Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

<div><p>The <i>C</i>. <i>elegans</i> AWC olfactory neuron pair communicates to specify asymmetric subtypes AWC^OFF and AWC^ON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWC^ON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that <i>slo-1</i> BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWC^ON. We also show for the first time that <i>slo-2</i> BK channels are important for AWC asymmetry and act redundantly with <i>slo-1</i> to inhibit calcium signaling. In addition, <i>nsy-5</i>-dependent asymmetric expression of <i>slo-1</i> and <i>slo-2</i> in the AWC^ON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, <i>slo-1</i> and <i>slo-2</i> regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of <i>bkip-1</i>, which encodes a previously identified auxiliary subunit of SLO-1, for <i>slo-1</i> and <i>slo-2</i> function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons.</p></div

<i>slo-1</i> and <i>slo-2</i> act downstream of <i>nsy-5</i> to antagonize the function of voltage-gated calcium channel-activated kinase cascade in promoting AWC^ON.

<p>(<b>A</b>) Double and triple mutant analysis of <i>slo-1(ky389gf)</i>, <i>slo-1(ky399gf)</i>, and <i>slo-1(eg142lf); slo-1(ok2214lf)</i> animals with mutants of known genes involved in establishment of AWC asymmetry. 2AWC^ON, both AWC cells express <i>str-2</i>; 1AWC^OFF/AWC^ON, only one of the two AWC cells expresses <i>str-2</i>; 2AWC^OFF, neither AWC cell expresses <i>str-2</i>. (<b>B</b>) The genetic pathway that demonstrates possible relationships between <i>slo-1</i>, <i>slo-2</i> and other genes required for AWC asymmetry. Genes in green represent AWC^OFF promoting, genes in red represent AWC^ON promoting, and those in grey represent less active or inactive genes.</p

SLO-1 and SLO-2 BK potassium channels are localized in the vicinity of UNC-2 voltage-gated calcium channels in AWC axons.

<p>(<b>A-C</b>) Images of wild-type L1 animals expressing single copy insertion transgenes <i>odr-3p</i>::<i>slo-1</i>::<i>TagRFP</i> and <i>odr-3p</i>::<i>GFP</i>::<i>unc-2</i> (A), <i>odr-3p</i>::<i>slo-2</i>::<i>TagRFP</i> and <i>odr-3p</i>::<i>GFP</i>::<i>unc-2</i> (B), as well as <i>odr-3p</i>::<i>slo-2</i>::<i>TagRFP</i> and <i>odr-3p</i>::<i>slo-1</i>::<i>GFP</i> (C) in AWC neurons. SLO-1::TagRFP (A), SLO-1::GFP (C), SLO-2::TagRFP (B, C), and GFP::UNC-2 (A, B) were localized in AWC cell bodies (arrows) and in a punctate pattern along AWC axons (arrowheads). In AWC axons, SLO-1::TagRFP was localized next to GFP::UNC-2 (A); SLO-2::TagRFP was adjacent to GFP::UNC-2 (B); and SLO-2::TagRFP was localized near SLO-1::GFP (C). Insets show higher magnification of the outlined areas that exemplify localization of two translational reporters in close proximity. Scale bar, 5 μm. Anterior is at left and ventral is at bottom. (D) Quantification of mean correlation coefficient between SLO-1 and UNC-2, SLO-2 and UNC-2, as well as SLO-1 and SLO-2 using three algorithms of the Coloc 2 plugin in Fiji: Pearson’s correlation coefficient, Spearman’s rank correlation coefficient, and Li’s ICQ. For each colocalization class, images of three animals were used for quantification. Positive values of each coefficient indicate positive correlation, values close to zero indicate no correlation, and negative values indicate anti-correlation. Pearson's correlation coefficient ranges from -1 to +1; Spearman’s rank correlation coefficient ranges from -1 to +1; Li's ICQ value ranges from -0.5 to +0.5. A schematic diagram of the AWC cell body, axon, dendrite, and cilia that represents the approximate region of images in A-C is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.s002" target="_blank">S2D Fig</a>.</p

<i>slo-1</i> and <i>slo-2</i> are expressed asymmetrically in the AWC^ON neuron.

