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Heightened sympathetic drive (dysautonomia) is a hallmark of several cardiovascular diseases including SARS-CoV-2. It is also a powerful prognostic predictor for arrhythmia and sudden cardiac death, especially in patients with channelopathies (long QT syndrome-LQTS, and catecholaminergic polymorphic ventricular tachycardia-CPVT). However, little is known about the molecular targets underlying this dysautonomia. We have identified a novel pathway using a combination of single cell and bulk RNAseq, neurochemistry, FRET imaging and single cell electrophysiology. This pathway involves impairment of cyclic nucleotide coupled phosphodiesterases (PDE) linked to enhanced intracellular calcium transients and exocytosis from rat sympathetic neurons. In particular, the adaptor protein Nos1-ap, Pde2A, and Ace2 are associated with sympathetic hyperexcitability. These proteins are also conserved in human stellates from patients with LQTS and CPVT, although their role in neuronal-myocyte cellular function is unknown. We have developed a unique human iPSC sympathetic-cardiac co-culture model for target discovery in LQTS and CPVT. The lecture will highlight the use of gene manipulation of these proteins to determine their role in driving abnormal transmission and arrhythmia.
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INTRODUCTION: Novel coronavirus SARS-COV-2 is primarily spread via droplets with respiratory involvement being the most common presentation. Approximately 14% of patients with COVID-19 pneumonia will progress to develop ARDS which is associated with high mortality. Pulmonary vasodilators have been shown to improve oxygenation in severe ARDS. Inhaled epoprostenol (iEPO) promote pulmonary vasodilation by decreasing intracellular calcium and possess anti-inflammatory and antiplatelet effects which may be beneficial in C-ARDS. METHOD(S): This was a retrospective cohort study that was IRB approved and included patients diagnosed with COVID-19 ARDS who received inhaled epoprostenol during period of 3/1/2020 and 12/31/2021. Patients that met study inclusion criteria were classified as responders or non-responders based on percent change of 10% or more in PaO2/FiO2 (PF) ratio post iEPO. The primary outcome was to compare the incidence of in-hospital mortality between both groups. Secondary outcomes included mechanical ventilation duration, ICU and hospital length of stay (LOS) and adverse events. RESULT(S): 148 patients were included in the analysis and based on post iEPO arterial blood gas (ABG) were classified as responders (n=87) and non-responders (n=61). Baseline characteristics were similar between both groups except for a lower weight and more prone positioning utilized in the responder group. Post iEPO PF ratio was significantly higher in the responder group compared to non-responder (158.69 vs. 107.67, p< 0.001) with similar duration of therapy in both groups (5.44 vs. 6.04 days, p=0.51). There was no difference in in-hospital mortality between responders and non-responders (69% vs. 72.1% p=0.68). There was no difference noted in ICU LOS, hospital LOS and duration of mechanical ventilation between both groups. A multivariable regression analysis was performed and noted no association between variables evaluated and response to inhaled epoprostenol therapy. CONCLUSION(S): Despite over 50% of patients in cohort responding to iEPO as demonstrated by significant improvement in oxygenation there was no difference in patient outcomes noted in responders as compared to nonresponders.
