) and DM1 (Hand > mblRNAi) flies (I′) are shown. Each spot represents a pool of dmiR‐1 transcripts detected in the same area. The zoom area in (I) and (I′) corresponds to pericardial cells.J, KScatter plot graph showing the signal intensity of dmiR‐1 levels quantified in pericardial cells of 1‐ and 5‐week‐old flies for controls (UAS‐Bru3, UAS‐mblRNAi) and DM1 contexts (Hand > Bru3, Hand > mblRNAi). n = 27 pericardial cells. Bars correspond to mean ± SD. Data information: Scale bar = 40 μm. P‐value < 0.05 considered statistically significant. (*)…

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  Figure 3. Multiplexin, a new cardiac dmiR‐1 target, is up‐regulated in DCM‐developing DM1 flies AUp‐regulated genes identified by heart‐specific transcriptomic approach (TU‐tagging) (Auxerre‐Plantié et al, 2019) in DM1 contexts developing DCM (Hand > mblRNAi and Hand > Bru3) aged of 5 weeks (top Venn diagrams). Lower Venn diagrams show DM1 up‐regulated genes identified in silico as potential dmiR‐1 targets.BAlignment of dmiR‐1 binding site with 3′UTR region of Multiplexin.C–F′ dmiR‐1 binding site in 3′UTRMp region is required to negatively regulate Mp expression in vivo. Adult hearts from transgenic GFP sensor lines carrying 3′UTRMp region with (UAS‐GFP‐3′UTRMp) or without dmiR‐1 seed site (UAS‐GFP ∆3′UTRMp). These lines were combined with the UAS‐dmiR‐1 line to generate double transgenic lines and tested for GFP expression in nontargeted context (crossed with w 1118 ) (C, E) or in heart‐targeted context (crossed with heart‐specific driver Hand‐Gal4) (D, F). When crossed with Hand‐Gal4, GFP expression in Hand > GFP‐3′UTRMp; dmiR‐1 line (carrying dmiR‐1 seed site) (D′) is attenuated in cardiac tube and pericardial cells compared with Hand > GFP ∆3′UTRMp; dmiR‐1 line (lacking dmiR‐1 seed…

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  Figure 4. Heart‐targeted Mp overexpression leads to DCM A–D′Adult hearts from 1‐week‐old flies (A, A′, B, B′) and their cross‐sections (C, C′, D, D′) labeled for Mp (green) and actin (red) for controls (UAS‐Mp) and Mp overexpression context (Hand > Mp). Highlighted regions in D correspond to areas used for quantifications of the fluorescent signals. Scale bar = 5 μm.E–GBox plots showing cardiac variables (diastolic (E) and systolic (F) diameters) and percent fractional shortening (G) for controls (Hand‐Gal4 and UAS‐Mp) and Mp overexpression (Hand > Mp) conditions at 1 and 5 weeks. Notice an increase in diastolic and systolic diameters with reduced cardiac contractility in Hand > Mp in comparison with controls. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile.H, H′M‐modes representing cardiac variables in 5‐week‐old control (UAS‐Mp) (H) and Mp‐overexpressing (Hand > Mp) (H′) flies.IScheme presenting cardiac role of dmiR‐1 and its target Mp in DCM‐developing Drosophila DM1 models. In wild‐type, Drosophila heart mbl promotes pre‐dmiR‐1 processing. Bru3 has a potential antagonistic role in…

