Muscle strength deficiency and mitochondrial dysfunction in a muscular dystrophy model of Caenorhabditis elegans and its functional response to drugs

Abstract

Muscle strength is a key clinical parameter used to monitor the progression of human muscular dystrophies, including Duchenne and Becker muscular dystrophies. Although Caenorhabditis elegans is an established genetic model for studying the mechanisms and treatments of muscular dystrophies, analogous strength-based measurements in this disease model are lacking. Here, we describe the first demonstration of the direct measurement of muscular strength in dystrophin-deficient C. elegans mutants using a micropillar-based force measurement system called NemaFlex. We show that dys-1(eg33) mutants, but not dys-1(cx18) mutants, are significantly weaker than their wild-type counterparts in early adulthood, cannot thrash in liquid at wild-type rates, display mitochondrial network fragmentation in the body wall muscles, and have an abnormally high baseline mitochondrial respiration. Furthermore, treatment with prednisone, the standard treatment for muscular dystrophy in humans, and melatonin both improve muscular strength, thrashing rate and mitochondrial network integrity in dys-1(eg33), and prednisone treatment also returns baseline respiration to normal levels. Thus, our results demonstrate that the dys-1(eg33) strain is more clinically relevant than dys-1(cx18) for muscular dystrophy studies in C. elegans This finding, in combination with the novel NemaFlex platform, can be used as an efficient workflow for identifying candidate compounds that can improve strength in the C. elegans muscular dystrophy model. Our study also lays the foundation for further probing of the mechanism of muscle function loss in dystrophin-deficient C. elegans, leading to knowledge translatable to human muscular dystrophy.This article has an associated First Person interview with the first author of the paper.

Keywords: C. elegans; Melatonin; Muscle strength; Muscular dystrophy; Prednisone.

Fig. 1.

Strength measurements of muscular dystrophy model mutants. (A) Baseline strength of the three different strains taken at three different time points. Measurements began in early adulthood once animals had been transferred to the microfluidic devices. Error bars represent s.e.m. dys-1(eg33), but not dys-1(cx18), is detectably weaker than the wild-type (WT) animal. This effect of dystrophin loss on animal strength is detected beginning on Day 3. (B) The differences in animal strength are not attributable to their diameters, as dys-1(eg33) animals are weaker, but not thinner, than the WT animals. [N for Day 1, 3, 5: WT, N=27, 23, 22; dys-1(cx18), N=29, 24, 22; dys-1(eg33), N=28, 21, 18. Single replicate analyzed with a two-sample t-test.] ****P<0.0001; n.s., nonsignificant.

Fig. 2.

Effect of pharmacological interventions on nematode strength. (A-F) The strengths for three different strains, each with a control and four treatments, are shown. Each strain was treated with melatonin and prednisone during development alone (M1, P1) or during development and adulthood (M2, P2); ‘C’ designates the control animals, which received no treatment. With the exception of wild-type animals undergoing development-only prednisone treatment, the wild type (A) and dys-1(cx18) (B) have no changes in strength in response to treatment. In contrast, all dys-1(eg33) animals improve their strength under any of the four treatments beginning on Day 3 (C). Worm diameters do not fluctuate much for wild type (D), dys-1(cx18) (E) or dys-1(eg33) (F) under the various treatments. In the case of dys-1(eg33), the diameter is not influenced at all by any treatments on Days 3 and 5, the time points at which strength improves drastically under treatment. These data indicate that strength improvements are not due to changes in animal size. Error bars represent s.e.m. [N for Day 1, 3, 5. WT: M1, N=27, 26, 25; M2, N=26, 25, 26; P1, N=26, 24, 23; P2, N=27, 24, 22. dys-1(cx18): M1, N=29, 27, 25; M2, N=30, 24, 24; P1, N=28, 28, 21; P2, N=28, 28, 26. dys-1(eg33): M1, N=27, 25, 23; M2, N=29, 25, 25; P1, N=29, 26, 23; P2, N=27, 25, 26. Single replicate analyzed with a two-sample t-test.] *P<0.05, **P<0.01, ***P<0.001; n.s., nonsignificant.

Fig. 3.

Swimming dystrophin mutants have lower thrashing rates than wild type, and both dys-1(cx18) and dys-1(eg33) respond positively to treatments. (A) Both dys-1(cx18) and dys-1(eg33) have lower thrashing rates than wild type (WT) across all ages. (B) WT, (C) dys-1(cx18) and (D) dys-1(eg33) have varying responses to drug treatments. The most prominent response is that of dys-1(eg33), which improves its thrashing rate drastically under both treatments at all time points. C, control; M, melatonin across life (similar to previous M2 condition); P, prednisone across life (similar to previous P2 condition). Error bars represent s.e.m. For all strains and treatments at each time point, N=10, with five replicates for each worm, with three independent biological replicates for a total of 150 data points per bar; results were analyzed with a two-sample t-test. *P<0.05, ****P<0.0001; n.s., nonsignificant.

Fig. 4.

dys-1(cx18) shows a temperature-sensitive phenotype, and dys-1(cx18) and dys-1(eg33) are levamisole resistant. (A) Day 1 adult dys-1(cx18) animals have lower thrashing rates when cultured at 25°C than at 20°C, whereas dys-1(eg33) is not affected by higher culture temperatures. Thus, dys-1(cx18) appears to be temperature sensitive. For all strains and treatments at each time point, N=10 with five replicates for each worm, with three independent biological replicates for a total of 150 data points per bar. Significances were analyzed using a two-way ANOVA with Tukey's multiple comparison test. (B,C) dys-1(cx18) has a mild levamisole resistance compared with wild type (WT), while dys-1(eg33) has a high resistance, both at 20°C and 25°C. At 20°C, n=50 for two independent biological replicates (total n=100 per strain); at 25°C, n=50 per strain. **P<0.01, ***P<0.001 and ****P<0.0001, for response to levamisole versus other strains tested. Two-way repeated measures ANOVA was used for statistical analysis.

Fig. 5.

There are no differences in sarcomere structure between dys-1 and wild-type worms; however, mitochondrial network defects are apparent, and pharmacological intervention prevents degradation from occurring. (A) Representative images of wild-type (WT), dys-1(cx18) and dys-1(eg33) worms stained with phalloidin on Day 1 of adulthood. (B) Representative images of PJ727, CC97 [dys-1(cx18)] and CC96 [dys-1(eg33)] worms on Day 3 of adulthood. Sarcomere defects are not apparent in either dys-1 mutant. Scale bar: 25 μm. (C) CB5600 (WT with GFP-tagged mitochondria) animals have a tubular mitochondrial network appearance, which is also maintained in animals treated with prednisone and melatonin. (D) CC90 animals [GFP-tagged mitochondria in dys-1(cx18)] exhibit minor fragmentation in the mitochondrial network, which is remedied by prednisone but not melatonin. (E) CC91 animals [GFP-tagged mitochondria in dys-1(eg33)] have noticeably fragmented mitochondrial networks. Animals treated with prednisone do not display this phenotype and instead have relatively WT-like appearance in the mitochondrial network. Animals treated with melatonin have slightly improved mitochondrial network integrity but are not improved to WT levels. Scale bar: 25 μm; the enlarged regions are an additional 1.7× magnification.

Fig. 6.

Mitochondrial dysfunction is also a phenotype of dys-1(eg33). (A) JC-10- and MitoTracker Red-stained mitochondria show moderate depolarization of the mitochondrial membrane in dys-1(cx18) and severe depolarization in dys-1(eg33). This defect is not remedied by treatment with prednisone in dys-1(eg33). Scale bar: 30 µm. (B) Compared with wild type (WT) and dys-1(cx18), dys-1(eg33) has an abnormally high basal oxygen consumption rate (OCR), while maximal respiratory capacity is unaffected. Significances were assessed using a one-way ANOVA and Bonferroni multiple corrections. (C) Treatment with prednisone restores basal OCR to WT levels in dys-1(eg33) animals. Significance was assessed using a one-way ANOVA with Tukey's multiple comparison test. All OCR data are based on 20 worms per well with five wells per strain/condition. *P<0.05, **P<0.01; n.s., nonsignificant.

Fig. 7.

Experimental protocol for testing the efficacy of pharmacological compounds and the microfluidic platform used from the beginning of adulthood. (A) A summary of the different treatments and associated abbreviation used to describe each treatment. (B) Animals start out on agar for the first 3 days, when development is occurring, and all animals except the control group of each strain receive a pharmacological treatment (pink). On the first day of adulthood, all animals are transferred to the devices in which they are fed and imaged over the next few days; animals receiving lifelong treatment continue to receive compounds in the microfluidic device (shown in pink). (C) A view of the 30-chamber microfluidic chip used to house the nematodes from Days 1-5 of adulthood. The device is bonded on a standard 75×50 mm glass slide. (D) An image of a microfluidic chamber used to house a single worm. The deflectable pillars enable force measurement. Scale bar: 300 μm. (E) A close-up view of some of the pillars being tracked for deflection via the NemaFlex image-processing software. Pillars currently in contact with the worm are shown in red; pillars that are deflected in a different frame of the image sequence are shown in blue. Scale bar: 100 μm.