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SESSION TITLE: COVID-19 Case Report Posters 2 SESSION TYPE: Case Report Posters PRESENTED ON: 10/19/2022 12:45 pm - 01:45 pm INTRODUCTION: Malignant hyperthermia (MH) is a hypermetabolic crisis where an increase in carbon dioxide is seen despite an increased minute ventilation with a proposed mechanism as a disturbance in calcium homeostasis. Commonly seen in volatile anesthetic agents and depolarizing neuromuscular blockers, rarely with nondepolarizing agents. There has been one reported case of cisatracurium-induced MH in the setting of ARDS. There have been two cases reported of nondepolarizing neuromuscular agents causing MH in the setting of COVID-19. CASE PRESENTATION: A 34-year-old man with severe COVID-19 complicated by ARDS on ventilator day 16, due to refractory fevers, ventilatory dyssynchrony, high minute ventilation and auto-PEEP phenomena, the decision was made to attempt neuromuscular paralysis. After one dose of cisatracurium, the patient became hyperthermic and end-tidal carbon-dioxide increased from 58-98 with inability to oxygenate. The patient developed high peak pressures, bedside ultrasound revealed no evidence of pneumothorax also confirmed with chest x-ray. The patient then received a dose of dantrolene with end-tidal improving to 60 and tachycardia also resolved. A creatinine kinase level drawn was elevated at 571. DISCUSSION: A proposed mechanism of MH is calcium release from sarcoplasmic reticulum, a mutation in skeletal muscle ryanodine receptor calcium release channels that can release IL-6 when activated leading to excessive muscular contraction. Proinflammatory cytokine IL-6, dantrolene may block IL-6 release which results in its therapeutic effect in the treatment of MH. IL-6 has been used to predict deterioration from COVID-19. Dantrolene in this sense has been proposed as a potential therapeutic agent against COVID-19, inhibiting intracellular calcium influx thus preventing the pathological feedback of viral entry into cells via endocytosis, as this is a calcium dependent process. Given the possible link between IL-6 release, calcium and MH, SARS-CoV-2 viral entry into cells may place patients at higher risk of MH. Patients with COVID-19 may be at higher risk of MH, even in rare agents such as non-depolarizing agents as seen in this case. Awareness of this potentially increased complication from these agents in those patients with COVID-19 is key as we continue in the ongoing global pandemic. CONCLUSIONS: Given the possible link between IL-6 release, calcium and MH, SARS-CoV-2 viral entry into cells may place patients at higher risk of MH. Patients with COVID-19 may be at higher risk of malignant hyperthermia, even in rare agents such as non-depolarizing agents as seen in this case. Awareness of this potentially increased complication from these agents in those patients with COVID-19 is key as we continue in the ongoing global pandemic. Reference #1: Sathyanarayanan SP, Hamza M, Hamid K, Groskreutz D. Cisatracurium-Associated Malignant Hyperthermia During Severe Sars-CoV-2 Infection. Am J Ther. 2021 Aug 10;28(5):e590-e591. doi: 10.1097/MJT.0000000000001437. PMID: 34387563;PMCID: PMC8415506. Reference #2: Chiba N, Matsuzaki M, Mawatari T, Mizuochi M, Sakurai A, Kinoshita K. Beneficial effects of dantrolene in the treatment of rhabdomyolysis as a potential late complication associated with COVID-19: a case report. Eur J Med Res. 2021 Feb 8;26(1):18. doi: 10.1186/s40001-021-00489-8. PMID: 33557936;PMCID: PMC7868892. Reference #3: Han H, Ma Q, Li C, Liu R, Zhao L, Wang W, Zhang P, Liu X, Gao G, Liu F, Jiang Y, Cheng X, Zhu C, Xia Y. Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors. Emerg Microbes Infect. 2020 Dec;9(1):1123-1130. doi: 10.1080/22221751.2020.1770129. PMID: 32475230;PMCID: PMC7473317. DISCLOSURES: No relevant relationships by Hira Bakhtiar No relevant relationships by Timothy DAmico no disclosure on file for Sarah Margolskee;No relevant relationships by Carlos Merino No relevant relationships by Joanna Moore
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Objective: Red blood cell (RBC) role is both passive action, oxygen delivery to the tissues as well as carbon dioxide to the lungs and active action involvement in the regulation of vascular tone. The aim was to investigate pathophysiological and ultrastructural changes of RBC in heart failure (HF) patients with hypertension (HT) and long Covid. Design and method: In total 12 patients with HF of Coronary Artery Disease origin, HT, and long Covid were examined. Mean age of patients was 62 ± 5.8 years. The control group consisted of 10 apparently healthy people. The functional state and ultrastructure of RBC were studied using electron microscopy. Results: During ultrastructure examination, structural pathologies of RBC in HF patients with HT and long Covid were revealed. RBC anisocytosis and poikilocytosis as structural damage variations in size and shape were found respectively. Reticulocytes were found much more often in HF patients with HT and long Covid than in the control group. In healthy control group, RBC had a typical discoid shape. In the presence of long Covid, both calcification as a marker of RBC apoptosis and destruction was also detected (Fig.1). Neutrophil extracellular traps (NETs) were found in RBC surrounding (Fig.1). Conclusions: Altered RBC function has important implications for HF patients with HT and long Covid. RBC has been shown to induce endothelial cell dysfunction and to increase cardiac injury as well as increased inflammatory processes in long Covid. The presence of HF, HT and long Covid leads to RBC calcification and activation of blood cell apoptosis. Prognostic role of RBC calcium distribution in combination with other important prognostic measures, such as biomarkers like Thrombospondin - 1, NT-proBNP and ST2 is subject of interest and requires further research.
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Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity. Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism. Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. Disclosures: Schwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees;Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.