Chondroitin sulfate proteoglycan 4,6 sulfation regulates sympathetic nerve regeneration after myocardial infarction
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Evaluation Summary:
In the present manuscript by Blake et al., the investigators show that myocardial infarction (MI) leads to increased sulfation of CSPGs in the cardiac scar. The investigators subsequently demonstrate that reducing sulfation with a sulfatase, arylsulfatase B (ARSB), promotes sympathetic neurite growth in vitro and ex vivo in a co-culture system. This paper provides interesting results regarding neural remodeling of the heart and has implications for visceral innervation in health and disease. This work is important in highlighting the role of neural-myocardial interactions after MI and offering a potential pathway to target in preventing post-MI sudden cardiac death.
(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. Reviewer #1, Reviewer #2 and Reviewer #4 agreed to share their name with the authors.)
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Abstract
Sympathetic denervation of the heart following ischemia/reperfusion induced myocardial infarction (MI) is sustained by chondroitin sulfate proteoglycans (CSPGs) in the cardiac scar. Denervation predicts risk of sudden cardiac death in humans. Blocking CSPG signaling restores sympathetic axon outgrowth into the cardiac scar, decreasing arrhythmia susceptibility. Axon growth inhibition by CSPGs can depend on the sulfation status of the glycosaminoglycan (CS-GAG) side chains. Tandem sulfation of CS-GAGs at the 4th (4S) and 6th (6S) positions of n-acetyl-galactosamine inhibits outgrowth in several types of central neurons, but we don’t know if sulfation is similarly critical during peripheral nerve regeneration. We asked if CSPG sulfation prevented sympathetic axon outgrowth after MI. Reducing 4S with the 4-sulfatase enzyme Arylsulfatase-B (ARSB) enhanced outgrowth of dissociated rat sympathetic neurons over CSPGs. Likewise, reducing 4S with ARSB restored axon outgrowth from mouse sympathetic ganglia co-cultured with cardiac scar tissue. We quantified enzymes responsible for adding and removing sulfation, and found that CHST15 (4S dependent 6-sulfotransferase) was upregulated, and ARSB was downregulated after MI. This suggests a mechanism for production and maintenance of sulfated CSPGs in the cardiac scar. We decreased 4S,6S CS-GAGs in vivo by transient siRNA knockdown of Chst15 after MI, and found that reducing 4S,6S restored tyrosine hydroxylase (TH) positive sympathetic nerve fibers in the cardiac scar. Reinnervation reduced isoproterenol induced arrhythmias. Our results suggest that modulating CSPG-sulfation after MI may be a therapeutic target to promote sympathetic nerve regeneration in the cardiac scar and reduce post-MI cardiac arrhythmias.
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Author Response
Reviewer #1 (Public Review):
Weaknesses:
• The causal relationship between ARSB treatment and neurite outgrowth phenomena the authors observe after MI are weak. Specifically, the co-culture assay does not seem to fully replicate the nerve/myocardial interface after MI. It is unclear why NGF needed to be added to the media to induce neurite outgrowth when it is established that multiple neurotrophic and neurotropic factors are already expressed in the myocardium after MI (Habecker et al., J Physiol 594.14 (2016) pp 3853-3875). By visual estimation, it also appears difficult to control for spatial distance between the sympathetic ganglion and myocardial explants in this culture system, a problem which may significantly affect the diffusion of axon guidance and other signaling molecules. Additionally, because ARSB is …
Author Response
Reviewer #1 (Public Review):
Weaknesses:
• The causal relationship between ARSB treatment and neurite outgrowth phenomena the authors observe after MI are weak. Specifically, the co-culture assay does not seem to fully replicate the nerve/myocardial interface after MI. It is unclear why NGF needed to be added to the media to induce neurite outgrowth when it is established that multiple neurotrophic and neurotropic factors are already expressed in the myocardium after MI (Habecker et al., J Physiol 594.14 (2016) pp 3853-3875). By visual estimation, it also appears difficult to control for spatial distance between the sympathetic ganglion and myocardial explants in this culture system, a problem which may significantly affect the diffusion of axon guidance and other signaling molecules. Additionally, because ARSB is added to both the sympathetic ganglion and the myocardial explants, it is unclear where exactly it is disrupting CSPG sulfation. It has been shown that glia also secrete CSPGs (Yiu & He, Nature Reviews Neuroscience volume 7, pp617-627 (2006)) after CNS injury, preventing axon regeneration. Thus, inhibition of 4-sulfation by ARSB within the sympathetic ganglion explant should be taken into account as well when considering experimental specificity. This particular experiment is perhaps the least convincing in this work overall.
