Evaluation of a fluorescence in situ hybridization (FISH)-based method for detection of SARS-CoV-2 in saliva.

The use of a non-invasive fluorescence in situ hybridization (FISH)-based method on saliva for the detection of SARS-CoV-2 is evaluated in a proof-of-concept study and thereafter utilized in an outpatient setting with the Biotrack-MED® analyzer. For a proof-of-concept study, saliva samples were obtained from 28 persons with mild or moderate COVID-19-related symptoms who were tested RT-PCR positive or negative for SARS-CoV-2. In an outpatient setting, 972 individual saliva samples were utilized. All saliva samples were FISHed with a Cy3-labeled SARS-CoV-2-specific DNA probe and were analyzed manually by fluorescence microscopy (proof-of-concept) or with the SARS-CoV-2 application of the Biotrack-MED® analyzer, a semi-autonomous multi-sample filter cytometer. The proof-of-concept study showed a sensitivity of 96.0% and a specificity of 98.5% and is therefore comparable to the RT-PCR analysis of nasopharyngeal swabs. The outpatient setting showed a sensitivity of 90.9% and a specificity of 94.5% and seems therefore a valid assay for the detection of SARS-CoV-2 in individuals that are healthy, mild or moderate symptomatic. In conclusion, the method evaluated in this study, the FISH-based SARS-CoV-2 application of the Biotrack-MED® analyzer, is a sensitive and reliable assay for the detection of SARS-CoV-2 in the general population.

Direct detection of SARS-CoV-2 antisense and sense genomic RNA in human saliva by semi-autonomous fluorescence in situ hybridization: A proxy for contagiousness?

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.

The Antibiotic Doxycycline Impairs Cardiac Mitochondrial and Contractile Function.

Tetracycline antibiotics act by inhibiting bacterial protein translation. Given the bacterial ancestry of mitochondria, we tested the hypothesis that doxycycline-which belongs to the tetracycline class-reduces mitochondrial function, and results in cardiac contractile dysfunction in cultured H9C2 cardiomyoblasts, adult rat cardiomyocytes, in Drosophila and in mice. Ampicillin and carbenicillin were used as control antibiotics since these do not interfere with mitochondrial translation. In line with its specific inhibitory effect on mitochondrial translation, doxycycline caused a mitonuclear protein imbalance in doxycycline-treated H9C2 cells, reduced maximal mitochondrial respiration, particularly with complex I substrates, and mitochondria appeared fragmented. Flux measurements using stable isotope tracers showed a shift away from OXPHOS towards glycolysis after doxycycline exposure. Cardiac contractility measurements in adult cardiomyocytes and Drosophila melanogaster hearts showed an increased diastolic calcium concentration, and a higher arrhythmicity index. Systolic and diastolic dysfunction were observed after exposure to doxycycline. Mice treated with doxycycline showed mitochondrial complex I dysfunction, reduced OXPHOS capacity and impaired diastolic function. Doxycycline exacerbated diastolic dysfunction and reduced ejection fraction in a diabetes mouse model vulnerable for metabolic derangements. We therefore conclude that doxycycline impairs mitochondrial function and causes cardiac dysfunction.

Blood-based 8-hydroxy-2'-deoxyguanosine level: A potential diagnostic biomarker for atrial fibrillation.

BACKGROUND: Recent research findings have revealed a key role of oxidative DNA damage in the pathogenesis of atrial fibrillation (AF). Therefore, the circulating oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) may represent a biomarker for staging AF and identifying patients at risk for AF recurrence and postoperative atrial fibrillation (POAF) after treatment. OBJECTIVE: The purpose of this study was to investigate whether serum levels of 8-OHdG correlate with the stage of AF, recurrence after AF treatment, and onset of POAF after cardiac surgery. METHODS: In this prospective observational study, 8-OHdG levels were detected by enzyme-linked immunosorbent assay in human serum samples. Blood samples were collected from control patients without AF history; patients with paroxysmal AF and persistent AF undergoing electrical cardioversion or pulmonary vein isolation (PVI); and patients with sinus rhythm (SR) undergoing cardiac surgery. AF recurrence was determined during 12-month follow-up. Univariate and multivariate analyses were used to identify changes in 8-OHdG levels between the groups. RESULTS: Compared to the control group, 8-OHdG levels in the patient groups gradually and significantly increased during arrhythmia progression. 8-OHdG levels in AF patients showing AF recurrence after PVI treatment were significantly increased compared to patients without AF recurrence. Moreover, in SR patients undergoing cardiac surgery, 8-OHdG levels were significantly elevated in those showing POAF compared to patients without POAF. CONCLUSION: 8-OHdG level may represent a potential diagnostic biomarker for AF staging as well as for predicting AF recurrence and POAF after treatment.

Evaluating Serum Heat Shock Protein Levels as Novel Biomarkers for Atrial Fibrillation.

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.

Cell-Free Circulating Mitochondrial DNA: A Potential Blood-Based Marker for Atrial Fibrillation.

