Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts
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Evaluation Summary:
This is an in-depth and rigorous analysis of transcriptomic changes in myogenic cells lacking dystrophin. Studies are made in both a mouse model and human subjects. the paper bears on possible roles of such alterations in pathogenesis of Duchenne muscular dystrophy. They draw attention to new therapeutic interventions for this condition.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts—the effector cells of muscle growth and regeneration—are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmd mdx myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD ( Myog, Mymk, Mymx , epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmd mdx-βgeo myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.
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Author Response
Reviewer #1 (Public Review):
In this work, Maxime R. and co-authors intended to investigate the consequence of dystrophin absence/alteration in myoblasts, the effector cells of muscle growth and regeneration, and the early role of such cells in the pathogenesis of the disease. They carried out a transcriptomic analysis, comparing transcripts expressed by dystrophic myoblasts isolated from two murine models of DMD (Dmdmdx and Dmdmdx-βgeo) and control healthy mice. The expression of a large number of genes, comprising key regulator of myogenic differentiation (Myod1, Myog, Pax3 etc.) resulted affected in comparison to control in both mouse lines.
We believe that the novelty and importance of these result lie in demonstrating for the first time that the loss of full-length dystrophin expression is both necessary and …
Author Response
Reviewer #1 (Public Review):
In this work, Maxime R. and co-authors intended to investigate the consequence of dystrophin absence/alteration in myoblasts, the effector cells of muscle growth and regeneration, and the early role of such cells in the pathogenesis of the disease. They carried out a transcriptomic analysis, comparing transcripts expressed by dystrophic myoblasts isolated from two murine models of DMD (Dmdmdx and Dmdmdx-βgeo) and control healthy mice. The expression of a large number of genes, comprising key regulator of myogenic differentiation (Myod1, Myog, Pax3 etc.) resulted affected in comparison to control in both mouse lines.
We believe that the novelty and importance of these result lie in demonstrating for the first time that the loss of full-length dystrophin expression is both necessary and sufficient to trigger molecular and functional abnormalities in myoblasts. The fundamental point is that, contrary to the prevailing belief, the dystrophin function may not be just to provide sarcolemma stability in myofibers but rather that there is a disease continuum: DMD defects in satellite cells (Dumont et al., 2015, Ref 45), cause myoblast dysfunctions diminishing muscle regeneration (this work), and also impairing myofiber differentiation (Shoji et al., Ref 4), with the resulting fibre being unstable and therefore degenerating. These data can better explain all the symptoms of dystrophic muscle pathology, where abnormalities in satellite cells, myoblasts and myofibers form the pathological vicious cycle. Moreover, we identify the key trigger behind these abnormalities in dystrophic myoblasts, which is MyoD downregulation. Furthermore, we demonstrate that the additional loss of short dystrophin isoforms, although these are expressed in myoblasts, do not exacerbate the phenotype. This latter finding is very important given the near complete lack of understanding of the pathology in dystrophin-null patients.
Authors highlighted similar gene expression modifications also in a myoblast cell line previously established from the mdx mouse.
Analogous alterations found in both primary myoblasts and in the established myoblast cell line demonstrate that this change is cell-autonomous and not evoked by the external factors in the dystrophic niche, e.g. inflammatory mediators. This also shows that the dystrophic phenotype resists the transcriptomic drift as it is maintained through numerous passages. This approach was praised later on in the review.
To assess the outcomes from the gene ontology analysis, which pointed on the alteration of muscle system and regulation of muscle system processes, authors evaluated the proliferative, chemotactic and differentiative capacities of dystrophic myoblasts. Myoblasts presented increased proliferation, reduced chemotaxis and quite surprisingly, improved differentiating capacity, if considering the transcriptomic data.
The key pathways (proliferation, migration and differentiation), that are essential for myoblast to evoke muscle regeneration, were confirmed to be altered in functional analyses, thus proving these transcriptomic alterations to be functional and biologically relevant. Our data showing accelerated differentiation in mdx myoblasts fully agree with findings by others, both in primary cultures and in isolated myofibers (Yablonka-Reuveni &Anderson, 2005, Ref 22).
Finally, Maxime R. and co-authors carried out a transcriptomic analysis in myoblasts from DMD human subjects. Even though the profile of altered gene expression resulted similar and the GO studies seemed to focus on the same pathway categories, a significative divergence was observed particularly at the level of gene expression.
