Apocynin Treatment Prevents Cardiac Connexin 43 Hemichannels Hyperactivity by Reducing Nitroso-Redox Stress in Mdx Mice

Round 1

Reviewer 1 Report

The manuscript presented by Vielma et al. shows that during DMD pathogenesis Cx43 form hemichannels that contribute to the progression of the disease under increase oxidative stress conditions.  Although the manuscript is well written, there are several items that should be addressed by the authors.

 

• Authors should pay special attention to figures format. Can the authors use the same graph type and stats nomenclature during the paper? As for example, when no significance is present sometimes authors use “ns” and sometimes nothing. Also, can the authors show independent points in the graphs troughout the manuscript? Use bar graphs from Figure 8 as a model.

 

• Number of experiments/animals should be included in all figure legends for each of the experiments.

 

 

• The authors show Cx43 lateralization in mdx mice (Fig 1A) correlated with increased levels of p22 and NOX2 (Fig 1B). However, 10 month BL10 mice have Cx43 lateratization (Fig 1A) but no correlation with p22 nor NOX2. Can the authors explain this results?

 

• In Western-blot from Figure 1C, loading control should be included.

 

 

• It is surprising the high variabily observed by the authors in 10month BL10 mice in Figure 1D. Can the authors explain the big error bars?

 

• Line 128-129 “TBARS levels were increased in mdx mice and restored to control values after 1 month of apocynin treatment” . This sentence should be changed or data should be analyzed different way (Fig 1a) since they do not show what it´s explained in the text. As they are presented now, apocyning treatment do not affect BL10 nor mdx mice.

 

 

• 10umol/L of carbenoxolone for 10 min blocked hemichannels activity completely? (Fig 3). The pictures are not representative of the data analysisi shown in the graph. Please provide representative pictures. Also, is there any structural differences between BL10 and mdx mice hearts structure? Authors should show hematoxilin-eosin staining to show heart structure.

 

• Can the authors show high magnification and merge images for tunnel assay? Also image magnification show be added to the pictures.

 

 

• Figure 5. Pictures should include nuclear staining (DAPI for example) and high magnification is required to better determine N-cad and Cx43 co-localization

 

• It would be necessary to check the levels of the dystrophin whenever working with MDX mice. They would also need to evaluate the expression of utrophin and whether the treatment increases the levels of this protein as utrophin could exert a compensatory effect to the lack of functional dystrophin.

 

• Is GAPDH a suitable loading control taking into account the topic of study?

 

 

 

 

