The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se

I thank the reviewer for taking the time to review the article and for the comments.

I realise that the role of neuregulin in muscle, especially in transcription of synapse specific genes and NMJ formation has been controversial. The reviewer suggests that in vivo NRG/ErbB signalling could be compensated by another signalling pathway (when ERBB2/4 receptors are ablated) to stimulate dystrophin expression¹ since, the reviewer comments, agrin compensates for this (NRG signalling through ERBB2/4) at the NMJ². However, in the article that is referred to by the reviewer and that describes the ErbB2/4 dKO mice², the aim was to determine if neuregulin (NRG) signaling through ERBB2/4 receptors in muscle was required for synapse specific transcription and NMJ formation. That Agrin plays an important role in acetylcholine receptor clustering and NMJ formation was demonstrated by the fact that mice carrying a mutation in the agrin gene lacked NMJs³. Thus the original article on the ErbB2/4 dKO mice concluded that NRG was not required for synapse-specific gene transcription at the NMJ and that development and maintenance of neuromuscular synapses were only marginally affected. Hence, the article does not conclude that NRG signalling is compensated for by Agrin, but rather that NRG is not required for synapse specific gene expression and NMJ formation. In addition, unlike NRG/ERBB signalling stimulating dystrophin expression in muscle¹, transcription of synapse specific genes and the formation of the NMJ involves reciprocal interactions between muscle fibers and motor neurons.

We subsequently demonstrated that neuromuscular synapses being marginally affected in the ErbB2/4 dKO mice was due to a lack of phosphorylation of alpha-dystrobrevin 1 (α-DB1)⁴. To demonstrate phosphorylation of α-DB1 by signalling through ERBB2/4 receptors, I used ErbB2/4 dKO myotubes  (as I did in this article) as well as myotubes formed from C2C12 and primary myotubes formed from myoblasts from wild type (wt) mice (Schmidt etal. Fig. 7 a & b)⁴. We observed phosphorylation of α-DB1 in samples prepared from myotubes formed from freshly purified primary myoblasts from ErbB 2/4 dKO mice. This phosphorylation of α-DB1 could be blocked by ERBB inhibitors (Schmidt et al. Fig. 7a)⁴ demonstrating  that: (i) signalling through ERBB2/4 receptors was responsible for phosphorylation of α-DB1, (ii) the observed phosphorylation of α-DB1 in myotubes formed from ErbB2/4 dKO primary myoblasts was likely caused by the presence of contaminating cells in the myoblast preparation. Contaminating cells in the myoblast preparation is obviously lost after extended culture periods as confirmed by the lack of phosphorylation of α-DB1 in ErbB2/4 dKO myotubes in the absence of any ERBB inhibitor (Schmidt et al. Fig. 7c)⁴ For the same reason I used the ErbB2/4 dKO myotubes instead of muscle tissue, to demonstrate a lack of dystrophin expression¹.

Once we discovered that signalling through ERBB2/4 receptors phosphorylated α-DB1 (Schmidt et al. Fig. 7 a-c)⁴ we realised that this explained the fragmentation of acetylcholine receptor (AChR) clusters at the neuromuscular synapse in ErbB2/4 dKO mice. This was  subsequently confirmed by experiments in cultured myotubes (wild type and α-DB1 KO) as well as with and without α-DB1 expression constructs (Schmidt et al. Fig. 6, results were not presented in order of discovery)⁴. We could not show absence of α-DB1 phosphorylation in vivo in the muscles of ErbB2/4 dKO mice for the same reasons mentioned above.

Due to the presence of tissues other than skeletal muscle in muscle lysates and cells other than myoblasts in primary myoblast purifications from ErbB2/4 dKO mice, it was not possible to resolve either the lack of phosphorylation of α-DB1 or the lack of dystrophin in the absence of ERBB2/4 signaling in the dKOs. In particular satellite cells present in muscle lysates (and in primary myoblast purifications) were shown to express a high level of dystrophin⁵ and will still express dystrophin in ErbB2/4 dKO mice since the HSA promoter driving CRE expression is not active⁶,and hence ERBB2/4 receptors would not be ablated in these cells. The juxtaposition of dystrophin expression in satellite cells and in myofibers makes it very difficult to distinguish dystrophin expression in satellite cells from that in muscle fibers⁵. It may well be possible that researchers who may have looked for the dystrophin expression in muscle specific ErbB2 and/or ErbB4 KO may have run into this problem and not observed a lack of dystrophin in muscle or muscle sections. Satellite cells, however, appear to have a limited potential to regenerate skeletal muscle⁶ and hence would not fully explain the lack of dystrophic symptoms in ErbB2/4 dKO mice.