NGF: It is standard practice to add NGF to these types of explant studies. We used neonatal ganglia, which require NGF for survival, and we did not want significant differences in NGF content in the culture to be the reason why axon growth differed between groups, as infarcted myocardium produces more NGF than control myocardium. We want sufficient NGF present to stimulate growth in all directions so we could assess the ability of explant derived CSPGs to inhibit growth. We have clarified this in the methods (lines 355-361 in the “clean” version of the revision).
Spatial variability: We marked TC dishes before plating the tissue and were as consistent as possible with the plating although it certainly was not perfect. We detected inhibition of outgrowth consistently in every ganglion co-cultured with infarcted heart, and ARSB treatment consistently abolished outgrowth inhibition. Thus, we believe this is a reliable assay for axon outgrowth and its inhibition by target-derived CSPGs. We added further detail to the methods (lines 355-356 in the “clean” version of the revision).
ARSB: It is true that multiple cell types, including glia and neurons, can produce CSPGs. Two different controls suggest ARSB is acting on CSPGs produced by the cardiac scar rather than ganglionic CSPGs. First, ganglia co-cultured with control heart tissue exhibited identical growth in the presence or absence of ARSB. Second, addition of ARSB did not change the growth of axons extending away from infarcted tissue explants, it only altered growth toward the scar tissue. We have clarified this in the text (lines 130-134).
• The underlying mechanism of the therapeutic potential of inhibiting 4-sulfation of CSPG is unclear. There is immunohistochemistry showing increased sympathetic nerve fibers by TH labeling, co-localized with fibronectin staining to delineate scar. However, how this phenomenon then leads to decreased arrhythmia is a bit of a black box, especially considering that scar tissue is electrophysiologically and mechanically discontinuous from working myocardium.
Overall, the authors demonstrated very interesting dynamics of CSPG sulfation after MI and its correlation with sympathetic innervation of the MI scar. They also demonstrated knockdown of CHST15 reduces PVCs in a mouse model of MI. The causal relationship between CSPG sulfation, reinnervation of scar, and arrhythmogenesis is less convincingly demonstrated as there is no clear functional pathway suggested by which reinnervating scar alters electrophysiology. The authors perhaps realize this issue and thus were careful to be measured in the title of their manuscript, which omits the pathophysiological consequences of sympathetic nerve regeneration.
We agree that this study has not identified the mechanisms by which reinnervation alters arrhythmia susceptibility. That was not the purpose of the study. Our purpose was to test the role of CSPG sulfation in preventing nerve regeneration into the cardiac scar. We agree that it will be very interesting to elucidate the mechanisms of arrhythmia suppression in another study.
Reviewer #2 (Public Review):
The study by Blake et al. tested an interesting hypothesis that chondroitin sulfate proteoglycan (CSPG) 4, 6 sulfation plays a critical role in mediating sympathetic denervation and cardiac arrhythmia post-ischemia-reperfusion (I/R) in a mouse model. They provided solid molecular evidence showing CSPG 4, 6 sulfation in cardiac scar tissues post-I/R. They also suggest upregulated CHST15 and downregulated ARSB as a mechanism for the production and maintenance of sulfated CSPGs. Most importantly, in vivo siRNA knockdown of chst15 at the early time window of I/R prevented sulfated CSPGs and sympathetic denervation in cardiac scar tissue, which eventually improved cardiac arrhythmia. The strengths of this study come from the focus on a novel CSPG pathway as well as the solid molecular/animal data. It is clear from this study that CSPGs could be a promising therapeutic target to treat cardiac arrhythmia post-myocardial damage.