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript describes an important aspect of Duchenne muscular dystrophy (DMD). It is a fatal disease that causes cardiomyopathy and is associated with oxidative stress. In the heart, oxidative stress interferes with the location of connexin 43 (Cx43) to the intercalated discs, causing lateralization to the plasma membrane, where Cx43 forms hemichannels. To probe into the heralded case in DMD, the authors investigated cardiomyopathy and examined the working hypothesis that it is associated with elevated NADPH oxidase 2 activities and formation of Cx43 hemichannels. To that end, the two species emerge as important biomarkers of oxidative stress. In that sense, the authors show that NADPH oxidase activity in DMD is associated with S-nitrosylation and opening of Cx43 hemichannels. The observed changes lead to apoptosis and cardiac dysfunction. Furthermore, NADPH oxidase inhibition appears to restore cardiac function.
The work was done competently, yet a number of issues arose that call for the attention of the authors and revisions that are expected to raise the quality of the presentation of the work done and the manuscript itself.
1. The manuscript suffers from linguistic mishaps in grammar and syntax, thereby rendering it difficult to follow.
a) In the introduction, the statement “After this study had begun, it was reported that nitric oxide synthase-mediated S-nitrosylation of Cx43 hemichannels is key to increase cardiomyocyte membrane permeability and electrical activity in mdx mice causing arrhythmias (38).” should be rewritten to read “After this study had begun, it was reported that nitric oxide synthase-mediated S-nitrosylation of Cx43 hemichannels is key to increasing cardiomyocyte membrane permeability and electrical activity in mdx mice, thus causing arrhythmias (38).”.
b) In the Results section, the statement “Therefore, we decided to investigate in mdx mice, an established animal model for DMD, the state of NADPH oxidase-derived oxidative stress and its impact on Cx43 subcellular location and redox state.” should be rephrased to read “Therefore, we decided to investigate the state of NADPH oxidase-derived oxidative stress and its impact on Cx43 subcellular location and redox state in mdx mice, an established animal model for DMD.”.
c) In the legend of figure 1, the letter E, designating the existence of the relevant figure, is missing.
d) In the case of Figure 2 and specifically the array of figures 2A, the figure on the far left hand side contains the word Apocynin as Apocinyn. It should be corrected.
e) In section 2.4, the statement “These data suggest that cardiomyocytes that present high Cx43 hemichannels activity are undergoing apoptosis, or at least are prone to undergo the apoptotic process.” should be corrected to read “These data suggest that cardiomyocytes, exhibiting high Cx43 hemichannel activity, are undergoing apoptosis or at least are prone to undergoing an apoptotic process.”.
f) In section 2.7, the statement “It has been reported that the dystrophic heart of mdx develop ventricular remodeling as a compensatory mechanism for the increased loss of cardiac function, involving cardiac hypertrophy, development of fibrosis and reduction in cardiac contractility (referencia LI AJP).” should be modified to read “It has been reported that dystrophic hearts in mdx mice develop ventricular remodeling as a compensatory mechanism for the increased loss of cardiac
function, involving cardiac hypertrophy, development of fibrosis, and reduction in
cardiac contractility (referencia LI AJP).”.
Furthermore, the reference should be placed appropriately in parenthesis and in
colloquial English.
g) In the Discussion section, the statement “This increased activity of Cx43
hemichannels has negative impact on cardiac contractility and rhythmicity, and
increases cardiomyocytes apoptosis and produced subsequent fibrosis.” should be
rephrased to read “This increased activity of Cx43 hemichannels has a negative
impact on cardiac contractility and rhythmicity. In fact, it increases
cardiomyocyte apoptosis and produces subsequent fibrosis.”.
h) In the same section, the statement “Our observations open a new line of research
for the role of Cx43 hemichannels in cardiac pathologies besides ischemic related
injuries, were Cx43 has been formerly associated [17, 19, 33, 42].” needs
revisions to read (at least form what it can be understood) “Our observations open
a new line of research into the role of Cx43 hemichannels in cardiac pathologies,
besides ischemic related injuries, with which Cx43 had been previously associated
[17, 19, 33, 42].”.
i) In the Limitations of the Study, the statement “These among, others, arise from
the lack of specificity of apocynin as NOX inhibitors, which precludes of
discarding other sources of oxidative stress to contribute to this
pathophysiological mechanism.” should be rewritten to read “These, among
others, arise from the lack of specificity of apocynin as a NOX inhibitor, which
precludes discarding other sources of oxidative stress from contributing to this
pathophysiological mechanism.”.
j) In the Materials and methods section, the statement “Male mdx C57BL/10ScSn-
DMDmdx/J, and control mice (C57BL/10SnJ) were purchased from Jackson
Laboratories (Bar Harbor, MA), maintained and breed at the animal facility of the
Pontificia Universidad Catolica de Chile (Santiago, Chile).” should be revised to
read “Male mdx C57BL/10ScSn-DMDmdx/J, and control mice (C57BL/10SnJ)
were purchased from Jackson Laboratories (Bar Harbor, MA), maintained and
bred at the animal facility of the Pontificia Universidad Catolica de Chile
(Santiago, Chile).”.
k) … and more of the same remarks spread throughout the manuscript.
2. In the case of Cx43 lateralization and S-nitrosylation in mdx hearts, the authors
present in Figure 1, the effect of isoproterenol in 2 month old and 10 month old mdx
mice. In the second case, the error bars are so high that essentially lie close to the
curve for the two month old mice. Why are the error bars so high in the second case
compared to the first case? What does that effectively signify in the experiments run?
Ample clarification should be provided.
3. In section 5 of the manuscript, there is an entire paragraph of empty statement(s) on
hypothetical contributions of the authors to the study presented here. Such a practice
should be avoided as it lowers the quality of the work and presentation made. It
should be amended
On the basis of the aforementioned remarks, the manuscript should be revised accordingly prior to any consideration.

Author Response

Please see the attachement.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The recommended changes have been implemented by the authors.