Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne muscular dystrophy
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Summary: The authors seek to tackle the question of exercise and inactivity in Duchenne muscular dystrophy, an important and unsolved issue. They use the zebrafish model system and two paradigms, one an inactivity paradigm (using tricaine) and the other an exercise paradigm using NMES. They find that inactivity worsens the dystrophic phenotype, and that different exercise paradigms impact the dystrophic phenotype differently. Overall this is an important study with exciting data and a potential to impact our understanding of exercise in DMD. However, as described below, all reviewers felt that several critical experimental considerations are necessary to consider in order to substantiate the data claims.
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Abstract
Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy ( dmd ) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.
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Reviewer #3:
This work by Kilroy et al., is a nice study on the role of inactivity on DMD zebrafish and the beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in these fish. The clinical presentation of muscular dystrophies is often variable which makes it difficult to predict the disease severity and progression. The key points of this work are (1) Same genetic defect could lead to phenotypic and functional variability (2) Inactivity in DMD deficiency worsens the disease progression in zebrafish (3) Neuromuscular electrical stimulation improves muscle structure and function. While this study summarizes these key points in a detailed manner, many of the mechanistic details leading to these observations are missing.
There have been many published natural history studies as well as longitudinal …
Reviewer #3:
This work by Kilroy et al., is a nice study on the role of inactivity on DMD zebrafish and the beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in these fish. The clinical presentation of muscular dystrophies is often variable which makes it difficult to predict the disease severity and progression. The key points of this work are (1) Same genetic defect could lead to phenotypic and functional variability (2) Inactivity in DMD deficiency worsens the disease progression in zebrafish (3) Neuromuscular electrical stimulation improves muscle structure and function. While this study summarizes these key points in a detailed manner, many of the mechanistic details leading to these observations are missing.
There have been many published natural history studies as well as longitudinal imaging studies performed in human DMD patients. How does phenotypic data in zebrafish compare with longitudinal phenotypic studies in human patients?
For data presented in figure 1: authors describe the birefringence phenotype in mild mutants as increased degeneration for three days and then increased regeneration. Could they provide any experimental evidence of "muscle regeneration" mentioned in this statement?. Similarly, they mention severe dmd mutant regenerated throughout this study, however, no experimental data is provided to support this statement. As myotome contains both normal and degenerating myofibers, could improvement in birefringence be a consequence of the growth of those normal myofibers vs regeneration of sick myofibers? The term regeneration has also been used later in NEMS studies and needs to be supplemented with the experimental evidence of regeneration.
DMD is caused by damage in sarcolemma and subsequent myofiber detachment. The authors didn't observe any effect on myofiber structure but still found reduced velocity in mutants that were subjected to intermittent inactivity. Could this be due to a slight increase in sarcolemma damage (not examined here) and/or changes in the calcium in muscle fibers? Similarly, what are the effects of extended inactivity on MTJ structure? While authors make good observations with their animal model (as also seen in human and other animal models previously), mechanistic details underlying these changes are lacking.
Authors show few transcripts in figure 10C that were restored to WT level in MT on eNMES treatment. What is the role of these genes in DMD pathology or muscle function? Why do authors think a change in these 5-6 genes out of several hundred genes is important?
While authors demonstrate proposed ECM modeling in response to eNMES, it will be helpful to present changes in ECM structure in response to eNMES treatment (EM or IF).
Previous studies in humans in other animal models have also shown that physical exertion or mild forms of exercise exacerbates the decline in muscle function in DMD deficiency. How are these results comparable to the previously published studies?
