Chondroitin Sulfate Proteoglycan 4,6 sulfation regulates sympathetic nerve regeneration after myocardial infarction

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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 is thought to depend on the sulfation status of the glycosaminoglycans (CS-GAGs) attached to the core protein. Tandem sulfation of CS-GAGs at the 4^th (4S) and 6^th (6S) positions of n-acetyl-galactosamine inhibits outgrowth in several types of neurons within the central nervous system, but it is not known if sulfation is similarly critical during peripheral nerve regeneration. We asked if CSPG sulfation prevented sympathetic axon outgrowth. Neurite outgrowth of dissociated rat sympathetic neurons across purified CSPGs is restored in vitro by reducing 4S with the 4-sulfatase enzyme Arylsulfatase-B (ARSB). Additionally, we co-cultured mouse cardiac scar tissue with mouse sympathetic ganglia ex vivo and found that reducing 4S with ARSB restored axon outgrowth to control levels. We examined levels of the enzymes responsible for adding and removing sulfation to CS-GAGs by western blot to determine if they were altered in the left ventricle after MI. We found that CHST15 (4S dependent 6-sulfotransferase) was upregulated, and ARSB was downregulated after MI. Increased CHST15 combined with decreased ARSB suggests a mechanism for production and maintenance of sulfated CSPGs in the cardiac scar. We altered tandem sulfated 4S,6S CS-GAGs in vivo by transient siRNA knockdown of Chst15 and found that reducing 4S,6S restored Tyrosine Hydroxylase (TH) positive sympathetic nerve fibers in the cardiac scar and reduced arrhythmias using a mouse model of MI. Overall, 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.

Data availability

All data generated during this study are included in the manuscript and supporting files. Source data for annotated Western blot images from figure 1, 4, 5, 6 and S1, S2, S3 have been made available in the zipped western blot source data folder. Images are not cropped and they are labeled such that the figure number and protein blotted in each image are in the file name.

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Matthew R Blake

Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-0584-7684
Diana C Parrish

Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.
Melanie A Staffenson

Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.
Shanice Sueda

Portland State University, Portland, United States

Competing interests
The authors declare that no competing interests exist.
William R Woodward

Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, United States

Competing interests
The authors declare that no competing interests exist.
Beth A Habecker

Department of Chemical Physiology and Biochemistry, Oregon Health and Science University, Portland, United States

For correspondence
habecker@ohsu.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-4658-8730

Funding

National Heart, Lung, and Blood Institute (HL093056)

Beth A Habecker

National Heart, Lung, and Blood Institute (F31HL152490)

Matthew R Blake

American Heart Association (20PRE35210768)

Matthew R Blake

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All procedures were approved by the OHSU Institutional Animal Care and Use Committee(IACUC# TR01_IP00001366) and comply with the Guide for the Care and Use of Laboratory Animals published by the National Academies Press (8th edition). All myocardial infarction surgeries were performed under anesthesia induced with 4% isoflurane and maintained with 2% isoflurane. After surgery, animals were treated with buprenorphine and meloxicam to minimize suffering and discomfort.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.