385-P: Blocking the Nicotinic Receptors of the Parasympathetic Nervous System Prevents Severe Hypoglycemia-Induced Fatal Cardiac Arrhythmias in Rats

In response to hypoglycemia, excessive stimulation of the parasympathetic nervous system (PNS) may induce bradycardia and fatal heart block. Since signaling via nicotinic receptors mediates the PNS response, it was hypothesized that these receptors may mediate hypoglycemia-induced cardiac arrhythmias. To test this hypothesis, mecamylamine (a nicotinic receptor antagonist; 7.5 mg/kg, n = 17) or saline (control; n = 20) was infused intravenously in Sprague Dawley rats during insulin-induced (0.2mU/kg/min) severe hypoglycemic (10-15 mg/dl) clamps for 3 hours with electrocardiogram recordings. Compared to controls, mecamylamine-treated rats required a 3-fold higher glucose infusion rate during severe hypoglycemia, consistent with lower peak epinephrine levels (5698±557 vs. 2418±396 pg/ml; p &amp;lt; 0.001). In control rats, hypoglycemia led to 2nd degree heart block (1.8±1.7/min), 3rd degree heart block (32%), and mortality (25%). However, mecamylamine treatment completely prevented 2nd and 3rd degree heart block resulting in 100% survival (*p &amp;lt; 0.05). In summary, blocking nicotinic receptors prevents cardiac arrhythmias and mortality during severe hypoglycemia. Clinically, targeting the parasympathetic nervous system could be a logical approach to prevent sudden death in people with insulin-treated diabetes at risk for hypoglycemia. Disclosure C.M. Reno: None. J. Bayles: None. Y. Huang: None. M.B. Oxspring: None. S. Fisher: None. Funding National Institutes of Health; National Institute of Diabetes and Digestive and Kidney Diseases; JDRF </jats:sec

Severe Hypoglycemia–Induced Fatal Cardiac Arrhythmias Are Mediated by the Parasympathetic Nervous System in Rats

The contribution of the sympathetic nervous system (SNS) versus the parasympathetic nervous system (PSNS) in mediating fatal cardiac arrhythmias during insulin-induced severe hypoglycemia is not well understood. Therefore, experimental protocols were performed in nondiabetic Sprague-Dawley rats to test the SNS with 1) adrenal demedullation and 2) chemical sympathectomy, and to test the PSNS with 3) surgical vagotomy, 4) nicotinic receptor (mecamylamine) and muscarinic receptor (AQ-RA 741) blockade, and 5) ex vivo heart perfusions with normal or low glucose, acetylcholine (ACh), and/or mecamylamine. In protocols 1–4, 3-h hyperinsulinemic (0.2 units/kg/min) and hypoglycemic (10–15 mg/dL) clamps were performed. Adrenal demedullation and chemical sympathectomy had no effect on mortality or arrhythmias during severe hypoglycemia compared with controls. Vagotomy led to a 6.9-fold decrease in mortality; reduced first- and second-degree heart block 4.6- and 4-fold, respectively; and prevented third-degree heart block compared with controls. Pharmacological blockade of nicotinic receptors, but not muscarinic receptors, prevented heart block and mortality versus controls. Ex vivo heart perfusions demonstrated that neither low glucose nor ACh alone caused arrhythmias, but their combination induced heart block that could be abrogated by nicotinic receptor blockade. Taken together, ACh activation of nicotinic receptors via the vagus nerve is the primary mediator of severe hypoglycemia–induced fatal cardiac arrhythmias.</jats:p

159-OR: Vitamin E Treatment Reduces Severe Hypoglycemia-Induced Fatal Cardiac Arrhythmias in Type 1 Diabetic Rats

Severe hypoglycemia can lead to fatal cardiac arrhythmias. Our studies have shown that diabetes, per se, increases the risk of hypoglycemia-induced mortality in rats. It was hypothesized that excess oxidative stress, associated with diabetes, increases the heart’s susceptibility to hypoglycemia-induced fatal arrhythmias. To test this hypothesis, Sprague Dawley rats were made diabetic (streptozotocin 65 mg/kg) and randomized to two treatment groups: 1) antioxidant Vitamin E (400 mg/kg/day; n=18) or 2) control (vehicle, n=16) injected subcutaneously over 8 days. Then, rats underwent hyperinsulinemic (0.4 units/kg/min) severe hypoglycemic (10-15 mg/dl) clamps for 3 hours with continuous electrocardiogram recording. Confirming its antioxidant properties, Vitamin E treatment significantly reduced oxidative stress in the heart 3.3-fold versus controls (Figure). As compared to hypoglycemia-induced mortality of 38% in controls, Vitamin E treatment significantly reduced mortality to 6% (p&amp;lt;0.05; Figure). Additionally, Vitamin E treatment reduced 3rd degree heart block (6%) vs. control (46%; Figure). Overall, these results suggest that 1) oxidative stress in diabetes increases the heart’s susceptibility to hypoglycemia-induced arrhythmias, and 2) Vitamin E treatment reduces oxidative stress and reduces both cardiac arrhythmias and mortality to insulin-induced severe hypoglycemia. Disclosure C.M. Reno: None. M.B. Oxspring: None. J. Bayles: None. I. Holiday: None. Y. Huang: None. S.J. Fisher: None. Funding National Institutes of Health (5T32DK091317, R01NS070235); JDRF (3-APF-2017-407-A-N) </jats:sec

158-OR: Calcium Channel Blockade Protects against Severe Hypoglycemia–Induced Fatal Cardiac Arrhythmias in Rats