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Reviewer #2:
In this paper, Kilroy, J.K. et al. Assess if inactivity in dmd zebrafish is deleterious for muscle structure and function. The authors first, categorized dmd fish into mild and severe phenotypic groups but by 8dpf this phenotypic variability disappears. Next, the authors devised two inactivity regimes: intermittent and extended and found that only fish undergoing extended inactivity exhibited improved muscle phenotype followed by rapid deterioration of muscle structures. Furthermore, these fish were more susceptible to contraction-induced injury. Finally, by varying the frequency, amplitude, and pulse of an electrical current, the authors developed four types of neuromuscular stimulation (NMES) aimed to mimic varying levels of strength training exercises. They found that endurance NMES improved muscle structure, reduced …
Reviewer #2:
In this paper, Kilroy, J.K. et al. Assess if inactivity in dmd zebrafish is deleterious for muscle structure and function. The authors first, categorized dmd fish into mild and severe phenotypic groups but by 8dpf this phenotypic variability disappears. Next, the authors devised two inactivity regimes: intermittent and extended and found that only fish undergoing extended inactivity exhibited improved muscle phenotype followed by rapid deterioration of muscle structures. Furthermore, these fish were more susceptible to contraction-induced injury. Finally, by varying the frequency, amplitude, and pulse of an electrical current, the authors developed four types of neuromuscular stimulation (NMES) aimed to mimic varying levels of strength training exercises. They found that endurance NMES improved muscle structure, reduced degeneration and increased fiber regeneration.
Major Concerns:
For the dmd phenotypic variability: the authors conclude that mild dmd phenotypic fish undergo extensive degeneration for the first three days followed by slight regeneration, while severe dmd fish undergo muscle regeneration throughout the study merit some caution. The authors should consider degeneration and regeneration rates. Compared to dmd fish exhibiting a mild muscle phenotype, dmd fish rate of degeneration early in development might exceed that of regeneration, while later in development, the rate of degeneration is probably lower compared to that of regeneration. To confirm that regeneration is the cause for increased muscle brightness over time in fish with severe muscle phenotype, assays showing degeneration, regeneration (and eventual failure of regeneration) should be performed.
Intermittent inactivity: zebrafish are diurnal, thus it is not surprising that sedating fish at night, when they are naturally at rest, resulted in no major effects on muscle organization. Authors should consider repeating this experiment with daytime sedation and/or alternating between day and night intermittent inactivity. It is not obvious if the authors are referring to fish with mild and/or severe muscle phenotype. This is particularly important because the authors are focusing their birefringence analysis between 5-8 dpf in which phenotypic variability was reported and the mild and severe phenotype have not yet converged. Please clarify.
Birefringence is one of two main assays used throughout the study. Birefringence is an assay that relies on polarized light bouncing from the anisotropic surfaces. Due to the anisotropic nature of the muscle this assay allows for visualizing the structure of the muscle. However, alignment of the fish is a critical part for this assay, if fish are not aligned with the direction of polarized light will exhibit a reduced and variable birefringence results. Thus, this might explain the discrepancy between muscle structure (birefringence assay) and muscle function (swimming behavior) in comparing the different NMES paradigms.
Perhaps a Western blot assays for quantifying either a muscle or housekeeping protein during 5, 6, 7, 8 dpf between wildtype, dmd and dmd NMES treated fish might provide a quantitative picture of degeneration and regeneration cycles based on protein mass of the fish. That is, if the muscles are degenerating, these fish will have less total protein to that of its control and treated counterparts.
Although the authors showed that inactive fish are more susceptible to NMES training. NMES was performed after the inactivity period. No experiments showing NMES treatments during extended inactivity will rule out if NMES could alleviate muscle wasting in relatively inactive fish.
Although the authors found differential gene expression between dmd and wildtype fish that have undergone eNMES treatment. The authors fail to show differential gene expression in dmd and wildtype fish not undergoing eNME treatment. This comparison is critical for determining if eNMES is the result of these changes in genes expressed between both strains.
Authors argue that eNMES improves cell adhesion based on % of fish exhibiting muscle detachment recovery. Authors should consider staining for ECM proteins in dmd and dmd plus eNMES fish to determine if indeed eNMES treatment improved cell adhesion.
