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At the beginning of the COVID-19 pandemic, diagnostic testing was not accessible for mildly ill or asymptomatic individuals. Military operational circumstances exclude the usage of reference laboratory tests. For that reason, at the beginning of the pandemic alternative test methods were needed in order to gain insight into the SARS-CoV-2 status of military personnel. The objectives of this study are to assess whether SARS-CoV-2 antibody rapid lateral flow assay (LFA) in combination with semi-autonomous SARS-CoV-2 antisense and sense genomic RNA fluorescence in situ hybridization (FISH) could establish disease status in military personnel in a fieldable setting, and to assess how this combination performed and to determine which type of sample performed best. A proof of concept sub-study regarding the SARS-CoV-2 application of the fieldable Biotrack-MED® FISH analyzer, a semi-autonomous multi-sample filter cytometer, preceded this observational prospective cohort pilot study. Dutch military personnel were included in the 26 June 2020-11 May 2021 period. Blood, nasopharyngeal and oropharyngeal swabs and saliva were tested at days 0 and 14. SPSS version 25 descriptive statistics and Cohen's kappa assessed agreement between test methods. Both the sensitivity and specificity of the field tests were calculated with ELISA and PCR as reference. Saliva appeared to be the preferred sample type for FISH, where blood was not useful. FISH analysis and LFA results had a concordance of 42% for testing negative, 30% for recovered from infection, 22% for ongoing-and 58% for acute infection in a reference laboratory lab result outcome (RT-PCR or ELISA respectively). The LFA results on serum and full blood corresponded with the ELISA-obtained results (kappa of 0.61 and 0.63 respectively at day 0 and 0.81 and 0.77 respectively at day 14). LFA (full blood-serum), FISH and RT-PCR on saliva did not reach the 90% sensitivity level advised by WHO, with 64-54, 38 and 71% at day 0 and 80-79, 53 and 24% at day 14 respectively.

Atrial fibrillation (AF) is accompanied by a detrimental loss of functional cardiomyocytes. Here, Zhang et al. show that AF-induced cardiomyocyte dysfunction is a consequence of DNA damage-mediated PARP1 activation, which leads to depletion of NAD+ and further oxidative stress and DNA damage, and identify PARP1 inhibition as a potential therapeutic strategy in the treatment of AF.

Saliva is a matrix which may act as a vector for pathogen transmission and may serve as a possible proxy for SARS-CoV-2 contagiousness. Therefore, the possibility of detection of intracellular SARS-CoV-2 in saliva by means of fluorescence in situ hybridization is tested, utilizing probes targeting the antisense or sense genomic RNA of SARS-CoV-2. This method was applied in a pilot study with saliva samples collected from healthy persons and those presenting with mild or moderate COVID-19 symptoms. In all participants, saliva appeared a suitable matrix for the detection of SARS-CoV-2. Among the healthy, mild COVID-19-symptomatic and moderate COVID-19-symptomatic persons, 0%, 90% and 100% tested positive for SARS-CoV-2, respectively. Moreover, the procedure allows for simultaneous measurement of viral load ('presence', sense genomic SARS-CoV-2 RNA) and viral replication ('activity', antisense genomic SARS-CoV-2 RNA) and may yield qualitative results. In addition, the visualization of DNA in the cells in saliva provides an additional cytological context to the validity and interpretability of the test results. The method described in this pilot study may be a valuable diagnostic tool for detection of SARS-CoV-2, distinguishing between 'presence' (viral load) and 'activity' (viral replication) of the virus. Moreover, the method potentially gives more information about possible contagiousness.