It was hypothesized that in response to insulin-induced severe hypoglycemia, excess calcium influx into the heart causes fatal cardiac arrhythmias. To test if blocking the calcium channels would decrease fatal cardiac arrhythmias during severe hypoglycemia, L-type calcium channel blocker (Verapamil; 1 mg/kg, n = 25) or saline (n = 24) were infused into Sprague Dawley rats during a hyperinsulinemic (0.2 mU/kg/min) severe hypoglycemic (10-15 mg/dl) clamp for 3 hours with ECG. During severe hypoglycemia, verapamil completely prevented mortality compared to 21% mortality in controls (p &amp;lt; 0.05; Figure). Decreased mortality was associated with a 99% decrease in 2nd degree heart block (Figure) and prevention of 3rd degree heart block compared to saline (p &amp;lt; 0.05). Glucagon and epinephrine were similar between the groups suggesting verapamil does not affect hypoglycemic counterregulation. Consistent with the notion of calcium-mediated arrhythmias, separate experiments demonstrated that pharmacological blockade of ryanodine receptor-mediated calcium signaling also reduced heart block by 97% and prevented mortality due to hypoglycemia. In summary, blocking calcium channels protects against severe hypoglycemia-induced fatal cardiac arrhythmias. Blockade of cardiac calcium channels could be a potential approach to prevent arrhythmias in people with diabetes at risk for hypoglycemia. Disclosure C.M. Reno: None. Y. Huang: None. C.G. Christensen: None. M.B. Oxspring: None. J. Bayles: None. S.J. Fisher: None. Funding National Institutes of Health (5T32DK091317, R01NS070235); JDRF (3-APF-2017-407-A-N) </jats:sec

Stratified analyses refine association between TLR7 rare variants and severe COVID-19

Summary: Despite extensive global research into genetic predisposition for severe COVID-19, knowledge on the role of rare host genetic variants and their relation to other risk factors remains limited. Here, 52 genes with prior etiological evidence were sequenced in 1,772 severe COVID-19 cases and 5,347 population-based controls from Spain/Italy. Rare deleterious TLR7 variants were present in 2.4% of young (<60 years) cases with no reported clinical risk factors (n = 378), compared to 0.24% of controls (odds ratio [OR] = 12.3, p = 1.27 × 10−10). Incorporation of the results of either functional assays or protein modeling led to a pronounced increase in effect size (ORmax = 46.5, p = 1.74 × 10−15). Association signals for the X-chromosomal gene TLR7 were also detected in the female-only subgroup, suggesting the existence of additional mechanisms beyond X-linked recessive inheritance in males. Additionally, supporting evidence was generated for a contribution to severe COVID-19 of the previously implicated genes IFNAR2, IFIH1, and TBK1. Our results refine the genetic contribution of rare TLR7 variants to severe COVID-19 and strengthen evidence for the etiological relevance of genes in the interferon signaling pathway

Whole-genome sequencing reveals host factors underlying critical COVID-19

: Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2-4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Whole-genome sequencing reveals host factors underlying critical COVID-19

AbstractCritical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease.</jats:p

Mapping the human genetic architecture of COVID-19

AbstractThe genetic make-up of an individual contributes to the susceptibility and response to viral infection. Although environmental, clinical and social factors have a role in the chance of exposure to SARS-CoV-2 and the severity of COVID-191,2, host genetics may also be important. Identifying host-specific genetic factors may reveal biological mechanisms of therapeutic relevance and clarify causal relationships of modifiable environmental risk factors for SARS-CoV-2 infection and outcomes. We formed a global network of researchers to investigate the role of human genetics in SARS-CoV-2 infection and COVID-19 severity. Here we describe the results of three genome-wide association meta-analyses that consist of up to 49,562 patients with COVID-19 from 46 studies across 19 countries. We report 13 genome-wide significant loci that are associated with SARS-CoV-2 infection or severe manifestations of COVID-19. Several of these loci correspond to previously documented associations to lung or autoimmune and inflammatory diseases3–7. They also represent potentially actionable mechanisms in response to infection. Mendelian randomization analyses support a causal role for smoking and body-mass index for severe COVID-19 although not for type II diabetes. The identification of novel host genetic factors associated with COVID-19 was made possible by the community of human genetics researchers coming together to prioritize the sharing of data, results, resources and analytical frameworks. This working model of international collaboration underscores what is possible for future genetic discoveries in emerging pandemics, or indeed for any complex human disease.</jats:p

Whole-genome sequencing reveals host factors underlying critical COVID-19

Altres ajuts: Department of Health and Social Care (DHSC); Illumina; LifeArc; Medical Research Council (MRC); UKRI; Sepsis Research (the Fiona Elizabeth Agnew Trust); the Intensive Care Society, Wellcome Trust Senior Research Fellowship (223164/Z/21/Z); BBSRC Institute Program Support Grant to the Roslin Institute (BBS/E/D/20002172, BBS/E/D/10002070, BBS/E/D/30002275); UKRI grants (MC_PC_20004, MC_PC_19025, MC_PC_1905, MRNO2995X/1); UK Research and Innovation (MC_PC_20029); the Wellcome PhD training fellowship for clinicians (204979/Z/16/Z); the Edinburgh Clinical Academic Track (ECAT) programme; the National Institute for Health Research, the Wellcome Trust; the MRC; Cancer Research UK; the DHSC; NHS England; the Smilow family; the National Center for Advancing Translational Sciences of the National Institutes of Health (CTSA award number UL1TR001878); the Perelman School of Medicine at the University of Pennsylvania; National Institute on Aging (NIA U01AG009740); the National Institute on Aging (RC2 AG036495, RC4 AG039029); the Common Fund of the Office of the Director of the National Institutes of Health; NCI; NHGRI; NHLBI; NIDA; NIMH; NINDS.Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care or hospitalization after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease