Expanded CCUG repeat RNA expression in Drosophila heart and muscle trigger Myotonic Dystrophy type 1-like phenotypes and activate autophagocytosis genes

Figure 1. Muscleblind is retained in ribonuclear foci in flies expressing expanded CUG or CCUG repeats. Representative fluorescent confocal images of IFMs (A–F) and heart cells (G–L) from flies expressing expanded CUG (A–C and G–I) or CCUG (D–F and J–L) repeats under the control of the Mhc-Gal4 and GMH5-Gal drivers, respectively. Ribonuclear foci retaining Muscleblind were present in flies expressing long CUG or CCUG repeats. Merged images in (C,F,I and L) include DAPI (blue) counterstaining of the nuclei. Scale bar = 10 µm.

Figure 2. CUG and CCUG expansions cause Muscleblind-dependent missplicing. (A,B) Representative semi-quantitative RT-PCR showing inclusion of Fhos exon 16′ in flies expressing the indicated constructs in muscle (A) or heart (B) under the control of Mhc-Gal4 or GMH5-Gal4, respectively. Endogenous Rp49 was used for normalization. Percentage of exon 16′ inclusion, revealed that expression of long CUG or CCUG repeats in the fly muscle (C) or heart (D), favored increased use of this exon. qRT-PCR results of Serca exon 13 expression relative to Rp49 expression, confirmed that the use of this exon in the flies expressing the expanded repeats and the control flies is significantly different in muscle (E) and heart tissues (F). The histograms show the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 3. The expression of autophagy-related genes is upregulated in flies expressing expanded CUG or CCUG repeats in muscle or heart. Relative expression levels of Atg4, Atg7, Atg8a, Atg9 and Atg12 measured by qRT-PCR in muscle (Mhc-Gal4 driver; A) and heart samples (GMH5-Gal4 driver; B), showed a significant upregulation of these autophagy-related genes in flies expressing expanded CUG (CUG(250)×) or CCUG repeats (CCUG(1100)×). The histograms show the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 4. Expression of expanded CUG or CCUG repeats in muscle induces similar levels of muscle area reduction and degeneration. (A–J) Dorsoventral sections of resin-embedded fly thoraces. In all images the dorsal side is displayed at the top. Mhc-Gal4 was used to drive the expression of the indicated constructs in muscle. (K) Quantification of the mean percentage of muscle area per genotype relative to the muscle area of the control flies (GFP), which is considered as 100%. While young flies (3 day-old, in A–E) expressing 20 CUG or CCUG repeats were not different from control flies expressing GFP, flies expressing expanded CUG or CCUG repeats have a 50% reduction in IFM muscle area. All aged flies (30 day-old, in F–J) displayed reduced muscle area compared to young flies of the same genotype. However, vacuolization (arrows) and occasional muscle splitting (asterisk) characteristic of degenerating muscles were present only in muscles expressing expanded repeats. The graph shows the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 5. Survival and locomotor function were reduced in flies expressing expanded CUG or CCUG repeats in muscle. (A) Average percentage of live flies versus age (in days). The Mhc-Gal4 driver was used to induce the expression of the indicated constructs in muscle. The horizontal dotted line marks the median survival. Whereas control and short-repeat-expressing flies had similar median survival (GFP; n = 90, CUG(20)×, n = 100 and CCUG(20)×, n = 95), long CUG and CCUG-expressing flies have reduced survival (CUG(250)×; n = 95 and CCUG(1100)×; n = 100). Differences in the survival curves were highly significant (p < 0.0001, log-rank test). (B) Histogram showing the climbing speed as the mean speed ± SEM in mm/s. Flies expressing long CUG or CCUG repeats had reduced climbing velocity compared to control flies or flies expressing the short versions of repeats. (C) Notched box plot showing the median and the distribution of the average landing height data obtained in the flight assay with the relevant genotypes. Flight disability was observed in flies expressing long CUG or CCUG repeats. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 6. Cardiac dysfunction in DM1 and DM2 model flies includes diastolic and systolic elongation, increased arrhythmicity, and reduced contractility. The mean heart period (HP, in A) was significantly increased in flies expressing expanded CUG or CCUG repeats in heart. This increase was caused by a prolongation of both diastolic and systolic intervals (DI, in B and SI, in C) in the model flies. Heart tube contractility and cardiac rhythm were also affected in these flies, because the percentage of fractional shortening (%FS in D) was reduced to only 20% and arrhythmia, measured as the arrhythmicity index (AI in E), was significantly increased to similar levels in both DM1 and DM2 model flies. Graph bars show the mean values and their standard errors (n = 18 to 29). *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Figure 7. Expression of expanded CUG or CCUG repeats in fly heart alters survival but not locomotion. (A) Average percentage of live flies, with the indicated genotypes, versus age (in days). The GMH5-Gal4 driver was used to induce expression of the indicated genotypes in cardiomyocytes. Horizontal dotted line marks the median survival. Flies expressing expanded CUG (CUG(250)×; n = 100) or CCUG (CCUG(1100)×; n = 97) repeats had a reduced lifespan compared to control flies (GFP; n = 100) or flies expressing the short versions of the repeats (CUG(20)×; n = 95 and CCUG(20)×; n = 99). The differences in survival curves were highly significant (p < 0.0001, log-rank test). (B) Histogram showing the climbing velocity of flies as the mean speed ± SEM in mm/s. Expression of long CUG or CCUG repeats in heart did not modify climbing velocity compared to control flies or flies expressing the short versions of repeats. (C) Notched box plot showing the median and the distribution of the average landing height data obtained in the flight assay using flies with the genotypes indicated. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t -test).