Dystrophin and utrophin were detected in loxP-flanked ErbB2/4 myotubes (Gajendran et al., Fig 1.)¹ formed from myoblasts, even after the extended culture period to generate them, and it was only after transient CRE transfection and ablation of ErbB2/4 receptors (confirmation of recombination checked by PCR) dystrophin expression was not observed but utrophin was, in different clones. Understandably, it was not possible to either demonstrate a lack of phosphorylation of α-DB1 or the lack of dystrophin expression when using lysates of total muscle tissues due to the presence of other tissues and cells, including the smooth muscle of blood vessels and satellite cells where ErbB2/4 receptors were not ablated (the HSA promoter driving CRE expression is not active there). In addition to dystrophin detection, any other component of the DAPC present in vivo in the muscle that we wanted to detect in myotubes such as utrophin, a-dystrobrevin, and a-syntrophin were also detected on western blots following immunoprecipitation of the dystrophin associated protein complex (DAPC) .

If the absence of NRG/ErbB2/4 signaling, and as a consequence absence of dystrophin in skeletal muscle, could be compensated in vivo by another mechanism, even possibly through another ligand other than NRG1, then this compensation mechanism would be independent of ERBB2/4 receptors as they are ablated in the skeletal muscles in these mice.

From experiments using α-DB1 KO mytobes in combination with α-DB1 expression constructs, it also became clear that signaling from α-DB1 downregulates dystrophin expression but not utrophin expression (results available at WO 2017/036852 A1 under documents “09.03.2017 Initial Publication with ISR (A1 10/2017)”)⁷. Thus opposing signals from α-DB1 (downregulation) and NRG/ERBB2/4 (upregulation) regulates dystrophin expression. This regulation appears to be mediated by the cleaved intracellular domain of ERBB4 (4ICD) since increased dystrophin levels in dystrobrevin KO myotubes are accompanied by a strong upregulation of ERBB4 expression most of which is cleaved to generate 4ICD (see figures at WO 2017/036852 A1)⁷.

Together with a wealth of published information, the data presented in this article, in my opinion, is essential and sufficient to conclude that expression of dystrophin is stimulated by NRG/ERBB signalling. It follows that in ErbB2/4 dKO mice that did not show dystrophic symptoms (see below), the absence of dystrophin in skeletal muscle but not in smooth muscle of blood vessels, clearly argues that the root cause of DMD is due to the lack of dystrophin in the smooth muscle of blood vessels. Naturally it is possible that dystrophin expression in the endothelial cells, between the smooth muscle and the lumen of blood vessels, in ErbB2/4 dKO mice also plays a role in vasodilation during exercise, assuming that the HSA promoter used to ablate ERBB2/4 receptors is not active in these cells.

Published data on DMD, together with the data presented in this article, would suggest that the lack of dystrophin in skeletal muscle exacerbates the onset of dystrophy caused by a lack of increased blood flow during exercise. This observation is also supported by a very early observation in mdx muscles that only some fibers, and not all, in skeletal muscle initially show dystrophic symptoms⁸ and also by the fact that expressing the full length dystrophin in smooth muscles in mdx mice, even though it was expressed at significantly lower levels than the level of dystrophin found wild type mice, improved aberrant vasoregulation⁹. The fact that skeletal muscle in those mice still showed some atrophy could be due to insufficient dystrophin expression in the smooth muscle causing dystrophic symptoms as a consequence of reduced blood flow during exercise and exacerbated due to the lack of dystrophin in skeletal muscle (Reviewed in Ennen JP et al.)¹⁰. The exacerbation of dystrophic symptoms in skeletal muscle, apart from structural defects, may also be as a result of disruption of signalling events from the DAPC due to the absence of dystrophin and this is further supported by the observation that α-DB KO results in increased dystrophin and ERBB4 expression in myotubes⁷.

As far as could be determined from the literature, there are no compensatory signals in vivo that can ameliorate the effects caused by deficient ERBB2 or ERBB4 signalling such as defective muscle spindle formation¹¹, dilated cardiomyopathy (DCM) following Herceptin (antibody against HER2/ErbB2) treatment¹², DCM in conditional ErbB2^13,14 or ErbB4¹⁵ KO mice, AChR cluster disintegration (also observed in mdx mice) in muscle specific ErbB2/4 dKO mice⁴, and the embryonic lethality observed in ErbB2 or ErbB4 KO mice^16,17. NRG and its ERBB receptors appear to exert their function in a spatio-temporal fashion in development and maintenance (in adults).

