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.

Carboxy terminus and pore-forming domain properties specific to Cx37 are necessary for Cx37-mediated suppression of insulinoma cell proliferation.

Connexin 37 (Cx37) suppresses cell proliferation when expressed in rat insulinoma (Rin) cells, an effect also manifest in vivo during vascular development and in response to tissue injury. Mutant forms of Cx37 with nonfunctional channels but normally localized, wild-type carboxy termini are not growth suppressive. Here we determined whether the carboxy-terminal (CT) domain is required for Cx37-mediated growth suppression and whether the Cx37 pore-forming domain can be replaced with the Cx43 pore-forming domain and still retain growth-suppressive properties. We show that despite forming functional gap junction channels and hemichannels, Cx37 with residues subsequent to 273 replaced with a V5-epitope tag (Cx37-273tr*V5) had no effect on the proliferation of Rin cells, did not facilitate G1-cell cycle arrest with serum deprivation, and did not prolong cell cycle time comparably to the wild-type protein. The chimera Cx43*CT37, comprising the pore-forming domain of Cx43 and CT of Cx37, also did not suppress proliferation, despite forming functional gap junctions with a permselective profile similar to wild-type Cx37. Differences in channel behavior of both Cx37-273tr*V5 and Cx43*CT37 relative to their wild-type counterparts and failure of the Cx37-CT to interact as the Cx43-CT does with the Cx43 cytoplasmic loop suggest that the Cx37-CT and pore-forming domains are both essential to growth suppression by Cx37.

Inducible coexpression of connexin37 or connexin40 with connexin43 selectively affects intercellular molecular transfer.

Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 µM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.

A functional channel is necessary for growth suppression by Cx37.

Connexin 37 (Cx37) profoundly suppresses the proliferation of rat insulinoma (Rin) cells by unknown mechanisms. To determine whether a functional pore domain is necessary for Cx37-mediated growth suppression, we introduced a mutation that converted threonine 154 into alanine (T154A). Like other connexins mutated at the homologous site, Cx37-T154A localized to appositional membrane but failed to form functional channels and exerted a dominant-negative effect on coexpressed wild-type Cx37 or Cx43. Unlike the wild-type protein, Cx37-T154A did not suppress the proliferation of Rin cells and did not, with serum deprivation, result in cell cycle arrest. Furthermore, progression through the cell cycle was unaffected by expression of Cx37-T154A. These results indicate that a pore-forming domain that is able to form functional channels is essential for the anti-proliferative, cell-cycle arrest and serum-sensitivity effects of Cx37, and furthermore that the normally localized C-terminal domain is not sufficient for these effects of Cx37.

Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells.

In addition to providing a pathway for intercellular communication, the gap junction-forming proteins, connexins, can serve a growth-suppressive function that is both connexin and cell-type specific. To assess its potential growth-suppressive function, we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed: cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression, and progression through the G(1)/S checkpoint was delayed, resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G(1). Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37, with decreases in G(1) and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G(1)/S checkpoint.