Given that myoblasts from individual DMD patients present heterogeneous phenotypes (Choi et al., 2016), such divergence at the level of individual gene expression between mouse and human is to be expected. Nevertheless, these changes become convergent in altered GO categories and pathways. In the revised manuscript we have included additional genome-scale metabolic analysis in human DMD myoblasts. This revealed significant alteration in specific metabolic pathways. These are consistent with the metabolic alterations found previously in dystrophic muscle and brain, thus confirming the commonality of dystrophic defects found here in myoblasts and described before in dystrophic tissues. Moreover, this analysis is an additional proof that DMD myoblasts are significantly altered when compared to healthy cells.
Authors link transcriptomic abnormalities and functional changes in proliferation, chemotaxis and differentiation of the dystrophic myoblasts with the alterations (probably epigenetic changes) occurring in satellite cells of dystrophic mice, consequent to the absence of the dystrophin protein. Such modifications in gene expression are supposed to be inherited by pathological myoblasts due to the division of the SC that is no longer asymmetric as occurring in healthy tissue.
Strengths
Transcriptomic data from samples of different sources are solid and rigorous statistical analyses have been carried out.
Transcriptomic and functional data from primary proliferating myoblasts of the two mouse models and from the myoblast cell line are similar. This is a convincing evidence that the transcriptomic alterations observed in primary myoblasts are not influenced by the exposure to the niche environment present in the dystrophic muscle, but that are cell autonomous.
Authors adopted a 3D culture for the functional analysis concerning myoblasts differentiations, in this way better mimicking the process occurring in vivo.
Weaknesses
The mdx mouse phenotype is mild in comparison to the severe symptoms and the rapid disease progression experimented by most of the human DMD subjects. Mdx mice is characterized by cycle of degeneration/regeneration initiating around the age of 6 weeks and continuing for several weeks. It was expected that authors discussed this point in detail, also considering that the animals used in this study were 8 weeks old.
The mdx mouse has a mutation resulting in the loss of full-length dystrophin expression, which reflects the molecular defect affecting the majority of DMD patients. Therefore, mdx is the most commonly used pre-clinical model in DMD studies. The intensity of myonecrosis during this active degeneration and regeneration period (starting at 12 days and not at 6 weeks) is as aggressive as in patients. In fact, it has been suggested that the intensity of myonecrosis seen in mdx mice would be lethal to DMD patients (Duddy et al., 2015). The difference between human and mdx mouse pathology is that, starting at 10 weeks of age, the fibre replacement in mdx leg muscles reduces gradually, due to an unknown mechanism. Therefore, we isolated myoblasts at 8 weeks, when mdx replicates the human pathology. To emphasise the relevance of our findings for the human pathology, we discuss this point in detail in the revised manuscript.
Furthermore, transcriptomic analysis of the human DMD myoblasts highlighted many differences as well as similarities when compared to mouse samples. Why do not focus more on this aspect? According to the authors, dystrophic abnormalities in myoblasts manifest irrespective of differences in genetic backgrounds and across species. The last one is a strong statement that should have been supported at least by functional data regarding chemotaxis proliferation and differentiation of human DMD myoblasts.
What we meant by: “dystrophic abnormalities in myoblasts manifest irrespective of differences in genetic backgrounds and across species” is that the lack of full-length dystrophin expressions results in identical molecular defects in mouse and human primary myoblasts and also in the dystrophic cell line, despite numerous gene expression alterations triggered by the long-term culture in the latter We agree that linking the functional alterations in human dystrophic myoblasts to the transcriptomic alteration that we identified is important. And indeed, altered proliferation, migration and differentiation of human DMD myoblasts have been described before (Witkowski and Dubovitz., 1985; Nesmith et al., 2016; Sun et al., 2020). In fact, these previous findings that were never fully investigated, prompted us to undertake this study. Thus, our data provide a molecular underpinning for these abnormalities. In the revised manuscript we have elaborated on the existing functional data supporting alterations in human myoblasts.
Further functional analyses will be needed to understand their consequences. It would require investigation of numerous parameters, including significant alterations in metabolic pathways, which we identified and described in the revised version of this manuscript. Given the aforementioned individual variability in patients’ population demonstrated by heterogeneous phenotypes in myoblasts, such functional analyses would need to involve a significant number of probands.
Therefore, a detailed study in a sufficiently large cohort of DMD myoblasts is a logical next step from the identification of specific pathway alterations described here. But it is an extensive new project beyond our immediate capability.
