Matt Blake, B.S. & Michael Berry, M.S.
November 23, 2021
12 p.m. to 1 p.m.
Matt Blake – Habecker Lab
Sympathetic denervation of the heart following Myocardial Infarction (MI), which is sustained by chondroitin sulfate proteoglycans (CSPGs) in the cardiac scar, predicts risk of sudden cardiac death.Previous work demonstrated that blocking CSPG signaling restored sympathetic axon outgrowth into the cardiac scar, decreasing arrhythmia susceptibility. Growth inhibition by CSPGs is thought to depend on the sulfation status of the glycosaminoglycans (GAGs) attached to the core protein. In the central nervous system, tandem sulfation of CSPGs at the 4th (4S) and 6th (6S) positions of n-acetyl-galactosamine is thought to be a primary inhibitor of axon outgrowth. It is not known if CSPG sulfation prevents sympathetic nerve regeneration. Our data suggest that CSPG sulfation prevents sympathetic axon outgrowth just as it alters regeneration in the CNS. Sympathetic neurite outgrowth across purified CSPGs is restored in vitro by removing 4S-CSPGs with the enzyme Arylsulfatase-B (ARSB). Neurite outgrowth across CSPGs is reduced to 69 % of control levels while pre-treatment of CSPGs with increasing amounts of ARSB restored axon outgrowth to control levels in a dose dependent manner. Additionally, we co-cultured cardiac scar tissue with sympathetic ganglia ex vivo and found that removing 4S-CSPGs with ARSB restored axon outgrowth to control levels. Axon outgrowth towards infarcted myocardium is reduced to 66% of control (healthy myocardium) levels while treatment of infarcted myocardium with ARSB restored axon outgrowth to 97% of control levels. We examined levels of the enzymes responsible for adding and removing sulfation to CSPG-GAGs by western blot to determine if they were altered in the left ventricle after MI. We found that CHST11 (4-sulfotranserase) was downregulated, CHST15 (4S dependent 6-sulfotransferase) was upregulated, and ARSB (4-sulfatase) was downregulated after MI. This change was preceded by increased expression of Galectin 3, a key regulator of the inflammatory fibrotic response after ischemic injury. Increased CHST15 combined with decreased ARSB suggest a mechanism for production and maintenance of sulfated CSPGs in the cardiac scar.
Michael Berry – Sivyer & Schultz Lab
Light-dependent shifts in biological rhythms are driven by intrinsically photosensitive retinal ganglion cells (ipRGCs) innervating the suprachiasmatic nucleus (SCN). The SCN is the body’s master circadian pacemaker, and synchronizes internal clocks to the external day night cycle. There are roughly 3000 ipRGCs, which contain their own photopigment, melanopsin however, only some of these innervate the SCN and their roles in driving circadian entrainment remain unclear. Using transgenic mice, we have identified a subclass of GlyT2 positive ipRGCs that tile the retina and are restricted to a dorsal region that encodes reflected luminance from the ground. These ipRGCs form the primary visual projection to the SCN shell and the lateral hypothalamus. Using multi-electrode array recordings and channel rhodopsin to ‘OptoTag’ these ipRGCs, we demonstrate they are the most sensitive ipRGCs amongst 8 functional clusters and encode sustained luminance over a wide range of light intensities from starlight to bright daylight. While only accounting for ~10% of the total ipRGCs, their selective ablation abolishes light-evoked circadian entrainment. Our results also challenge the theory that ipRGCs overlap as a dense meshwork to optimize photon capture.