The reviewer is correct that no comments were made about dystrophin in the ErbB2/4 dKO mice². However in ErbB2/4 dKO mice nuclei in muscle fibers were examined extensively (see figures in supplemental data of that publication)² and centralized nuclei, a hallmark of DMD^18,19 were not observed. It was also reported in the supporting online text (supplemental data)² that atrophic fibers could not be observed. Hence, based on these observations, there was no indication to look for dystrophin expression. It was also mentioned in the article that sustained muscle strength was not affected and that the ErbB2/4 dKO mice did not have a myasthenic condition. However these mice showed a hind limb extension reflex which was not reported in the article.

I trust that I have addressed the reviewers concern sufficiently, and if the reviewer would be willing to review a revised version of this article, I would welcome the opportunity to submit one that would include some of the explanations/clarifications presented here.

Bibliography
1.      Gajendran, N. The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se [version 1; referees: 1 not approved]. F1000Res. 7, 1321 (2018).
2.      Escher, P. et al. Synapses form in skeletal muscles lacking neuregulin receptors. Science 308, 1920–1923 (2005).
3.      Gautam, M. et al. Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85, 525–535 (1996).
4.      Schmidt, N. et al. Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin. J. Cell Biol. 195, 1171–1184 (2011).
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9.      Ito, K. et al. Smooth muscle-specific dystrophin expression improves aberrant vasoregulation in mdx mice. Hum. Mol. Genet. 15, 2266–2275 (2006).
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The author has responded with some reasonable arguments but the issue with the paper is lack of evidence. No new evidence is provided.  In vivo rescue of the dystrophic phenotype in dystrophic deficient mice and dogs by delivery to skeletal muscle is a compelling argument for the role of dystrophin in skeletal muscle. If there is a role in smooth muscle, then this needs to be shown by in vivo experiments not a cell line.

I thank the reviewer for responding to my comments.

Although the reviewer does not question the discovery described in the manuscript, that NRG signalling is required for dystrophin expression as demonstrated in myotube cultures, concerns remain regarding in vivo evidence to confirm that the root cause of muscular dystrophy is due to the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle.

The reviewer’s concern is understandable given the vast number of publications that focused on the lack of dystrophin in skeletal muscle as the cause of muscular dystrophy. These reports are indeed in contrast to our observation that  ErbB2/4 dKO mice don’t show dystrophic symptoms¹. In these mice, NRG signalling, and as a consequence also dystrophin expression (based on our in vitro results/ new findings), is abolished in skeletal muscles but not in vascular smooth muscle (VSM).

Ideally, to address the reviewers concern, a conditional transgenic mouse where either ERBB2 (or ERBB4) is knocked out in both skeletal and VSM should be examined to have a situation where dystrophin would be lacking in both.

On the other hand it would also be good to demonstrate that dystrophin absence in skeletal muscle does not cause dystrophic symptoms, confirming our observations on the ErbB2/4 dKO mouse.

I address below the reviewers comments.

In vivo evidence for a role for dystrophin in smooth muscle:

Mice where ErbB2 is conditionally knocked out in skeletal muscle, HSA-CRE/ErbB2, following CRE expression driven by the HSA promoter² show a similar phenotype as ErbB2/4 dKO¹ mice mentioned in this article (in both mouse strains the same HSA promoter is used to express CRE). As the HSA promoter is not active in smooth muscle, ERBB2 receptors would not be ablated and dystrophin will still be expressed in VSM, in contrast to skeletal muscles in these mice. The loxP flanked ErbB2 mouse is the same mouse that was used in breeding with an ERBB4 conditional  KO mouse and an HSA-CRE transgenic to finally generate the ErbB2/4 dKO mouse¹. The abnormal muscle spindle formation reported for the HSA-CRE/ErbB2 mouse was not looked at in the ErbB2/4 dKO mouse probably because it had already been described in the HSA-CRE/ErbB2 KO mouse. In addition the study describing the ErbB2/4 dKO mouse was focused on the role of NRG/ERBB signaling in synapse specific transcription and NMJ formation.

Interestingly an ErbB2 conditional knock-out mouse with CRE expression driven by the muscle creatine kinase (MCK) promoter, MCK-CRE/ErbB2 was made that was different from the one mentioned above  as it showed impaired muscle regeneration and a requirement of ERBB2 for survival of muscle spindles and myoblasts³. As VSM express both the brain and muscle isoforms of creatine kinase⁴, the MCK promoter would be active in VSM and hence ERBB2 would be ablated resulting in a loss of dystrophin expression in VSM in these mice, explaining the different histopathology characters between the two ErbB2 KO mice that differ  in the promoter driving CRE expression. The muscle phenotype described for the MCK-CRE/ErbB2 KO mouse was not observed in the HSA-CRE/ERBB2 KO mouse where the HSA promoter is not active in VSM, and hence ErbB2 would not be ablated and therefore dystrophin would be expressed. MCK-CRE/ERBB2 KO mouse primary myoblasts lacking ErbB2, were reported to undergo extensive apoptosis when differentiating into myofibers³, an abnormality that I did not observe that when using ErbB2/4 dKO myotubes⁵.