Background: Staging of atrial fibrillation (AF) is essential to understanding disease progression and the accompanied increase in therapy failure. Blood-based heat shock protein (HSP) levels may enable staging of AF and the identification of patients with higher risk for AF recurrence after treatment. Objective: This study evaluates the relationship between serum HSP levels, presence of AF, AF stage and AF recurrence following electrocardioversion (ECV) or pulmonary vein isolation (PVI). Methods: To determine HSP27, HSP70, cardiovascular (cv)HSP and HSP60 levels, serum samples were collected from control patients without AF and patients with paroxysmal atrial fibrillation (PAF), persistent (PeAF) and longstanding persistent (LSPeAF) AF, presenting for ECV or PVI, prior to intervention and at 3-, 6- and 12-months post-PVI. Results: The study population (n = 297) consisted of 98 control and 199 AF patients admitted for ECV (n = 98) or PVI (n = 101). HSP27, HSP70, cvHSP and HSP60 serum levels did not differ between patients without or with PAF, PeAF or LSPeAF. Additionally, baseline HSP levels did not correlate with AF recurrence after ECV or PVI. However, in AF patients with AF recurrence, HSP27 levels were significantly elevated post-PVI relative to baseline, compared to patients without recurrence. Conclusions: No association was observed between baseline HSP levels and the presence of AF, AF stage or AF recurrence. However, HSP27 levels were increased in serum samples of patients with AF recurrence within one year after PVI, suggesting that HSP27 levels may predict recurrence of AF after ablative therapy.

Atrial fibrillation (AF), the most common, progressive tachyarrhythmia is associated with serious complications, such as stroke and heart failure. Early recognition of AF, essential to prevent disease progression and therapy failure, is hampered by the lack of accurate diagnostic serum biomarkers to identify the AF stage. As we previously showed mitochondrial dysfunction to drive experimental and human AF, we evaluated whether cell-free circulating mitochondrial DNA (cfc-mtDNA) represents a potential serum marker. Therefore, the levels of two mtDNA genes, COX3 and ND1, were measured in 84 control patients (C), 59 patients undergoing cardiac surgery without a history of AF (SR), 100 paroxysmal (PAF), 116 persistent (PeAF), and 20 longstanding-persistent (LS-PeAF) AF patients undergoing either cardiac surgery or AF treatment (electrical cardioversion or pulmonary vein isolation). Cfc-mtDNA levels were significantly increased in PAF patients undergoing AF treatment, especially in males and patients with AF recurrence after AF treatment. In PeAF and LS-PeAF, cfc-mtDNA levels gradually decreased. Importantly, cfc-mtDNA in serum may originate from cardiomyocytes, as in vitro tachypaced cardiomyocytes release mtDNA in the medium. The findings suggest that cfc-mtDNA is associated with AF stage, especially in males, and with patients at risk for AF recurrence after treatment.

BackgroundDerailment of proteostasis, the homeostasis of production, function, and breakdown of proteins, contributes importantly to the self‐perpetuating nature of atrial fibrillation (AF), the most common heart rhythm disorder in humans. Autophagy plays an important role in proteostasis by degrading aberrant proteins and organelles. Herein, we investigated the role of autophagy and its activation pathway in experimental and clinical AF. Methods and ResultsTachypacing of HL‐1 atrial cardiomyocytes causes a gradual and significant activation of autophagy, as evidenced by enhanced LC3B‐II expression, autophagic flux and autophagosome formation, and degradation of p62, resulting in reduction of Ca2+ amplitude. Autophagy is activated downstream of endoplasmic reticulum (ER) stress: blocking ER stress by the chemical chaperone 4‐phenyl butyrate, overexpression of the ER chaperone‐protein heat shock protein A5, or overexpression of a phosphorylation‐blocked mutant of eukaryotic initiation factor 2α (eIF2α) prevents autophagy activation and Ca2+‐transient loss in tachypaced HL‐1 cardiomyocytes. Moreover, pharmacological inhibition of ER stress in tachypaced Drosophila confirms its role in derailing cardiomyocyte function. In vivo treatment with sodium salt of phenyl butyrate protected atrial‐tachypaced dog cardiomyocytes from electrical remodeling (action potential duration shortening, L‐type Ca2+‐current reduction), cellular Ca2+‐handling/contractile dysfunction, and ER stress and autophagy; it also attenuated AF progression. Finally, atrial tissue from patients with persistent AF reveals activation of autophagy and induction of ER stress, which correlates with markers of cardiomyocyte damage. ConclusionsThese results identify ER stress–associated autophagy as an important pathway in AF progression and demonstrate the potential therapeutic action of the ER‐stress inhibitor 4‐phenyl butyrate.

Atrial fibrillation (AF), the most common progressive tachyarrhythmia, results in structural remodeling which impairs electrical activation of the atria, rendering them increasingly permissive to the arrhythmia. Previously, we reported on endoplasmic reticulum stress and NAD+ depletion in AF, suggesting a role for mitochondrial dysfunction in AF progression. Here, we examined mitochondrial function in experimental model systems for AF (tachypaced HL-1 atrial cardiomyocytes and Drosophila melanogaster) and validated findings in clinical AF. Tachypacing of HL-1 cardiomyocytes progressively induces mitochondrial dysfunction, evidenced by impairment of mitochondrial Ca2+-handling, upregulation of mitochondrial stress chaperones and a decrease in the mitochondrial membrane potential, respiration and ATP production. Atrial biopsies from AF patients display mitochondrial dysfunction, evidenced by aberrant ATP levels, upregulation of a mitochondrial stress chaperone and fragmentation of the mitochondrial network. The pathophysiological role of mitochondrial dysfunction is substantiated by the attenuation of AF remodeling by preventing an increased mitochondrial Ca2+-influx through partial blocking or downregulation of the mitochondrial calcium uniporter, and by SS31, a compound that improves bioenergetics in mitochondria. Together, these results show that conservation of the mitochondrial function protects against tachypacing-induced cardiomyocyte remodeling and identify this organelle as a potential novel therapeutic target.

Due to ageing of the population, the incidence of cardiovascular diseases will increase in the coming years, constituting a substantial burden on health care systems. In particular, atrial fibrillation (AF) is approaching epidemic proportions. It has been identified that the derailment of proteostasis, which is characterized by the loss of homeostasis in protein biosynthesis, folding, trafficking, and clearance by protein degradation systems such as autophagy, underlies the development of common cardiac diseases. Among various safeguards within the proteostasis system, autophagy is a vital cellular process that modulates clearance of misfolded and proteotoxic proteins from cardiomyocytes. On the other hand, excessive autophagy may result in derailment of proteostasis and therefore cardiac dysfunction. Here, we review the interplay between autophagy and proteostasis in the healthy heart, discuss the imbalance between autophagy and proteostasis during cardiac diseases, including AF, and finally explore new druggable targets which may limit cardiac disease initiation and progression.

The heat shock response (HSR) is an ancient and highly conserved program of stress-induced gene expression, aimed at reestablishing protein homeostasis to preserve cellular fitness. Cells that fail to activate or maintain this protective response are hypersensitive to proteotoxic stress. The HSR is mediated by the heat shock transcription factor 1 (HSF1), which binds to conserved heat shock elements (HSE) in the promoter region of heat shock genes, resulting in the expression of heat shock proteins (HSP). Recently, we observed that hyperactivation of RhoA conditions cardiomyocytes for the cardiac arrhythmia atrial fibrillation. Also, the HSR is annihilated in atrial fibrillation, and induction of HSR mitigates sensitization of cells to this disease. Therefore, we hypothesized active RhoA to suppress the HSR resulting in sensitization of cells for proteotoxic stimuli.Stimulation of RhoA activity significantly suppressed the proteotoxic stress-induced HSR in HL-1 atrial cardiomyocytes as determined with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A) promoter and HSP protein expression by Western Blot analysis. Inversely, RhoA inhibition boosted the proteotoxic stress-induced HSR. While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE. Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress.These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.