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.

Imbalance of ER and Mitochondria Interactions: Prelude to Cardiac Ageing and Disease?

Cardiac disease is still the leading cause of morbidity and mortality worldwide, despite some exciting and innovative improvements in clinical management. In particular, atrial fibrillation (AF) and heart failure show a steep increase in incidence and healthcare costs due to the ageing population. Although research revealed novel insights in pathways driving cardiac disease, the exact underlying mechanisms have not been uncovered so far. Emerging evidence indicates that derailed proteostasis (i.e., the homeostasis of protein expression, function and clearance) is a central component driving cardiac disease. Within proteostasis derailment, key roles for endoplasmic reticulum (ER) and mitochondrial stress have been uncovered. Here, we describe the concept of ER and mitochondrial stress and the role of interactions between the ER and mitochondria, discuss how imbalance in the interactions fuels cardiac ageing and cardiac disease (including AF), and finally assess the potential of drugs directed at conserving the interaction as an innovative therapeutic target to improve cardiac function.

Mitochondrial Dysfunction Underlies Cardiomyocyte Remodeling in Experimental and Clinical Atrial Fibrillation.

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.

DNA damage-induced PARP1 activation confers cardiomyocyte dysfunction through NAD+ depletion in experimental atrial fibrillation.

Atrial fibrillation (AF) is the most common clinical tachyarrhythmia with a strong tendency to progress in time. AF progression is driven by derailment of protein homeostasis, which ultimately causes contractile dysfunction of the atria. Here we report that tachypacing-induced functional loss of atrial cardiomyocytes is precipitated by excessive poly(ADP)-ribose polymerase 1 (PARP1) activation in response to oxidative DNA damage. PARP1-mediated synthesis of ADP-ribose chains in turn depletes nicotinamide adenine dinucleotide (NAD+), induces further DNA damage and contractile dysfunction. Accordingly, NAD+ replenishment or PARP1 depletion precludes functional loss. Moreover, inhibition of PARP1 protects against tachypacing-induced NAD+ depletion, oxidative stress, DNA damage and contractile dysfunction in atrial cardiomyocytes and Drosophila. Consistently, cardiomyocytes of persistent AF patients show significant DNA damage, which correlates with PARP1 activity. The findings uncover a mechanism by which tachypacing impairs cardiomyocyte function and implicates PARP1 as a possible therapeutic target that may preserve cardiomyocyte function in clinical AF.

Untying the knot: protein quality control in inherited cardiomyopathies.

Mutations in genes encoding sarcomeric proteins are the most important causes of inherited cardiomyopathies, which are a major cause of mortality and morbidity worldwide. Although genetic screening procedures for early disease detection have been improved significantly, treatment to prevent or delay mutation-induced cardiac disease onset is lacking. Recent findings indicate that loss of protein quality control (PQC) is a central factor in the disease pathology leading to derailment of cellular protein homeostasis. Loss of PQC includes impairment of heat shock proteins, the ubiquitin-proteasome system, and autophagy. This may result in accumulation of misfolded and aggregation-prone mutant proteins, loss of sarcomeric and cytoskeletal proteins, and, ultimately, loss of cardiac function. PQC derailment can be a direct effect of the mutation-induced activation, a compensatory mechanism due to mutation-induced cellular dysfunction or a consequence of the simultaneous occurrence of the mutation and a secondary hit. In this review, we discuss recent mechanistic findings on the role of proteostasis derailment in inherited cardiomyopathies, with special focus on sarcomeric gene mutations and possible therapeutic applications.

Role of Autophagy in Proteostasis: Friend and Foe in Cardiac Diseases.

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.

Torpor-arousal cycles in Syrian hamster heart are associated with transient activation of the protein quality control system.

Hibernation consists of torpor, with marked suppression of metabolism and physiological functions, alternated with arousal periods featuring their full restoration. The heart is particularly challenged, exemplified by its rate reduction from 400 to 5-10 beats per minute during torpor in Syrian hamsters. In addition, during arousals, the heart needs to accommodate the very rapid return to normal function, which lead to our hypothesis that cardiac function during hibernation is supported by maintenance of protein homeostasis through adaptations in the protein quality control (PQC) system. Hereto, we examined autophagy, the endoplasmic reticulum (ER) unfolded protein (UPRER) response and the heat shock response (HSR) in Syrian hamster hearts during torpor and arousal. Transition from torpor to arousal (1.5 h) was associated with stimulation of the PQC system during early arousal, demonstrated by induction of autophagosomes, as shown by an increase in LC3B-II protein abundance, likely related to the activation of the UPRER during late torpor in response to proteotoxic stress. The HSR was not activated during torpor or arousal. Our results demonstrate activation of the cardiac PQC system - particularly autophagosomal degradation - in early arousal in response to cardiac stress, to clear excess aberrant or damaged proteins, being gradually formed during the torpor bout and/or the rapid increase in heart rate during the transition from torpor to arousal. This mechanism may enable the large gain in cardiac function during the transition from torpor to arousal, which may hold promise to further understand 'hibernation' of cardiomyocytes in human heart disease.

