Objective The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti-SARS-CoV-2 antibody levels. Methods In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA-and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. Results The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R 2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. Conclusions With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination.
Excessive β-adrenergic stimulation and tachycardia are potent triggers of cardiac remodeling; however, their exact cellular effects remain elusive. Here, we sought to determine the potency of β-adrenergic stimulation and tachycardia to modulate gene expression profiles of cardiomyocytes. Using neonatal rat ventricular cardiomyocytes, we showed that tachycardia caused a significant upregulation of sodium–calcium exchanger (NCX) and the activation of calcium/calmodulin-dependent kinase II (CaMKII) in the nuclear region. Acute isoprenaline treatment ameliorated NCX-upregulation and potentiated CaMKII activity, specifically on the sarcoplasmic reticulum and the nuclear envelope, while preincubation with the β-blocker propranolol abolished both isoprenaline-mediated effects. On a transcriptional level, screening for hypertrophy-related genes revealed tachycardia-induced upregulation of interleukin-6 receptor (IL6R). While isoprenaline prevented this effect, pharmacological intervention with propranolol or NCX inhibitor ORM-10962 demonstrated that simultaneous CaMKII activation on the subcellular Ca2+ stores and prevention of NCX upregulation are needed for keeping IL6R activation low. Finally, using hypertensive Dahl salt-sensitive rats, we showed that blunted β-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of β-adrenergic signaling, which is compromised in cardiac pathologies, in preventing IL6R overactivation under increased workload. A better understanding of these processes may contribute to refinement of therapeutic options for patients receiving β-blockers.
The nucleus of a cardiomyocyte has been increasingly recognized as a morphologically distinct and partially independent calcium (Ca2+) signaling microdomain, with its own Ca2+-regulatory mechanisms and important effects on cardiac gene expression. In this review, we (1) provide a comprehensive overview of the current state of research on the dynamics and regulation of nuclear Ca2+ signaling in cardiomyocytes, (2) address the role of nuclear Ca2+ in the development and progression of cardiac pathologies, such as heart failure and atrial fibrillation, and (3) discuss novel aspects of experimental methods to investigate nuclear Ca2+ handling and its downstream effects in the heart. Finally, we highlight current challenges and limitations and recommend future directions for addressing key open questions.
Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): BioTechMed Graz Objective Cardiac remodelling encompasses changes at the molecular, cellular and gene expression level following pathologic insult to the heart. Initially, it maintains cardiovascular homeostasis and allows patients to remain asymptomatic, but if untreated, it eventually progresses to symptomatic heart failure. Excessive β-adrenergic stimulation and tachycardia are potent triggers of cardiac remodelling; however, the underlying mechanisms of their cellular effects are not fully understood. Using neonatal rat ventricular cardiac myocytes (NRVCMs), we studied individual and synergistic potency of ß-adrenergic stimulation and tachycardia to modulate pathological gene expression profiles, as well as the effectiveness of ß-blockers (BB) in preventing these alterations. Methods Primary NRVCMs were isolated from 1-day-old neonatal Wistar rats, cultured for 3 days and subsequently stimulated for 3h at basal (1Hz) and tachycardia (8Hz) conditions either in (1) cell culture medium to determine the sole effect of tachycardia, (2) cell culture medium supplemented with ß-adrenergic agonist isoprenaline (ISO; 10µM) to investigate the influence of ß-adrenergic stimulation and signalling or (3) cell culture medium supplemented with ISO following 1h preincubation with propranolol (ISO+BB; 1µM) to assess the potential of BB in preventing gene reprogramming. Screening of relative mRNA levels of hypertrophic marker genes and regulators of ion homeostasis in cardiomyocytes was performed by qPCR and calculated using the 2-ΔΔCt quantification method. Results qPCR screening of the known hypertrophic marker genes revealed that tachycardia caused significant transcriptional upregulation of regulator of calcineurin 1 (RCAN1) and interleukin-6 receptor (IL6R). Treatment with ISO additionally upregulated RCAN1, while preincubation with BB resulted in a return towards baseline expression of both genes, completely blocking the effects of tachycardia alone or when combined with ISO stimulation. Interestingly, two potassium channel genes, KCNH2 and KCNJ2, responsible for expression of hERG and Kir2.1 channels, respectively, were unchanged with tachycardia alone but significantly downregulated upon additional stimulation with ISO. Preincubation with BB could - at least partially - reverse the effect. Conclusion In conclusion, we could show that apart from the well-documented effect of excessive ß-adrenergic stimulation on hypertrophic signalling in cardiomyocytes, it also has a direct, non-tachypacing mediated effect on the expression levels of hERG and Kir2.1 potassium channels, which may be causally involved in inducing early cardiac remodelling. Thus, a previously unidentified benefit of BB therapy may be restoring potassium homeostasis contributing to the prevention of adverse cardiac remodelling and its progression to heart failure.
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