We appreciate the careful reading of the manuscript and the positive comments. We added data to the supplement and revised the text to address the specific issues raised in the detailed review.
Reviewer #3 (Public Review):
The authors' prior work demonstrated that inhibiting CSPG signaling following myocardial infarction (MI) enabled sympathetic axon outgrowth into the post-MI scar and reduced ventricular arrhythmogenesis. In this study, the authors sought to determine if CSPG sulfation prevented sympathetic axon outgrowth into the post-MI scar.
The strengths of the study include its depth, multimodal tools, in vitro and ex vivo experimentation, and translatability of its findings to large animals and humans.
Minor weaknesses include limited rigor in experimental design supporting some of the conclusions.
The impact of this work is likely to be in the translation to large animals and humans, where scar modifying therapies may come to the forefront of post-MI treatments. The antiarrhythmic potential of this approach is potentially significant, as no current therapies improve the innervation of myocardial scar.
We appreciate the positive comments, and hope that our revised text clarifies issues related to experimental rigor.
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Evaluation Summary:
In the present manuscript by Blake et al., the investigators show that myocardial infarction (MI) leads to increased sulfation of CSPGs in the cardiac scar. The investigators subsequently demonstrate that reducing sulfation with a sulfatase, arylsulfatase B (ARSB), promotes sympathetic neurite growth in vitro and ex vivo in a co-culture system. This paper provides interesting results regarding neural remodeling of the heart and has implications for visceral innervation in health and disease. This work is important in highlighting the role of neural-myocardial interactions after MI and offering a potential pathway to target in preventing post-MI sudden cardiac death.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
In the present manuscript by Blake et al., the investigators show that myocardial infarction (MI) leads to increased sulfation of CSPGs in the cardiac scar. The investigators subsequently demonstrate that reducing sulfation with a sulfatase, arylsulfatase B (ARSB), promotes sympathetic neurite growth in vitro and ex vivo in a co-culture system. This paper provides interesting results regarding neural remodeling of the heart and has implications for visceral innervation in health and disease. This work is important in highlighting the role of neural-myocardial interactions after MI and offering a potential pathway to target in preventing post-MI sudden cardiac death.
(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. Reviewer #1, Reviewer #2 and Reviewer #4 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This is an interesting study of how CSPG sulfation states are altered after MI and how this relates to sympathetic nerve regrowth in the infarct. The authors first demonstrate that 4,6-sulfation of CSPG is increased in the heart after ischemia/reperfusion injury. They then demonstrate that reducing 4-sulfation of CSPG with ARSB increases sympathetic neurite outgrowth from sympathetic ganglion explants in vitro. The authors then treated co-cultures of scar tissue from myocardial infarcts with superior cervical ganglion explants with ARSB and showed increased neurite outgrowth on the side of the scar after ARSB. Changes in CSPG sulfation enzymes were then shown to be altered after MI by Western blot, specifically an increase in CHST15. Finally, siRNA knockdown of CHST15 expression after MI in mice reduced CSPG …
Reviewer #1 (Public Review):
This is an interesting study of how CSPG sulfation states are altered after MI and how this relates to sympathetic nerve regrowth in the infarct. The authors first demonstrate that 4,6-sulfation of CSPG is increased in the heart after ischemia/reperfusion injury. They then demonstrate that reducing 4-sulfation of CSPG with ARSB increases sympathetic neurite outgrowth from sympathetic ganglion explants in vitro. The authors then treated co-cultures of scar tissue from myocardial infarcts with superior cervical ganglion explants with ARSB and showed increased neurite outgrowth on the side of the scar after ARSB. Changes in CSPG sulfation enzymes were then shown to be altered after MI by Western blot, specifically an increase in CHST15. Finally, siRNA knockdown of CHST15 expression after MI in mice reduced CSPG sulfation and was associated with increased sympathetic reinnervation of the scar. This was also associated with fewer PVCs induced by isoproterenol and caffeine.
Strengths:
• This is an impressive body of experiments with a logical progression to show CSPG sulfation changes after MI and the enzymes regulating this sulfation.