In the discussion, the authors suggest two possible mechanisms as responsible for alterations in the behavior of the SC that ultimately affect the functionality of myoblasts, an RNA-mediated pathological process or an alteration in epigenetic regulation. They consider the latter mechanism more likely. This is based in particular on transcriptomic data showing the downregulation of important genes involved in histone modifications, normally linked to transcriptional activation. They also reported from the literature that HDAC inhibitors upregulate MyoD, a gene that is effectively downregulated in this study. Since the authors postulate that the epigenetic dysregulation of Myod1 expression is responsible for the pathological cascade of gene downregulation, ultimately leading to the pathological phenotype, it would have been interesting to evaluate the impact of HDACi on this pathways or the overexpression of enzymes responsible for H3K4 methylation as Smid1 (downregulated in this study).
We have presented several hypotheses regarding the mechanism in which loss of full-length dystrophin expression could affect myoblasts, including restricted spatio-temporal requirement for small amounts of full-length dystrophin and an RNA-based mechanism. The notion that epigenetic dysregulation of Myod1 expression causes a pathological cascade of transcription downregulation of genes controlled by MyoD was based on our finding that transcripts downregulated in dystrophic myoblasts exhibit overrepresentation of MyoD binding sites. We discussed this as a likely mechanism, supported by a body of literature on the known alterations of epigenetic regulation found in DMD (fifteen papers in total). We also offered a hypothesis that since treatment of mdx mice with histone deacetylase inhibitors (HDACi) promoted myogenesis (Saccone et al., 2014) and HDACi upregulate Myod1 (Mal et al., 2001), HDACi could increase myogenesis by counteracting the changes we found in dystrophic myoblast. However, while evaluation of the impact of HDACi or of the overexpression of enzymes responsible for H3K4 methylation would prove or disprove this one of the working hypotheses we made in the Discussion, it would, in no way, alter the key discovery of this study, which is that loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts. Thus, if preferred, this Discussion paragraph could be shortened not to detract the reader from the main findings of this manuscript.
Reviewer #2 (Public Review):
This study is one of many that explore various abnormalities in the mononuclear myogenic cell compartments in DMD. Although the aim has been extensively investigated in the last several decades, it is still relevant.
It is correct that abnormalities of proliferation, migration and differentiation in dystrophic myogenic cells have been reported over decades, but these were not followed up and often disregarded. Certainly, their causative link to DMD mutations and their consequences for the pathology were never investigated. Our study is the first to provide the comprehensive molecular underpinning for these abnormalities, demonstrating that the loss of full-length dystrophin expression directly and significantly affects myoblasts.
The biggest limitation of this study is that it relies on the RNAseq analyses of extensively cultured myoblasts. While the computation analyses are profound, the study lacks any mechanistic explanation for the relevance of the transcriptional differences seen in the DMD myoblasts.
We are not sure where this opinion had originated from. In fact, we used freshy isolated primary myoblasts in RNAseq experiments and then confirmed the key alterations functionally in primary myoblasts freshy isolated from two strains of DMD mice. Furthermore, we performed the mechanistic analyses, where we linked process alterations to functional defects, in which we focussed on proliferation, migration and differentiation, as processes known to impact the DMD pathology.
In an approach considered as one of the strengths of our work by the other Reviewer, these findings in primary myoblasts were then reproduced in myoblast cell line, to demonstrate that alterations observed are not evoked by the exposure to the niche environment present in the dystrophic muscle, but that are cell-autonomous. Importantly, DMD mutant cells show these alterations despite being extensively cultured in vitro, demonstrating expressivity of this mutation. Finally, alterations were confirmed in human primary myoblasts.
Cell purity, the myogenic status of the cells, passage number, and the period that cells were in culture are not well described. This study's cell isolation method allows contamination with non-myogenic cells that can significantly influence the RNAseq analyses. Immunostaining for myogenic markers, for example, MyoD, would indicate the purity of the cell culture. Extensive culturing of the primary myoblasts promotes clonal selection and introduces numerous molecular alterations; thus, the passage number and duration of the culture are significant factors. It looks that some assays were conducted with cells in the high passage. For example, in myogenic differentiation assay where they needed one million cells for each pellet. Maybe that is the reason for the low differentiation rate presented in Sup. Fig 2.