In another study, siRNA mediated silencing of dystrophin expression in the muscles of adult mice was meticulously analysed⁶. The authors concluded that in spite of the clear absence of dystrophin in the skeletal muscle, they did not observe any of the histopathology characters observed in mdx mice⁶. This paper describes silencing of dystrophin expression in skeletal muscles of adult mice, which results in a delay before the existing dystrophin is depleted, and the authors suggest that the dystrophic pathology observed in dystrophin deficiency may be developmentally regulated. However in the ERBB2/4 dKO mice, dystrophin would be ablated early during development in skeletal muscles since the HSA promoter driving CRE expression is active on E9.5, when dystrophin would also start to be expressed⁷, which would argue against the dystrophic pathology being developmentally regulated.

In vivo rescue of the dystrophic phenotype in mice and dogs may be due to delivery of the therapeutic (construct, anti-sense oligoribonucleotides, gene editing components) into skeletal and vascular smooth muscle

There have been numerous attempts to express dystrophin in skeletal muscle that ameliorated the dystrophic pathology to some extent, but none have led to applicable therapy. As the focus of most therapies are to restore  skeletal, and sometimes also cardiac muscle it was not of concern whether VSM would or would not be targeted during therapy. Gene therapy using Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoters⁸ would also result in the  expression of dystrophin, or smaller versions of it, in surrounding VSM after intra-muscular (IM) injection or particle bombardment⁹. In a Phase I study of gene therapy for Duchenne/Becker muscular dystrophy (D/BDM) the CMV promoter was also used¹⁰ and hence also here dystrophin would be restored  in VSM. Systemic delivery of antisense oligoribonucleotides¹¹, or restoration of dystrophin expression by (intravenous) stem cell transplantation¹², would also result in dystrophin restoration in VSM.

Although in vivo rescue of the dystrophic phenotype in dystrophin deficient mice and dogs focused on delivery of the construct to skeletal muscle, it would also target VSM and hence it is neither possible to exclude the role of VSM nor to conclude that delivery to skeletal muscle is sufficient, to rescue the dystrophic phenotype. Furthermore, simply expressing dystrophin in skeletal muscle fibers lacking dystrophin would restore the dystrophin associated protein complex (DAPC) without saying much about whether muscle function is restored. If indeed muscle function is restored, then it should be looked at whether dystrophin may also have been expressed in VSM through leaking of the therapeutic into those tissues, sometimes through the circulation especially when using viral based therapies as described below.
Gene editing of mutated dystrophin using adeno-associated viruses (AAV) to deliver CRISPR components is a promising therapeutic strategy and has recently been demonstrated to function in a canine model of Duchenne muscular dystrophy¹³. But it still needs to be determined if the gene editing will be sustainable over long term. In this study an AAV serotype 9 that shows tropism for heart and skeletal muscle was used to deliver the CRISPR components. To drive the expression of Cas9 (one of the gene editing components), a muscle specific creatine kinase regulatory cassette was used that should also result in gene editing of dystrophin in VSM especially when the viruses are injected intravenously for systemic delivery. In a second experiment in the same study, 6 weeks following delivery through direct injection into muscles, dystrophin expression and assembly of the DAPC in dystrophic muscles was observed. Dystrophin expression in contralateral muscles that were not injected was also observed and it was attributed to leakage of AAV9 into circulation.

In contrast to the siRNA silencing of dystrophin mentioned earlier, which did not affect muscle function, a very high amount of virus carrying the gene editing components was used in the case of CRISPR mediated gene editing in the canine, which will increase the likelihood for some of it leaking into the circulation and transducing other tissues, including VSM.

Given that therapy for Duchenne patients is largely focused on skeletal muscle, this article would suggest that development of therapeutics should (also) focus on VSM (and possibly also vascular endothelial cells) for any long term therapeutic benefit for patients with Duchenne and Becker muscular dystrophy. In addition the demonstration that NRG/ERBB signalling stimulates dystrophin expression would help to improve treatment regimens with Herceptin/Trastuzumab to ameliorate cardiomyopathy. Finally, this article suggests that investigation of changes in functional dystrophin levels as a possible cause of disease states associated with NRG/ERBB signalling is warranted.

I trust I have addressed the concerns the reviewer raised.

Bibliography
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2.      Leu, M. et al. Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development. Development 130, 2291–2301 (2003).
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