• The authors have utilized in vitro and in vivo models to test their hypothesis that CSPG 4-sulfation suppresses innervation of the MI scar.
• There is an important disease model and therapeutic aim to this work.Weaknesses:
• The causal relationship between ARSB treatment and neurite outgrowth phenomena the authors observe after MI are weak. Specifically, the co-culture assay does not seem to fully replicate the nerve/myocardial interface after MI. It is unclear why NGF needed to be added to the media to induce neurite outgrowth when it is established that multiple neurotrophic and neurotropic factors are already expressed in the myocardium after MI (Habecker et al., J Physiol 594.14 (2016) pp 3853-3875). By visual estimation, it also appears difficult to control for spatial distance between the sympathetic ganglion and myocardial explants in this culture system, a problem which may significantly affect the diffusion of axon guidance and other signaling molecules. Additionally, because ARSB is added to both the sympathetic ganglion and the myocardial explants, it is unclear where exactly it is disrupting CSPG sulfation. It has been shown that glia also secrete CSPGs (Yiu & He, Nature Reviews Neuroscience volume 7, pp617-627 (2006)) after CNS injury, preventing axon regeneration. Thus, inhibition of 4-sulfation by ARSB within the sympathetic ganglion explant should be taken into account as well when considering experimental specificity. This particular experiment is perhaps the least convincing in this work overall.
• The underlying mechanism of the therapeutic potential of inhibiting 4-sulfation of CSPG is unclear. There is immunohistochemistry showing increased sympathetic nerve fibers by TH labeling, co-localized with fibronectin staining to delineate scar. However, how this phenomenon then leads to decreased arrhythmia is a bit of a black box, especially considering that scar tissue is electrophysiologically and mechanically discontinuous from working myocardium.Overall, the authors demonstrated very interesting dynamics of CSPG sulfation after MI and its correlation with sympathetic innervation of the MI scar. They also demonstrated knockdown of CHST15 reduces PVCs in a mouse model of MI. The causal relationship between CSPG sulfation, reinnervation of scar, and arrhythmogenesis is less convincingly demonstrated as there is no clear functional pathway suggested by which reinnervating scar alters electrophysiology. The authors perhaps realize this issue and thus were careful to be measured in the title of their manuscript, which omits the pathophysiological consequences of sympathetic nerve regeneration.
This work will be important in highlighting the importance of neural/myocardial interactions in structural heart disease. It will also draw attention and effort to understanding neural remodeling after myocardial infarction and altering this remodeling for therapeutic benefit.
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Reviewer #2 (Public Review):
The study by Blake et al. tested an interesting hypothesis that chondroitin sulfate proteoglycan (CSPG) 4, 6 sulfation plays a critical role in mediating sympathetic denervation and cardiac arrhythmia post-ischemia-reperfusion (I/R) in a mouse model. They provided solid molecular evidence showing CSPG 4, 6 sulfation in cardiac scar tissues post-I/R. They also suggest upregulated CHST15 and downregulated ARSB as a mechanism for the production and maintenance of sulfated CSPGs. Most importantly, in vivo siRNA knockdown of chst15 at the early time window of I/R prevented sulfated CSPGs and sympathetic denervation in cardiac scar tissue, which eventually improved cardiac arrhythmia. The strengths of this study come from the focus on a novel CSPG pathway as well as the solid molecular/animal data. It is clear …
Reviewer #2 (Public Review):
The study by Blake et al. tested an interesting hypothesis that chondroitin sulfate proteoglycan (CSPG) 4, 6 sulfation plays a critical role in mediating sympathetic denervation and cardiac arrhythmia post-ischemia-reperfusion (I/R) in a mouse model. They provided solid molecular evidence showing CSPG 4, 6 sulfation in cardiac scar tissues post-I/R. They also suggest upregulated CHST15 and downregulated ARSB as a mechanism for the production and maintenance of sulfated CSPGs. Most importantly, in vivo siRNA knockdown of chst15 at the early time window of I/R prevented sulfated CSPGs and sympathetic denervation in cardiac scar tissue, which eventually improved cardiac arrhythmia. The strengths of this study come from the focus on a novel CSPG pathway as well as the solid molecular/animal data. It is clear from this study that CSPGs could be a promising therapeutic target to treat cardiac arrhythmia post-myocardial damage.