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Reviewer #1:
The manuscript by Kilroy and colleagues centers on demonstrating that inactivity is deleterious for DMD zebrafish and that electrical stimulation is highly beneficial in the model. The authors identify a subpopulation of inactive DMD (sapje) zebrafish that progress faster in dystrophic disease muscle breakdown. They use tricaine to restrict movement and show a faster myofiber breakdown in the severe DMD fish cohorts. The authors then use neuromuscular electrical stimulation (NMES) to improve muscle pathologies and overall DMD zebrafish outcomes. The authors go into extensive details in characterizing the consequences of NMES on normal and DMD zebrafish muscle growth, health, and overall function. Transcriptomic analysis reveals fibrotic and regenerative genes are modulated by NMES.
Overall, this is a strong manuscript on …
Reviewer #1:
The manuscript by Kilroy and colleagues centers on demonstrating that inactivity is deleterious for DMD zebrafish and that electrical stimulation is highly beneficial in the model. The authors identify a subpopulation of inactive DMD (sapje) zebrafish that progress faster in dystrophic disease muscle breakdown. They use tricaine to restrict movement and show a faster myofiber breakdown in the severe DMD fish cohorts. The authors then use neuromuscular electrical stimulation (NMES) to improve muscle pathologies and overall DMD zebrafish outcomes. The authors go into extensive details in characterizing the consequences of NMES on normal and DMD zebrafish muscle growth, health, and overall function. Transcriptomic analysis reveals fibrotic and regenerative genes are modulated by NMES.
Overall, this is a strong manuscript on the effects of NMES/electrical stimulation on DMD muscle growth. It does lay several parameters for evaluation of NMES in the zebrafish model. The manuscript is fairly well-written and most of the experiments are presented in a straight-forward manner with clear interpretations. I do have some issues with one or two points that the authors try to extrapolate from their studies. I have significant issues with the description and use of tricaine as an inactivity paradigm in these studies as there are multiple interpretations of these findings. I have a few points about the NMES stimulation protocol and NMJ contribution that should be addressed. This is a good manuscript and can be an important addition to the field if these points are addressed.
- The inactivity paradigm (e.g. figure 2) using tricaine as a means of inducing inactivity has pluses and minuses. There are issues with comparing it to rodent and human inactivity experiments (which usually involve hindlimb/limb immobilization), as the authors here are using chemical inhibition. Tricaine has systemic effects on multiple tissue types and organ systems including neurological and respiratory systems. I would be careful to call this model an inactivity model as a more appropriate model would be to physically restrain the zebrafish larvae to prevent movement. While technically challenging this experiment can be done and would likely be more reflective of the consequences of physical inactivity in the DMD fish than tricaine anesthesia. Mdx mice have respiratory consequences due to pulmonary muscle weakness, independent of an inactivity (Burns et al., J.Physiol., 2017).
The authors need to rule out if the consequences of tricaine administration is due to inactivity or pulmonary/secondary dystrophic pathology issues (e.g. swim bladder or respiration).
The NMES protocol is more extensively established by the authors and has a clearer interpretation. That being said, the main benefit of NMES is to stimulate muscle force/function in the absence of proper innervation by the NMJ, which is also disrupted in DMD. The authors do an excellent job in demonstrating that the NMJ does not change in morphology via immunofluorescence and anatomical observations. Can/have the authors evaluated the functional output of the NMJ in the NMES-treated DMD zebrafish? Were any electrophysiological measurements performed on the NMES treated DMD fish, independent of any therapeutic experimental protocol?
Hmox1 overexpression has been pursued as a strategy for DMD in mice by the Zoltan Arany and Joseph Dulak's groups, so the findings in figure 10 are supported. Have the authors evaluated whether or not the entire Hmox1 pathway was affected in the NMES-treated DMD fish?
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Summary: The authors seek to tackle the question of exercise and inactivity in Duchenne muscular dystrophy, an important and unsolved issue. They use the zebrafish model system and two paradigms, one an inactivity paradigm (using tricaine) and the other an exercise paradigm using NMES. They find that inactivity worsens the dystrophic phenotype, and that different exercise paradigms impact the dystrophic phenotype differently. Overall this is an important study with exciting data and a potential to impact our understanding of exercise in DMD. However, as described below, all reviewers felt that several critical experimental considerations are necessary to consider in order to substantiate the data claims.
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