Cell homogeneity across genotypes was fully confirmed by sample-based hierarchical clustering, clearly segregating transcripts into groups corresponding to genotypes. Furthermore, the same alterations were found in corresponding myoblast cell lines, which purity and myogenic potential was demonstrated previously (Onopiuk et al., 2015). Therefore, varying contamination with non-myogenic cells could not significantly influence these results. However, for completeness, in the revised manuscript (Supplementary Figure 8) we described cell characterisation using MyoD as a marker, proving that the well-established myoblast isolation procedure used by us produces pure myoblast cultures.
As for the differentiation assay, isolated myoblasts were never passaged extensively (one passage only) but sufficient numbers were obtained through the efficient isolation. Moreover, cells from every genotype were maintained and treated identically. Therefore, under these given conditions, any differences were the result of the DMD gene mutation and not culturing.
It is hard to explain how DMD myoblasts differentiate better than the WT controls if they have a suppressed myogenic program in the proliferation stage. Even at day 0 of differentiation, DMD myoblasts differentiated better according to the RT-qPCR presented in Figure 5c. Additionally, it is unusual that the marker of differentiation Myog and Myh1 reached the peak at day 2 of differentiation for the WT myoblasts.
In fact, our data fully agree with findings by others, that mdx cells display accelerated differentiation both in primary cultures and in isolated myofibers (Yablonka-Reuveni &Anderson, 2005). Our team recently demonstrated that DMD mutations evoke marked transcriptome and miRNome dysregulations early in human muscle cell development (Mournetas et al, 2021). Expression of key coordinators of muscle differentiation was dysregulated in proliferating dystrophic myoblasts, the differentiation of which was subsequently found to be altered, in line with the mouse cells studied here. Clearly, further studies into the mechanisms of this and numerous other alterations described by us here are urgently needed, as these may uncover new therapeutic targets.
As to whether it is unusual for these differentiation markers to peak at that time, we cannot comment, as no reference for this statement was given and the expressions can vary depending on the experimental conditions used – in our case the 3D culture could make the difference. Yet, again, cells from every genotype were maintained and treated identically and so any differences reflect the impact of the DMD mutation.
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Evaluation Summary:
This is an in-depth and rigorous analysis of transcriptomic changes in myogenic cells lacking dystrophin. Studies are made in both a mouse model and human subjects. the paper bears on possible roles of such alterations in pathogenesis of Duchenne muscular dystrophy. They draw attention to new therapeutic interventions for this condition.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this work, Maxime R. and co-authors intended to investigate the consequence of dystrophin absence/alteration in myoblasts, the effector cells of muscle growth and regeneration, and the early role of such cells in the pathogenesis of the disease. They carried out a transcriptomic analysis, comparing transcripts expressed by dystrophic myoblasts isolated from two murine models of DMD (Dmdmdx and Dmdmdx-βgeo) and control healthy mice. The expression of a large number of genes, comprising key regulator of myogenic differentiation (Myod1, Myog, Pax3 etc.) resulted affected in comparison to control in both mouse lines. Authors highlighted similar gene expression modifications also in a myoblast cell line previously established from the mdx mouse. To assess the outcomes from the gene ontology analysis, which …
Reviewer #1 (Public Review):
In this work, Maxime R. and co-authors intended to investigate the consequence of dystrophin absence/alteration in myoblasts, the effector cells of muscle growth and regeneration, and the early role of such cells in the pathogenesis of the disease. They carried out a transcriptomic analysis, comparing transcripts expressed by dystrophic myoblasts isolated from two murine models of DMD (Dmdmdx and Dmdmdx-βgeo) and control healthy mice. The expression of a large number of genes, comprising key regulator of myogenic differentiation (Myod1, Myog, Pax3 etc.) resulted affected in comparison to control in both mouse lines. Authors highlighted similar gene expression modifications also in a myoblast cell line previously established from the mdx mouse. To assess the outcomes from the gene ontology analysis, which pointed on the alteration of muscle system and regulation of muscle system processes, authors evaluated the proliferative, chemotactic and differentiative capacities of dystrophic myoblasts. Myoblasts presented increased proliferation, reduced chemotaxis and quite surprisingly, improved differentiating capacity, if considering the transcriptomic data. Finally, Maxime R. and co-authors carried out a transcriptomic analysis in myoblasts from DMD human subjects. Even though the profile of altered gene expression resulted similar and the GO studies seemed to focus on the same pathway categories, a significative divergence was observed particularly at the level of gene expression.