-
Reviewer #3 (Public Review):
The authors' prior work demonstrated that inhibiting CSPG signaling following myocardial infarction (MI) enabled sympathetic axon outgrowth into the post-MI scar and reduced ventricular arrhythmogenesis. In this study, the authors sought to determine if CSPG sulfation prevented sympathetic axon outgrowth into the post-MI scar.
The strengths of the study include its depth, multimodal tools, in vitro and ex vivo experimentation, and translatability of its findings to large animals and humans.
Minor weaknesses include limited rigor in experimental design supporting some of the conclusions.
The impact of this work is likely to be in the translation to large animals and humans, where scar modifying therapies may come to the forefront of post-MI treatments. The antiarrhythmic potential of this approach is …
Reviewer #3 (Public Review):
The authors' prior work demonstrated that inhibiting CSPG signaling following myocardial infarction (MI) enabled sympathetic axon outgrowth into the post-MI scar and reduced ventricular arrhythmogenesis. In this study, the authors sought to determine if CSPG sulfation prevented sympathetic axon outgrowth into the post-MI scar.
The strengths of the study include its depth, multimodal tools, in vitro and ex vivo experimentation, and translatability of its findings to large animals and humans.
Minor weaknesses include limited rigor in experimental design supporting some of the conclusions.
The impact of this work is likely to be in the translation to large animals and humans, where scar modifying therapies may come to the forefront of post-MI treatments. The antiarrhythmic potential of this approach is potentially significant, as no current therapies improve the innervation of myocardial scar.
-
Reviewer #4 (Public Review):
Sympathetic neural remodeling following myocardial infarction (MI) plays a critical role in the pathogenesis of lethal ventricular arrhythmias and progression to heart failure. Thus, one of the cornerstones of treating ventricular arrhythmias is beta-adrenergic blockade. After an MI, there is sympathetic denervation of the infarct scar and hyperinnervation of the infarct border zone. This heterogeneity in sympathetic innervation is thought to contribute to electrical instability and arrhythmogenesis. One mechanism for the lack of nerve regeneration in the infarct scar following MI has previously been shown to be the presence of chondroitin sulfate proteoglycans. In the present manuscript, the investigators show that MI leads to increased sulfation of chondroitin sulfate proteoglycans in the cardiac scar. The …
Reviewer #4 (Public Review):
Sympathetic neural remodeling following myocardial infarction (MI) plays a critical role in the pathogenesis of lethal ventricular arrhythmias and progression to heart failure. Thus, one of the cornerstones of treating ventricular arrhythmias is beta-adrenergic blockade. After an MI, there is sympathetic denervation of the infarct scar and hyperinnervation of the infarct border zone. This heterogeneity in sympathetic innervation is thought to contribute to electrical instability and arrhythmogenesis. One mechanism for the lack of nerve regeneration in the infarct scar following MI has previously been shown to be the presence of chondroitin sulfate proteoglycans. In the present manuscript, the investigators show that MI leads to increased sulfation of chondroitin sulfate proteoglycans in the cardiac scar. The investigators subsequently demonstrate that reducing sulfation with a sulfatase promotes sympathetic neurite growth in vitro and ex vivo in a co-culture system. Furthermore, molecular studies showed that there was an alteration in the expression of enzymes involved in the sulfation of chondroitin sulfate proteoglycans, specifically increased expression of a sulfotransferase and decreased expression of a sulfatase. The investigators went on to show that knockdown of the gene for this sulfotransferase with a silencing RNA not only restores sympathetic innervation of the infarct scar but also reduces susceptibility to arrhythmias post-MI in vivo. A series of compelling experimental manipulations dissect this pathway, conclusively supporting the key claims of the paper. In addition, the fact that a silencing RNA against this sulfotransferase is already in clinical trials for another indication increases the translational potential of this study.
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