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

Introduction

Signaling from neuregulin 1 (NRG), through its epidermal growth factor (EGF) family of receptors ERBB1-4, has major functions in several organs such as heart, breast, and nervous system including central and peripheral synapses. The role of NRG signaling in skeletal muscle has been controversial. To investigate signaling events in muscle fibers, myotubes formed by fusion of myoblasts, are routinely used as the in vitro equivalent of muscle fibers. We already reported that in myotubes, formed from C2C12 myoblasts, NRG signaling through ERBB2/4 heterodimeric receptors phosphorylates α-dystrobrevin 1 (α-DB1)¹, one of the components of the dystrophin associated protein complex (DAPC). DAPC links the intracellular actin cytoskeleton to the extracellular matrix and is thereby thought to provide structural stability during muscular activity. DAPC, apart from containing dystrophin, a 427 kDa protein, consists of several other proteins such as α- and β-dystrobrevins, dystroglycans, sarcoglycans, sarcospan, syntrophins, and laminins. At the neuromuscular synapse, the DAPC is also formed with utrophin, also a 427 kDa protein, instead of dystrophin. The phosphorylation of α-DB1 through NRG/ERBB signaling stabilized acetylcholine receptors (AChRs) at the neuromuscular synapse^1,2.

Duchenne and Becker muscular dystrophy (D/BMD) patients have mutation(s) in the dystrophin gene, resulting in the expression of a truncated dystrophin protein^3–6. The main body of research on DMD argues for the lack of dystrophin in skeletal muscle as the cause for DMD. In mice, apart from muscular dystrophy, absence of dystrophin causes neuromuscular junction (NMJ) fragmentation⁷ similar to the NMJ fragmentation associated with a loss of NRG/ERBB signaling¹. Lack of dystrophin, besides causing muscular dystrophies, results in cardiomyopathy⁸ and is also responsible for several disease states in the brain⁶. The importance of NRG signaling for normal cardiac development in mice was firmly established by the fact that ablation of NRG, ERBB4, or ERBB2 resulted in premature death during midgestation^9–11. In cardiac muscle NRG/ERBB4 signaling is sufficient for cardiomyocyte proliferation and repair of heart injury¹², but knowledge of the detailed signaling mechanisms and the target proteins through which this was achieved are lacking. The aim of this investigation was to identify the function of NRG/ERBB signaling in muscle and, as it phosphorylated α-DB1 in the DAPC complex, determine if it had a functional role in muscular dystrophy by identifying downstream signaling targets.

Methods

Western analysis

Myotubes from 10-cm culture dishes were harvested in 600 μl lysis buffer and protein complexes were immunoprecipitated as described previously^1,14 with modifications. In brief, myotubes harvested in ice-cold lysis buffer (10 mM Na₃PO₄, pH 7.8, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitor mixture (Roche), and phosphatase inhibitors Pic1 and Pic2 (Sigma-Aldrich)) were homogenized in a Dounce homogenizer and incubated for 3 h at 4°C with protein G-coupled mouse monoclonal syntrophin antibody 1351 (4 μl/80 μl protein G beads, Abcam, catalog number ab11425). Beads were then washed in lysis buffer containing protease inhibitors, but without Triton X-100, resuspended in 3 x SDS loading buffer (150 mM Tris-HCl [pH 6.8], 300 mM dithiothreitol [added just before use], 6% SDS, 0.3% bromophenol blue, and 30% glycerol), and denatured (94°C, 3 min) before loading on an 8% acrylamide/0.8% bis-acrylamide (Figure 1 A & B) or 6–8% gradient/0.8% bis-acrylamide (Figure 2B) SDS-PAGE gels buffered with Tris-glycine.

Figure 1. Dystrophin, and utrophin levels in Erbb2/4 dKO myotubes.

(A and B), Western blots of immunoprecipitated DAPC proteins from myotubes with loxP flanked exons of Erbb2 and Erbb4 gene (lanes 1 to 3) and Cre mediated knock-out of Erbb2 and Erbb4 genes (lanes 4 to 6) detected with dystrophin (A) and utrophin (B) antibodies. Lanes 1 to 3 and 4 to 6 each represent the same experiment performed independently and loaded on the same gel. The lower part of the blot shows detection of syntrophin that served as a loading control. Immunoglobulin G (IgG) detected is the syntrophin antibody used for the immunoprecipitation. As the western blot in (A) was stripped of antibodies and used in (B), the loading control in (B) applies to both A and B. (C and D) qPCR data of dystrophin (C) and utrophin (D) levels in C2C12 myotubes, relative to Erbb2/4 dKO (erbb2/4^-/-) myotubes. Expression levels were normalized to ribosomal protein L8 (rL8) expression. This experiment was performed at least twice with similar results. This figure was previously published in a patent (Patent Link: WO 2017/036852 A1), but the copyright is the author’s own.

Figure 2. Dystrobrevin 1 expression levels in Erbb2/4 dKO myotubes and association with the DAPC.

(A) qPCR data of α-dystrobrevin 1 expression in Erbb2/4 dKO (erbb2/4^-/-) myotubes relative to C2C12 myotubes. Expression levels were normalized to ribosomal protein L8 (rL8) expression. This experiment was performed at least twice and gave similar results. (B) Western blots of control and transfected C2C12 and dystrobrevin KO (db^-/-) myotubes following IP of the DAPC and detection of dystrobrevin and EGFP-dystrobrevins (GFP-DB1 & GFP-DB1-P3 with 3 mutated tyrosine phosphorylation sites) with anti-α-dystrobrevin 1 antibodies. The EGFP-DB1 transfected dystrobrevin KO myotubes appears to have lost the expression construct however a repeat of the experiment showed expression of the transfected construct (see raw data). Following syntrophin detection (lower panel), the blot was stripped and used for dystrobrevin detection (upper panel). The lower panel in figure 2b (syntrophin detection) was previously published in a patent (Patent Link: WO 2017/036852 A1), but the copyright is the author’s own.

Gels were transferred onto PVDF membranes (Millipore) and subject to ECL (Thermo Fisher Scientific) development after incubation with primary and secondary antibodies. BSA (3%) was used as a blocking reagent. The following primary antibodies were used: rabbit anti-dystrophin (H300) polyclonal (diluted 1:400, catalog number sc-15376) and mouse anti-utrophin (55) monoclonal (diluted 1:400, catalog number sc-136116) were from Santa Cruz Biotechnology, Inc., mouse monoclonal anti-syntrophin 1351 (4 μl antibody/80 μl protein G beads for lysate from a 10 cm culture dish of myotubes, catalog number ab11425) from abcam, rabbit anti-α-dystrobrevin (1:1,500; a kind gift of D.J. Blake and R. Nawrotzki, University of Cardiff, Wales, UK) and rabbit anti-α-syntrophin 258 (5 μg/ml for Westerns; a kind gift from Stanly C. Froehner and Marvin Adams, University of Washington, Seattle, WA). Goat anti-mouse IgG-HRP (catalog number sc-2005) and goat anti-rabbit IgG-HRP (catalog number sc-2004) secondary antibodies (Santa Cruz Biotechnology, Inc.) were used at a 1:5,000 dilution.

Results

NRG/ErbB signaling is required for dystrophin expression

We previously reported that there were two isoforms of α-DB1 associated with the DAPC, a 75 kDa and an 89 kDa protein¹. These two α-DB1 proteins correspond to the previously reported proteins between 66-97 kDa with the smaller protein containing more phosphotyrosine¹⁶. We also demonstrated that ablation of ERBB2/4 receptors resulted in a lack of phosphorylation of the 75 kDa protein¹ and that lower levels of this α-DB1 isoform associated with the DAPC compared to the 89 kDa isoform. However in myotubes with intact ERBB2/4 receptors, such as in C2C12 myotubes, we did not observe such a difference in the levels of these two isoforms¹. In the absence of NRG/ErbB signalling we also observed AChR cluster fragmentation both in vitro in myotubes and in vivo in muscle fibers. Similar observations were made in mdx mice deficient in dystrophin. Mdx mice have a reduction in all the dystrobrevin isoforms at the sarcolemma¹⁷, and thus in the DAPC, as well as AChR cluster fragmentation⁷ in muscle compared to wild-type mice. These observations suggested that a lack or reduced level of dystrophin may have caused the reduction in the 75 kDa α-DB1 protein associated with the DAPC, and AChR cluster fragmentation, in the Erbb2/4 dKO myotubes implying that one of the targets of NRG/ERBB signaling is dystrophin.

To investigate this, Erbb2/4 dKO myotubes derived from immortalized Erbb2/4 dKO myoblasts¹ and myotubes formed from myoblasts containing loxP flanked Erbb2/4 genes, before transfection with Cre recombinase, were used. Both, ERBB2 and ERBB4 receptors were ablated in skeletal muscle to eliminate NRG signaling¹, because ERBB4 receptors, apart from forming heterodimers, can also form homodimers and ERBB2 can heterodimerize with ERBB3¹⁸. Furthermore, as cultured myotubes secrete NRG¹⁹, the external addition of NRG was not necessary, as demonstrated for phosphorylation of α-dystrobrevin 1 by NRG/ERBB¹ signaling.

In three independent experiments (Figure 1A) each using myotubes formed from a different myoblast clone with loxP-flanked Erbb2/4 genes, dystrophin was clearly detected (lanes 1–3). However dystrophin was not detected in Erbb2/4 dKO myotubes (lanes 4–6) where ERBB2/4 receptors were ablated after Cre mediated recombination of loxP flanked Erbb2/4 genes. Utrophin on the other hand was detected in myotubes with and without ERBB2/4 receptors (Figure 1B) demonstrating that NRG/ERBB signaling selectively regulates dystrophin expression (Figure 3).

Figure 3. Schematic drawing of neuregulin (NRG) signaling to stimulate dystrophin expression.

Under normal circumstances, NRG signaling through ERBB2/4 (HER2/4) receptors stimulates dystrophin expression, allowing the formation of a normal dystrophin-associated protein complex (DAPC). If either the dystrophin gene contains mutations or NRG signaling is blocked, then in the absence of functional dystrophin, a normal DAPC is not formed, resulting in various disease states such as dilated cardiomyopathy, Duchenne/Becker muscular dystrophy (D/BMD), and neuromuscular synapse instability.

The estimation by qPCR of dystrophin and utrophin mRNA levels (Figure 1C, D) confirm that not only the protein but also dystrophin mRNA expression is absent in Erbb2/4 dKO (shown in Figure 1 as erbb2/4^-/- ) myotubes. Signals in qPCR were only observed above threshold at about 34 cycles which is essentially detection of non-specific amplification or background signal, whereas detection of rL8, used to normalize expression of dystrophin and utrophin, was above threshold at about 22 cycles in Erbb2/4 dKO and C2C12 samples. Detection of dystrophin mRNA in C2C12 cells by qPCR confirmed that the primers used to amplify dystrophin functioned. It is only possible to conclude that dystrophin is present in C2C12 and absent in Erbb2/4 dKO myotubes. The level cannot be estimated. This is because the level of dystrophin in C2C12 was relative to that in Erbb2/4 dKO myotubes, for which essentially background non-specific values were obtained in qPCR due to the absence of dystrophin mRNA. Utrophin detection (Figure 1D), using the same RNA/cDNA preparation used for dystrophin detection, confirmed that the cDNA preparation from Erbb2/4 dKO myotubes was intact. Utrophin expression was reduced to only less than half the amount (Figure 1D) in Erbb2/4 dKO myotubes compared to C2C12 myotubes which may be due to the lack of NRG signaling since NRG was reported to stimulate utrophin expression to some extent²⁰. Myotubes formed from C2C12 myoblasts were used as a control for qPCR instead of myotubes containing loxP flanked Erbb2/4 genes (used for the western blots in Figure 1A, B) as the loxP-flanked Erbb4 gene²¹ is a hypomorph due to the insertion of the neo selection cassette and therefore levels of dystrophin mRNA may have been affected (NRG signaling through ERBB2/4 stimulate dystrophin expression and reduced Erbb4 expression may have affected this). This is not a problem for dystrophin protein detection (not estimation) in immunoprecipitated samples from myotubes containing loxP flanked Erbb2/4 genes. qPCR on Erbb2/4 dKO myotubes confirmed the absence of dystrophin expression (Figure 1C) as observed on the western blot (Figure 1A). Hence NRG/ERBB signaling is necessary for dystrophin expression. However, the Erbb2/4 dKO mice did not show dystrophic symptoms¹.

