Sympathetic remodeling and altered angiotensin-converting enzyme 2 localization occur in patients with cardiac disease but are not exacerbated by severe COVID-19.

PURPOSE: Remodeling of sympathetic nerves and ACE2 has been implicated in cardiac pathology, and ACE2 also serves as a receptor for SARS-CoV-2. However, there is limited histological knowledge about the transmural distribution of sympathetic nerves and the cellular localization and distribution of ACE2 in human left ventricles from normal or diseased hearts. Goals of this study were to establish the normal pattern for these parameters and determine changes that occurred in decedents with cardiovascular disease alone compared to those with cardiac pathology and severe COVID-19. METHODS: We performed immunohistochemical analysis on sections of left ventricular wall from twenty autopsied human hearts consisting of a control group, a cardiovascular disease group, and COVID-19 ARDS, and COVID-19 non-ARDS groups. RESULTS: Using tyrosine hydroxylase as a noradrenergic marker, we found substantial sympathetic nerve loss in cardiovascular disease samples compared to controls. Additionally, we found heterogeneous nerve loss in both COVID-19 groups. Using an ACE2 antibody, we observed robust transmural staining localized to pericytes in the control group. The cardiovascular disease hearts displayed regional loss of ACE2 in pericytes and regional increases in staining of cardiomyocytes for ACE2. Similar changes were observed in both COVID-19 groups. CONCLUSIONS: Heterogeneity of sympathetic innervation, which occurs in cardiac disease and is not increased by severe COVID-19, could contribute to arrhythmogenesis. The dominant localization of ACE2 to pericytes suggests that these cells would be the primary target for potential cardiac infection by SARS-CoV-2. Regional changes in ACE2 staining by myocytes and pericytes could have complex effects on cardiac pathophysiology.

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19 F NMR.

R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry-related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N-termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N-terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4- and 5-fluoroindole labeled protein and severe line broadening for 6- and 7-fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the 19 F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.