Validation of muscle degeneration due to miRNA loss

The progression of muscle degeneration phenotypes upon stress, due to miRNA loss was further followed up in greater detail for three miRNAs (miR-137, miR-927, and miR-966).

The experiment was done in three independent biological replicates and the appearance of MD phenotypes was quantified in miRNA mutants as mild and strong muscle degeneration (Figure 9A).

Additionally, muscle atrophy was scored in these animals at normal and different stress conditions. Muscle atrophy, in general, is described as a loss of muscle mass that is driven by an increase in protein degradation or a decrease in protein synthesis. It is an integral feature of systemic diseases including cancer, cachexia, cardiac failure, AIDS, and sepsis.

Loss of muscle mass due to aging, also known as sarcopenia is often associated with muscle disuse, fasting (starvation), extrinsic changes in innervation, stem cell function, and endocrine regulation of muscle homeostasis (Demontis et al., 2013). Since most miRNA mutants were responsive to various stresses, including aging, muscle atrophy was also studied in miRNA mutants to dissect miRNA involvement in muscle maintenance.

The frequency of muscle degeneration and muscle atrophy in miRNA mutants kept at normal and different stress conditions, such as young, temperature stress, sugar starvation, and aging were normalized to the frequency of muscle degeneration and atrophy of young control animals. Muscle degeneration phenotypes were scored as mild and strong muscle degeneration as shown with blue and red arrows, respectively and muscle atrophy was scored as shown in green arrows (Figure 9A). As observed before, all animals, miRNA mutants and control were sensitive to stress and develop muscle degeneration and muscle atrophy in response to unfavorable conditions. However, the severity of stress-dependent muscle maintenance defects was different in different mutants. Interestingly, even change in genetic background in controls resulted in different frequencies of muscle degeneration, implying that in general, the muscle tissue is extremely sensitive to stress. For example in Figure 8, heterozygous Canton-S/OR were used as control and the frequency of muscle degeneration was never higher than 10%, while in this experiment (Figure 9), we used w¹¹¹⁸ mutants crossed to wildtype OR as control. This resulted in the dramatic increase in muscle degeneration phenotypes. A similar observation was made on miR-137 mutants that were back-crossed in the w¹¹¹⁸ genetic background for 8 generations.

Upon stress and aging, controls themselves showed approximately 1.5-2.5 fold increase in the incidence of muscle degeneration relative to young non-stressed animals. MiR-137 mutants had approximately 2 fold increase in muscle degeneration already at young age when compared to control flies of the same age. This phenotype was even more enhanced upon temperature stress and aging. Similarly, miR-966 mutants showed muscle degeneration phenotypes as early as in young age (1.5 fold higher than control) and the phenotype was enhanced upon aging (3.5 fold compared to 2.5 fold in control). However, 927 and miR-966 mutants did not show temperature stress or sugar starvation response (Supplementary

Table 4). MiR-927 mutants were responsive only to aging with approximately 4 fold increase in muscle degeneration compared to control flies (Figure 9B).

A comparison of muscle atrophy showed that upon various stresses control flies had an increase in the frequency of muscle atrophy when compared to young non-stressed animals (approximately 2.5-4 fold increase). Young miRNA mutants had muscle atrophy already at 7 day, the frequency of which was approximately three times higher than in control. In addition, miRNA mutants showed loss of muscle integrity in response to sugar starvation and during aging, and the frequency of atrophic muscle appearance was six times higher than in young controls. This tendency of being extremely sensitive to aging was also observed in miR-927 and miR-966 mutants that also showed 6-fold increase in the frequency of atrophic muscle appearance in comparison to young controls (Supplementary Table 4). The frequency of muscle atrophy was observed in higher rate compared to the frequency of muscle degeneration. MiR-927 and miR-966 mutants did not show any differences in muscle degeneration phenotypes during temperature stress and sugar starvation, while an increase in 3-4 fold difference of atrophic muscles was observed compared to young control. Since miR-137 mutants showed an increase in muscle degeneration and muscle atrophy phenotypes already in young animals and these phenotypes were significantly progressing during aging, it indicates that this miRNA can be an especially good candidate to study further in order to elucidate the common mechanisms between MD and age-dependent muscle loss.

Loss of miRNA affects muscle maintenance

(A) Representative images of muscle degeneration and atrophic muscle phenotypes in adult Drosophila IFMs. Blue and the red arrows represent mild and strong muscle degeneration, while the green arrows represent muscle atrophy. (B) A bar graph representing relative muscle degeneration and (C) relative muscle atrophy in different stresses compared to young control flies (w¹¹¹⁸/OR). Stress enhances muscle degeneration and atrophy even in wild type flies. Control and miR-137 mutants were responsive to all stresses when compared to young age (significance shown by red stars). In general, miR-137 mutants had ~2 times more muscle degeneration in young and sugar starvation and ~3 times more muscle degeneration in temperature stress and aging compared to young controls. Similarly, an increase in muscle atrophy by ~3-5.5 fold was observed in miR-137 mutants when compared to young controls.

Values are obtained from the averages of 3 biological replicates. Error bars represent AVE±SEM and statistical significance was determined by two-tailed Student’s t-test.

*P<0.05, **P<0.01, ***P<0.001 represent comparisons to control at the same condition, while *P<0.05, **P<0.01, ***P<0.001 represent comparisons within the same genotype at a young age. D7= young, 7 day old, TS= temperature stress, SS= sugar starvation, and D30=

aging, 30 day old. Scale bar 100 μm. See also Supplementary Table 4.

3.3 Conserved predicted targets of miRNAs are associated