Due to the presence of tissues and cells other than skeletal muscle and myoblasts, such as vascular smooth muscles (VSM) and satellite cells, in muscle lysates and in primary myoblast purifications from Erbb2/4 dKO mice, it is not possible to resolve the lack of dystrophin in skeletal muscle in these mice. In particular satellite cells were shown to express a high level of dystrophin²² and will still express dystrophin in Erbb2/4 dKO mice since the HSA promoter driving Cre recombinase expression is not active²³, 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 fibers²². Satellite cells, however, appear to have a limited potential to regenerate skeletal muscle²³ and hence it would not fully explain the lack of dystrophic symptoms in Erbb2/4 dKO mice.

Discussion

We previously reported that in HSA-Cre/Erbb2/4 dKO mice, loss of NRG signaling in skeletal muscles led to a lack of α-DB1 phosphorylation and its reduced association with the DAPC, resulting in AChR cluster fragmentation¹. The current study shows abolishing NRG/ErbB signaling in myotubes results in the loss of dystrophin expression and that the reduced association of α-DB1 with the DAPC is independent of its phosphorylation state.

Abolishment of NRG/ERBB signaling in skeletal muscle results in the specific loss of dystrophin and this is supported by: 1) Erbb2/4 dKO myotubes fail to express dystrophin whereas utrophin is still expressed; 2) Loss of NRG/ERBB signaling abolishes phosphorylation of the 75 kDa α-DB1 and results in lower amounts of the 75 kDa α-DB1 associated with the DAPC¹. Similarly in dystrophin deficient mdx mice less dystrobrevin isoforms are detected at the sarcolemma¹⁷; 3) The reduced association of α-DB1 with the DAPC is not dependent on the phosphorylation state of α-DB1 (Figure 2B) and hence is not due to a lack of NRG/ErbB mediated phosphorylation of α-DB1 in Erbb2/4 dKO myotubes, but more likely due to the loss of dystrophin; 4) The reduced association of α-DB1 with the DAPC is not due to downregulation of α-DB1 expression (Figure 2A) as a consequence of the loss of NRG/ErbB signaling; 5) Loss of NRG/ERBB signaling in vitro and in vivo results in AChR cluster fragmentation¹, implying that the loss of NRG/ERBB signaling is not compensated for in vivo by another signaling pathway.

Reduced localization of dystrobrevin at the sarcolemma and AChR cluster fragmentation due to a lack of dystrophin are also seen in dystrophin deficient mdx mice. However in contrast to mdx mice, Erbb2/4 dKO mice do not show dystrophic symptoms²⁴. In these mice²⁴, muscle fibers were examined extensively and centralized nuclei, a hallmark of DMD^25,26 were not observed. Also atrophic fibers could not be observed in these mice (supplementary information of reference 24)²⁴. Finally, sustained muscle strength was also not affected in the Erbb2/4 dKO mice and neither did they have a myasthenic condition²⁴. However these mice showed a hind limb extension reflex (personal observation) which was also reported for the HSA-CRE/Erbb2 KO mouse²⁷. The loxP flanked Erbb2 mouse is the same mouse that was used in breeding to finally generate the HSA-Cre/Erbb2/4 dKO mouse²⁴. The abnormal muscle spindle formation reported for the HSA-Cre/Erbb2 mouse was not looked at in the HSA-Cre/Erbb2/4 dKO mouse probably because it had already been described for the HSA-Cre/Erbb2 KO mouse.

The reason why Erbb2/4 dKO mice do not show a dystrophic muscle phenotype as seen in mdx mice, despite the absence of dystrophin, could be due to the specificity of the promoter that drives the expression of Cre recombinase in these conditional KO mice. In both, HSA-Cre/Erbb2 mice²⁷ and HSA-Cre/Erbb2/4 dKO mice²⁴ used in this study, Cre recombinase expression is driven by the HSA promoter that is active in the striated muscles, skeletal and heart muscle but not in vascular smooth muscle (VSM)^17,28,29. Hence, ERBB2 receptors would not be ablated and dystrophin will still be expressed in VSM, in contrast to skeletal muscles in these mice.

Interestingly an Erbb2 conditional knock-out mouse with Cre expression driven by the muscle creatine kinase (MCK) promoter, MCK-Cre/Erbb2 did show impaired muscle regeneration and a requirement of ERBB2 for survival of muscle spindles and myoblasts³⁰. As MCK is expressed in both, skeletal muscle and VSM³¹, the MCK promoter would be active in these tissues. Hence in mice where Cre recombinase expression is driven by the MCK promoter, ERBB2 would be ablated in both skeletal muscle and VSM, resulting in a loss of dystrophin expression in both muscle types, explaining the different histopathology with HSA-Cre/Erbb2/4 dKO mice.

In the HSA-Cre/Erbb2/4 dKO mice, ERBB2/4 receptors and dystrophin in smooth muscle of blood vessels would still be expressed, as Cre recombinase would not be expressed in VSM, allowing the formation of a normal functional DAPC. Hence VSM in these mice still allows for increased blood flow to skeletal and cardiac muscle during exercise. In healthy VSM, neuronal nitric oxide synthase (nNOS) associates with dystrophin and generates nitric oxide (NO) that signals to soluble guanylate cyclase, generating cyclic guanosine 3’,5’-monophosphate (cGMP) in VSM, causing vasodilation enabling exercise-induced increase of blood flow and thereby preventing muscle ischemia³². It cannot be excluded from the results presented here, that dystrophin expression in endothelial cells also plays a role in vasodilation during exercise, assuming that the HSA promoter used to drive Cre recombinase expression to ablate ERBB2/4 receptors is not active in those cells in HSA-Cre/Erbb2/4 dKO mice.

The absence of obvious dystrophic pathology in HSA-Cre/Erbb2/4 dKO mice despite the lack of dystrophin in skeletal muscle strongly suggests that the main cause of muscular dystrophy is not the lack of dystrophin in skeletal muscle per se but systemic lack of functional dystrophin, especially in smooth muscle of blood vessels, resulting in impaired sympatholysis and muscle ischemia during exercise³². This hypothesis is consistent with published data describing the effects of phosphodiesterase type 5 (PDE5) inhibitors, which interfere with breakdown of NO by PDE5 and thereby prolong the half-life of cGMP, the target of NO³³. Treatment with PDE5 inhibitors alleviated the dystrophic phenotype in mdx mice³² and also in DMD patients. PDE5 inhibition with either tadalafil or sildenafil treatment in Duchenne muscular dystrophy boys restored normal blood vessel function and blood flow during exercise³³. However, a phase 3 randomized trial of tadalafil for DMD did not slow down the decline in ambulatory ability in boys with DMD³⁴. The authors suggested that the boys in the trial may not have engaged in sufficient daily ambulation, something they could not keep track of due to the large number of patients enrolled and geographical locations of the study. Tadalafil regulates blood flow through its target NO-cGMP signaling in skeletal muscle, only when muscles are active. Since smooth muscle cells are also lining the lymph vessels, ambulation would also help lymphatic drainage and in patients where ambulation is limited, lymph drainage massage may be beneficial for skeletal muscle health.

The observation that absence of dystrophin in skeletal muscle does not result in a dystrophic phenotype is supported by a study where siRNA mediated silencing of dystrophin expression in the muscles of adult mice resulted in a clear absence of dystrophin in skeletal muscle without any of the histopathology characteristics observed in mdx mice³⁵. Because in this study adult mice were used, the authors suggested that the dystrophic pathology normally 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 recombinase expression is active on embryonic day 9.5 (E9.5), when dystrophin would also start to be expressed³⁶, which would argue against the dystrophic pathology being developmentally regulated.

Taken together, the following studies support the conclusion that the lack of dystrophin in VSM is the root cause of DMD: 1) Skeletal muscle specific HSA-Cre/Erbb2/4 dKO mice, based on the results described here, lack dystrophin in skeletal muscle but would still express dystrophin in VSM and do not show a dystrophic pathology²⁴; 2) HSA-Cre/Erbb2 KO²⁷ mice show a similar pathology to the HSA-Cre/Erbb2/4 dKO mice; 3) MCK-Cre/Erbb2 mice³⁰ where NRG/ErbB signaling and hence dystrophin expression is ablated in both, skeletal muscle and VSM (as MCK promoter is active in both), suffered from impaired muscle regeneration; 4) Loss of dystrophin in skeletal muscle fibers through RNAi knock down did not result in an overt dystrophic pathology³⁵; 5) expression of dystrophin in VSM, even at significantly lower levels compared to wild type (wt) controls, improved aberrant vasoregulation in mdx mice³⁷. Taken together, all these studies support the notion that aberrant vasoregulation, when dystrophin is lacking in VSM, is likely the cause of the onset of DMD and that lack of dystrophin in skeletal muscle exacerbates the dystrophic pathology.

There have been numerous attempts to restore dystrophin in skeletal muscle that ameliorated the dystrophic pathology to some extent, but none have led to an applicable therapy. As the focus of most therapies is to restore skeletal, and sometimes also cardiac muscle, it was not of concern whether VSM would or would not be targeted during therapy. In a study where dystrophin overexpression in transgenic mdx mice eliminated dystrophic symptoms³⁸, the MCK promoter was used to drive dystrophin expression and hence dystrophin would have been expressed in both, skeletal muscle and VSM. Gene therapy using Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoters³⁹ would also result in the expression of a functional dystrophin in surrounding VSM after intra-muscular (IM) injection or particle bombardment⁴⁰. In a Phase I study of gene therapy for Duchenne/Becker muscular dystrophy (D/BDM) the CMV promoter was also used⁴¹ which would also restore dystrophin expression in VSM. Systemic delivery of antisense oligoribonucleotides⁴², or restoration of dystrophin expression by (intravenous) stem cell transplantation⁴³, would also result in dystrophin restoration in VSM.

Similarly, even though in vivo rescue of the dystrophic phenotype in dystrophin deficient mice and dogs focused on delivery of the therapeutic to skeletal muscle (see example below), these therapies would also target VSM. Furthermore, simply expressing dystrophin in skeletal muscle fibers lacking dystrophin would restore the dystrophin associated protein complex (DAPC) but it does not imply impaired skeletal muscle regeneration and function is restored. The VSM should be analysed to determine whether dystrophin may also have been expressed through leaking of the therapeutic into VSM, 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 dystrophy⁴⁴. However, 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, 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 indicating a broad distribution. Therefore, from what is known so far, it is neither possible to exclude the role of VSM in the onset of DMD, nor to conclude that expression of dystrophin in skeletal muscle only, is sufficient for the rescue of the dystrophic phenotype in DMD.

Published data on DMD, together with the data presented in this article, suggests that the lack of dystrophin in skeletal muscle exacerbates the onset of dystrophy caused by a lack of increased blood flow during exercise. This is also supported by an observation in mdx muscles that only some fibers, and not all, in skeletal muscle initially show dystrophic symptoms⁴⁵ 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 in wt mice, improved aberrant vasoregulation³⁷. 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.)⁴⁶. The exacerbation of dystrophic symptoms in skeletal muscle, apart from structural defects, may also be as a result of disruption of signaling events from the DAPC due to the absence of dystrophin.

We previously reported that the blockade of NRG signaling through ERBB2/4 receptors prevented phosphorylation of α-dystrobrevin1 and hence affected NMJ stability¹. In the current study it is shown that ablation of ERBB2/4 receptors, and thus elimination of NRG signaling, results in a lack of dystrophin expression (Figure 1). Thus NRG/ERBB signaling maintains NMJ stability through at least two pathways, one where it phosphorylates α-dystrobrevin 1^1,2 and the other where it stimulates dystrophin expression thereby allowing the formation of a functional DAPC that stabilizes acetylcholine receptors (Figure 3).

If the absence of NRG/ERBB signaling, and as a consequence absence of dystrophin in skeletal muscle and lack of phosphorylation of α-DB1, 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 skeletal muscles of Erbb2/4 dKO mice. However there is probably no compensatory signal for NRG/ERBB in vivo as AChR cluster fragmentation is observed in vivo as well. 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 formation²⁷, dilated cardiomyopathy (DCM) following Herceptin (antibody against HER2/ERBB2) treatment⁴⁷, DCM in conditional Erbb2^28,48 or Erbb4⁴⁹ KO mice, AChR cluster fragmentation (also observed in mdx mice) in muscle specific Erbb2/4 dKO mice¹, and the embryonic lethality observed in Erbb2¹¹ or Erbb4¹⁰ KO mice. Based on what is known so far, NRG and its ERBB receptors appear to exert their function in a spatio-temporal fashion in development and maintenance (in adults).