Authors link transcriptomic abnormalities and functional changes in proliferation, chemotaxis and differentiation of the dystrophic myoblasts with the alterations (probably epigenetic changes) occurring in satellite cells of dystrophic mice, consequent to the absence of the dystrophin protein. Such modifications in gene expression are supposed to be inherited by pathological myoblasts due to the division of the SC that is no longer asymmetric as occurring in healthy tissue.
Strengths
Transcriptomic data from samples of different sources are solid and rigorous statistical analyses have been carried out.
Transcriptomic and functional data from primary proliferating myoblasts of the two mouse models and from the myoblast cell line are similar. This is a convincing evidence that the transcriptomic alterations observed in primary myoblasts are not influenced by the exposure to the niche environment present in the dystrophic muscle, but that are cell autonomous.
Authors adopted a 3D culture for the functional analysis concerning myoblasts differentiations, in this way better mimicking the process occurring in vivo.Weaknesses
The mdx mouse phenotype is mild in comparison to the severe symptoms and the rapid disease progression experimented by most of the human DMD subjects. Mdx mice is characterized by cycle of degeneration/regeneration initiating around the age of 6 weeks and continuing for several weeks. It was expected that authors discussed this point in detail, also considering that the animals used in this study were 8 weeks old. Furthermore, transcriptomic analysis of the human DMD myoblasts highlighted many differences as well as similarities when compared to mouse samples. Why do not focus more on this aspect?
According to the authors, dystrophic abnormalities in myoblasts manifest irrespective of differences in genetic backgrounds and across species. The last one is a strong statement that should have been supported at least by functional data regarding chemotaxis proliferation and differentiation of human DMD myoblasts.
In the discussion, the authors suggest two possible mechanisms as responsible for alterations in the behavior of the SC that ultimately affect the functionality of myoblasts, an RNA-mediated pathological process or an alteration in epigenetic regulation. They consider the latter mechanism more likely. This is based in particular on transcriptomic data showing the downregulation of important genes involved in histone modifications, normally linked to transcriptional activation. They also reported from the literature that HDAC inhibitors upregulate MyoD, a gene that is effectively downregulated in this study. Since the authors postulate that the epigenetic dysregulation of Myod1 expression is responsible for the pathological cascade of gene downregulation, ultimately leading to the pathological phenotype, it would have been interesting to evaluate the impact of HDACi on this pathways or the overexpression of enzymes responsible for H3K4 methylation as Smid1 (downregulated in this study). -
Reviewer #2 (Public Review):
This study is one of many that explore various abnormalities in the mononuclear myogenic cell compartments in DMD. Although the aim has been extensively investigated in the last several decades, it is still relevant.
The biggest limitation of this study is that it relies on the RNAseq analyses of extensively cultured myoblasts. While the computation analyses are profound, the study lacks any mechanistic explanation for the relevance of the transcriptional differences seen in the DMD myoblasts.
• Cell purity, the myogenic status of the cells, passage number, and the period that cells were in culture are not well described. This study's cell isolation method allows contamination with non-myogenic cells that can significantly influence the RNAseq analyses. Immunostaining for myogenic markers, for example, MyoD, …
Reviewer #2 (Public Review):
This study is one of many that explore various abnormalities in the mononuclear myogenic cell compartments in DMD. Although the aim has been extensively investigated in the last several decades, it is still relevant.
The biggest limitation of this study is that it relies on the RNAseq analyses of extensively cultured myoblasts. While the computation analyses are profound, the study lacks any mechanistic explanation for the relevance of the transcriptional differences seen in the DMD myoblasts.
• Cell purity, the myogenic status of the cells, passage number, and the period that cells were in culture are not well described. This study's cell isolation method allows contamination with non-myogenic cells that can significantly influence the RNAseq analyses. Immunostaining for myogenic markers, for example, MyoD, would indicate the purity of the cell culture. Extensive culturing of the primary myoblasts promotes clonal selection and introduces numerous molecular alterations; thus, the passage number and duration of the culture are significant factors. It looks that some assays were conducted with cells in the high passage. For example, in myogenic differentiation assay where they needed one million cells for each pellet. Maybe that is the reason for the low differentiation rate presented in Sup. Fig 2.
• It is hard to explain how DMD myoblasts differentiate better than the WT controls if they have a suppressed myogenic program in the proliferation stage. Even at day 0 of differentiation, DMD myoblasts differentiated better according to the RT-qPCR presented in Figure 5c. Additionally, it is unusual that the marker of differentiation Myog and Myh1 reached the peak at day 2 of differentiation for the WT myoblasts. -
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