<p>(<b>A, C</b>) Images of wild-type L1 animals showing expression of <i>slo-1p</i>::<i>GFP</i> (A) and <i>slo-2p</i>::<i>GFP</i> (C) at a higher level in AWCR (bottom panels) than in AWCL (top panels). Both AWCL and AWCR were labeled by <i>odr-1p</i>::<i>TagRFP</i>. The cell body of both AWC cells is outlined by dashed lines. Scale bar, 5 μm. Anterior is at left and ventral is at bottom. (<b>B, D</b>) Quantification of asymmetric expression of <i>slo-1p</i>::<i>GFP</i> (B) and <i>slo-2p</i>::<i>GFP</i> (D) in AWCL and AWCR in wild type and mutants defective in AWC asymmetry. The single focal plane with the brightest fluorescence in each AWC was selected from the acquired image stack and compared for fluorescence intensity. The fluorescence intensity of <i>slo-1p</i>::<i>GFP</i> and <i>slo-2p</i>::<i>GFP</i> was compared using visual quantitative scoring between AWCL and AWCR in each animal, as previously performed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.ref007" target="_blank">7</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.ref022" target="_blank">22</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.ref058" target="_blank">58</a>]. If no obvious difference in fluorescence intensity between the two AWC cells was observed, the animal was categorized as AWCL = AWCR. If an obvious difference in fluorescence intensity was observed between AWCL and AWCR, the animal was assigned to AWCL > AWCR or AWCR > AWCL. For both <i>slo-1p</i>::<i>GFP</i> and <i>slo-2p</i>::<i>GFP</i>, the visual quantification of fluorescence was performed by the same individual. Only animals with visible expression in both AWC neurons were used in the analysis. <i>p</i> values were calculated using Fisher’s exact test. ns, not significant. Error bars indicate standard error of proportion. (<b>E, G</b>) Representative images of wild-type L1 animals expressing <i>slo-1p</i>::<i>2xnlsGFP</i> (E) and <i>slo-2p</i>::<i>GFP</i> (G) in AWC^ON (bottom panel) but not in AWC^OFF (top panel). Both AWC neurons were marked with <i>ceh-36p</i>::<i>myrTagRFP</i>. AWC^ON cells were marked by <i>str-2p</i>::<i>2xnlsTagRFP</i>, and AWC^OFF neurons were defined by lack of <i>str-2p</i>::<i>2xnlsTagRFP</i>. Scale bar, 5 μm. Anterior is at left and ventral is at bottom. (<b>F, H</b>) Quantification of <i>slo-1p</i>::<i>2xnlsGFP</i> (F) and <i>slo-2p</i>::<i>GFP</i> (H) expression in AWC^ON and AWC^OFF. The single focal plane with the brightest fluorescence in each AWC was selected from the acquired image stack and compared for fluorescence intensity. The fluorescence intensity of <i>slo-1p</i>::<i>2xnlsGFP</i> and <i>slo-2p</i>::<i>GFP</i> was compared using visual quantitative scoring between AWC^ON and AWC^OFF in each animal, as previously performed [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.ref007" target="_blank">7</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.ref022" target="_blank">22</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.ref058" target="_blank">58</a>]. Each animal was categorized into one of three categories: AWC^ON = AWC^OFF, AWC^ON > AWC^OFF, and AWC^OFF > AWC^ON based on the comparison of GFP intensities between AWC^ON and AWC^OFF cells of the same animal. <i>p</i> values were calculated using a <i>Z</i>-test. Error bars indicate standard error of proportion.</p

<i>slo-1</i> and <i>slo-2</i> regulate the subcellular localization of synaptic markers in AWC neurons.

<p><b>(A</b>) Left panels: Images of wild type, <i>slo-1(ky399gf)</i>, and <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants expressing the single copy insertion transgene <i>odr-3p</i>::<i>GFP</i>::<i>unc-2</i> (the same transgene as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005654#pgen.1005654.g005" target="_blank">Fig 5A and 5B</a>) in AWC cell bodies (arrows) and axons (arrowheads) in L1. Right panel: Quantification of GFP::UNC-2 fluorescence intensity in AWC axons and cell bodies. <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants displayed a significant decrease in GFP::UNC-2 intensity in AWC axons and cell bodies. (<b>B</b>) Left panels: Images of wild-type, <i>slo-1(ky399gf)</i>, and <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants expressing the single copy insertion transgene <i>odr-3p</i>::<i>YFP</i>::<i>rab-3</i> in AWC cell bodies (arrows) and axons (arrowheads) in L1. Right panel: Quantification of YFP::RAB-3 fluorescence intensity in AWC axons and cell bodies. <i>slo-1(eg142lf); slo-1(ok2214lf)</i> mutants had a significant decrease in YFP::RAB-3 intensity in AWC axons and cell bodies. (<b>A</b>, <b>B</b>) Anterior is at left and ventral is at bottom. Scale bar, 5 μm. Student’s <i>t-</i>test was used for statistical analysis. ns, not significant. Error bars, standard error of the mean. AU, arbitrary unit.</p

Model of <i>slo-1</i> and <i>slo-2</i> function in AWC asymmetry.

<p>AWC asymmetry is stochastic, and this figure illustrates the case when AWC^ON is on the left side of the head. Molecules in green represent AWC^OFF promoting, molecules in red represent AWC^ON promoting, and those in grey represent less active or inactive molecules. In the AWC^OFF neuron (right), calcium enters the cell through voltage-gated calcium channels (UNC-2/UNC-36 and EGL-19/UNC-36) and stimulates a MAP kinase cascade consisting of UNC-43 (CaMKII), TIR-1 (Sarm1) adaptor protein, and NSY-1 (MAPKKK). This leads to expression of the AWC^OFF marker <i>srsx-3</i> and suppression of the AWC^ON marker <i>str-2</i>. In the AWC^ON cell (left), NSY-5 gap junctions activate SLO-1 and SLO-2 voltage- and calcium-activated potassium channels, which antagonize the function of UNC-2/UNC-36 and EGL-19/UNC-36 calcium channels by suppressing the calcium-activated CaMKII-MAP kinase cascade. NSY-4 (claudin) acts in parallel with NSY-5, SLO-1, and SLO-2 to inhibit calcium channel-mediated signaling, resulting in de-repression of <i>str-2</i> expression.</p

<i>bkip-1</i> modulates <i>slo-1</i> and <i>slo-2</i> activity in AWC neurons.

<p>(<b>A</b>) Genetic analysis of known modulators of SLO-1 in AWC asymmetry. (<b>B</b>) Images of wild type and <i>bkip-1(zw2)</i> L1 animals expressing <i>odr-3p</i>::<i>slo-1</i>::<i>GFP</i> in AWC axons and cell bodies. Scale bar, 5 μm. (<b>C, D</b>) Quantification of SLO-1::GFP fluorescence intensity in AWC axons (C) and AWC cell body (D). In <i>bkip-1(zw2)</i> mutants, SLO-1::GFP intensity is significantly decreased in AWC axons, but is not significantly affected in AWC cell body. Anterior is at left and ventral is at bottom. Student’s <i>t-</i>test was used for statistical analysis. ns, not significant. Error bars, standard error of the mean. AU, arbitrary unit.</p