NRG/ErbB signaling also induces cardiomyocyte proliferation and repairs heart injury¹² and is essential for normal cardiac development^9–11, whereas dystrophin deficiency does not impair cardiac development but does result in dilated cardiomyopathy (DCM)⁸. ERBB4 can heterodimerize with ERBB2 and a function blocking ERBB2/HER2 antibody treatment results in DCM in mice²⁸ and in cancer patients⁴⁷, consistent with our observations that NRG/ERBB signaling is required for dystrophin expression since DMD patients and mdx mice lacking functional dystrophin, develop DCM (Figure 3). Hence the data presented here suggests that a target protein for the reported beneficial effects of signalling through ERBB4 in repairing heart injury is dystrophin whose expression is regulated through NRG/ERBB2/4 signaling. Thus NRG/ERBB signaling carries out different functions, through different signaling targets, in cardiac development and maintenance, with the latter being carried out through regulating dystrophin expression.

Although normal blood flow is important for prevention of the onset of dystrophic symptoms, the general consensus is that exercise would cause damage to skeletal muscle and in the absence of dystrophin, muscle regeneration would be impaired. However, the Erbb2/4 dKO mice lacking dystrophin in skeletal muscle only, do not show any obvious dystrophic pathology which challenges this general consensus.

There are two studies that may give an explanation as to why a dystrophic pathology is not observed in skeletal muscles when dystrophin is lacking as in HSA-Cre/Erbb2/4 dKO mice. It has been demonstrated that the host environment is critical for controlling the function of satellite cells⁵⁰. Hence normal dystrophin expression in VSM may prevent or delay the onset of dystrophy in skeletal muscle and thereby maintain a host environment that is permissive to muscle regeneration by satellite cells. Alternatively muscle regeneration in response to injury could be mediated by myofibers derived from circulating mature myeloid cells (blood cells) that migrate to skeletal muscle in response to inflammatory cues⁵¹. Hence, upon muscle damage in HSA-Cre/Erbb2/4 dKO mice where dystrophin is absent only in skeletal muscle, satellite cells and/or circulating myeloid cells could help regenerate mature myofibers underscoring the importance of the vasculature and increased blood flow during exercise.

Although current therapy for Duchenne patients is largely focused on skeletal muscle, the present study suggests 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. Dystrophin deficient mdx mice show a milder dystrophic pathology and have a shorter life span compared to humans. It thus needs to be determined whether in humans, restoring dystrophin in VSM (and endothelial cells) and thereby a functional DAPC, is sufficient to ameliorate the dystrophic pathology.

The current study also suggests that increasing NRG signaling through ERBB2/4 receptors, especially in the smooth muscle of blood vessels, could be a way to increase truncated dystrophin expression in D/BMD patients. This would ameliorate dystrophic symptoms in those patients where the mutation in dystrophin does not affect association of nNOS⁵² and thereby enabling normal blood flow during exercise. Apart from its implications for muscular dystrophy, the demonstration that NRG/ERBB signaling stimulates dystrophin expression should help to improve treatment regimens with the anti-HER2 antibody, Herceptin/Trastuzumab, to prevent cardiomyopathy. Furthermore, as dystrophin is also present in all regions of the brain, being most abundant in the cerebellum⁵³, and since NRG/ERBB signaling regulates dystrophin expression, changes in functional dystrophin levels could be the underlying cause of some of the disease states such as schizophrenia, associated with abnormal NRG/ERBB signaling in the brain.

Conclusions

NRG signaling through ERBB2/4 receptors is necessary for stimulation of dystrophin expression. However, when ERBB2/4 receptors are lacking in skeletal muscle only, mice do not exhibit dystrophic symptoms supporting the notion that lack of dystrophin expression in smooth muscle of blood vessels is the root cause of the onset of D/BMD.

Data availability

F1000Research: Dataset 1. Uncropped western blot images and raw Ct values from qPCR. DOI: https://dx.doi.org/10.5256/f1000research.15889.d214628⁵⁴.

F1000Research: Dataset 2. Raw data for figure 2. DOI: https://doi.org/10.5256/f1000research.15889.d226797⁵⁵