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  Figure EV4. Mp is expressed in the adult fly heart and up‐regulated in pericardial cells of DCM‐developing DM1 lines A–C″(A) Adult heart of w 1118 line labeled for Mp (green), actin (red), and DAPI (blue). (C) The yellow arrows indicate ventral longitudinal muscles (VLM). (C′) The yellow arrows indicate circular fibers (within cardioblast cells). (A″, B″) Cross‐section of w 1118 cardiac tube after 3D reconstruction with Imaris software, showing the expression of Mp at the internal and the external surface of the cardiac tube.D–G′Adult heart of 1 and 5 weeks of age labeled for Mp (green) for controls (UAS‐mblRNAi, UAS‐Bru3) (D–G) and DM1 context (Hand > mblRNAi, Hand > Bru3) (D′–G′). Encircled region in E′ corresponds to an example of area used for quantifications of the fluorescent signal in pericardial cells using the CTCF method.H, IFluorescence signal intensity quantification for Mp expression in pericardial cells in adult heart of 1 and 5 weeks of age for controls (UAS‐mblRNAi, UAS‐Bru3) and DM1 context (Hand > mblRNAi, Hand > Bru3) using the CTCF method. For each genotype, 9 hearts were analyzed and 3 pericardial cells were analyzed from each heart. Bars correspond to mean ±…

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  Figure 5. Mp loss‐of‐function leads to reduced heart size A–B′Adult heart of 1 week of age labeled for Mp (green) and actin (red) for control (UAS‐Mp RNAi) (A, A′) and mutant (Hand > Mp RNAi) contexts (B, B′).CFluorescence signal intensity quantification for Mp expression in cardioblasts in adult heart of 1 week for control (UAS‐Mp RNAi) and mutant (Hand > Mp RNAi) contexts using the CTCF method. For each genotype, 9 hearts were analyzed and fluorescence intensity was measured in two regions from segment A3 and two regions from segment A4. Bars correspond to mean ± SD.D–FBox plots showing cardiac size analyses (diastolic (D) and systolic diameters (E)) and percent fractional shortening (F) performed by SOHA approach for controls (Hand‐Gal4 and UAS‐Mp RNAi) and mutant (Hand > Mp RNAi) contexts at 1 and 5 weeks of age, showing the reduced size of cardiac tube in Hand > Mp RNAi context in comparison to the Hand‐Gal4 control. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: Scale bar = 20 μm. P‐value < 0.05 considered statistically significant. (*) P‐value = 0.033, (**)…

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  Figure 6. Increased cardiac expression of human Mp ortholog Col15A1 and low expression of miR‐1 are associated with DCM in DM1 patients A, B(A) Col15A1 transcript levels tested by RT–qPCR and (B) Col15A1 protein levels tested by Western blot in cardiac samples from healthy donors and from DM1 patients with (DCM+) and without DCM (DCM−).C miR‐1 levels tested by RT–qPCR in cardiac samples from healthy donors and from DM1 patients.D, FBox plots showing cardiac variables (diastolic (D) and systolic (E) diameters and percent fractional shortening (F)) assessed by SOHA approach for controls (UAS‐Bru3; Mp RNAi and UAS‐UPRT; Bru3), Mp rescue (Hand > Bru3; Mp RANi) and DM1 context (Hand > UPRT; Bru3) at 5 weeks of age showing rescue of diastolic and systolic diameters in Hand > Bru3; Mp RNAi in comparison with controls (D, E) and rescue of cardiac contractility (F) n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile.G–ICross‐sections of cardiac tubes 3D‐reconstructed using Imaris software from 5‐week‐old control (UAS‐Bru3; MpRNAi) (G), DM1 (Hand > UPRT; Bru3) (H) and Mp rescue (Hand > Bru3; Mp…

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  Figure EV5. The young DM1 flies with reduced Mp present normal heart size A–CBox plots showing cardiac size analyses (diastolic (A) and systolic (B) diameters) and percent fractional shortening (C) performed by SOHA approach for controls (UAS‐Bru3; UAS‐MpRNAi and UAS‐UPRT; Bru3) and Mp rescue (Hand > Bru3; Mp RNAi) and DM1 (Hand > UPRT; Bru3) at 1 week of age. n = 20 hearts. Central band corresponds to median. Whiskers correspond to Min to Max. Box corresponds to interquartile range from 25th to 75th percentile. Data information: P‐value < 0.05 considered statistically significant. (*) P‐value = 0.033, (**) P‐value = 0.021. The nonparametric Mann–Whitney test was performed to compare control samples and samples of interest.