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 4th (4S) and 6th (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.
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.
- Beth A Habecker
- Matthew R Blake
- Matthew R Blake
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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.
- Kalyanam Shivkumar, UCLA Health, United States
© 2022, Blake et al.
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.
The functional complementarity of the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) allows for optimal combined gaze stabilization responses (CGR) in light. While sensory substitution has been reported following complete vestibular loss, the capacity of the central vestibular system to compensate for partial peripheral vestibular loss remains to be determined. Here, we first demonstrate the efficacy of a 6-week subchronic ototoxic protocol in inducing transient and partial vestibular loss which equally affects the canal- and otolith-dependent VORs. Immunostaining of hair cells in the vestibular sensory epithelia revealed that organ-specific alteration of type I, but not type II, hair cells correlates with functional impairments. The decrease in VOR performance is paralleled with an increase in the gain of the OKR occurring in a specific range of frequencies where VOR normally dominates gaze stabilization, compatible with a sensory substitution process. Comparison of unimodal OKR or VOR versus bimodal CGR revealed that visuo-vestibular interactions remain reduced despite a significant recovery in the VOR. Modeling and sweep-based analysis revealed that the differential capacity to optimally combine OKR and VOR correlates with the reproducibility of the VOR responses. Overall, these results shed light on the multisensory reweighting occurring in pathologies with fluctuating peripheral vestibular malfunction.
Genuinely new discovery transcends existing knowledge. Despite this, many analyses in systems neuroscience neglect to test new speculative hypotheses against benchmark empirical facts. Some of these analyses inadvertently use circular reasoning to present existing knowledge as new discovery. Here, I discuss that this problem can confound key results and estimate that it has affected more than three thousand studies in network neuroscience over the last decade. I suggest that future studies can reduce this problem by limiting the use of speculative evidence, integrating existing knowledge into benchmark models, and rigorously testing proposed discoveries against these models. I conclude with a summary of practical challenges and recommendations.