Application of kinomic array analysis to screen for altered kinases in atrial fibrillation remodeling.

BACKGROUND: Dysregulation of protein kinase-mediated signaling is an early event in many diseases, including the most common clinical cardiac arrhythmia, atrial fibrillation (AF). Kinomic profiling represents a promising technique to identify candidate kinases. OBJECTIVE: In this study we used kinomic profiling to identify kinases altered in AF remodeling using atrial tissue from a canine model of AF (atrial tachypacing). METHODS: Left atrial tissue obtained in a previous canine study was used for kinomic array (containing 1024 kinase pseudosubstrates) analysis. Three groups of dogs were included: nonpaced controls and atrial tachypaced dogs, which were contrasted with geranylgeranylacetone-treated dogs with AF, which are protected from AF promotion, to enhance specificity of detection of putative kinases. RESULTS: While tachypacing changed activity of 50 kinases, 40 of these were prevented by geranylgeranylacetone and involved in differentiation and proliferation (SRC), contraction, metabolism, immunity, development, cell cycle (CDK4), and survival (Akt). Inhibitors of Akt (MK2206) and CDK4 (PD0332991) and overexpression of a dominant-negative CDK4 phosphorylation mutant protected against tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes. Moreover, patients with AF show down- and upregulation of SRC and Akt phosphorylation, respectively, similar to findings of the kinome array. CONCLUSION: Contrasting kinomic array analyses of controls and treated subjects offer a versatile tool to identify kinases altered in atrial remodeling owing to tachypacing, which include Akt, CDK4, and SRC. Ultimately, pharmacological targeting of altered kinases may offer novel therapeutic possibilities to treat clinical AF.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

Endoplasmic Reticulum Stress Is Associated With Autophagy and Cardiomyocyte Remodeling in Experimental and Human Atrial Fibrillation.

BACKGROUND: Derailment 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 RESULTS: Tachypacing 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. CONCLUSIONS: These 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.

The protective role of small heat shock proteins in cardiac diseases: key role in atrial fibrillation.

Atrial fibrillation (AF) is the most common tachyarrhythmia which is associated with increased morbidity and mortality. AF usually progresses from a self-terminating paroxysmal to persistent disease. It has been recognized that AF progression is driven by structural remodeling of cardiomyocytes, which results in electrical and contractile dysfunction of the atria. We recently showed that structural remodeling is rooted in derailment of proteostasis, i.e., homeostasis of protein production, function, and degradation. Since heat shock proteins (HSPs) play an important role in maintaining a healthy proteostasis, the role of HSPs was investigated in AF. It was found that especially small heat shock protein (HSPB) levels get exhausted in atrial tissue of patients with persistent AF and that genetic or pharmacological induction of HSPB protects against cardiomyocyte remodeling in experimental models for AF. In this review, we provide an overview of HSPBs as a potential therapeutic target for normalizing proteostasis and suppressing the substrates for AF progression in experimental and clinical AF and discuss HSP activators as a promising therapy to prevent AF onset and progression.

Derailed Proteostasis as a Determinant of Cardiac Aging.

Age comprises the single most important risk factor for cardiac disease development. The incidence and prevalence of cardiac diseases, which represents the main cause of death worldwide, will increase even more because of the aging population. A hallmark of aging is that it is accompanied by a gradual derailment of proteostasis (eg, the homeostasis of protein synthesis, folding, assembly, trafficking, function, and degradation). Loss of proteostasis is highly relevant to cardiomyocytes, because they are postmitotic cells and therefore not constantly replenished by proliferation. The derailment of proteostasis during aging is thus an important factor that preconditions for the development of age-related cardiac diseases, such as atrial fibrillation. In turn, frailty of proteostasis in aging cardiomyocytes is exemplified by its accelerated derailment in multiple cardiac diseases. Here, we review 2 major components of the proteostasis network, the stress-responsive and protein degradation pathways, in healthy and aged cardiomyocytes. Furthermore, we discuss the relation between derailment of proteostasis and age-related cardiac diseases, including atrial fibrillation. Finally, we introduce novel therapeutic targets that might possibly attenuate cardiac aging and thus limit cardiac disease progression.

RhoA Activation Sensitizes Cells to Proteotoxic Stimuli by Abrogating the HSF1-Dependent Heat Shock Response.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

Diagnosis and Therapy of Atrial Fibrillation: The Past, The Present and The Future.

Atrial fibrillation (AF) is the most common age-related cardiac arrhythmia. It is a progressive disease, which makes treatment difficult. The progression of AF is caused by the accumulation of damage in cardiomyocytes which makes the atria more vulnerable for AF. Especially structural remodeling and electrical remodeling, together called electropathology are sustainable in the atria and impair functional recovery to sinus rhythm after cardioversion. The exact electropathological mechanisms underlying persistence of AF are at present unknown. High resolution wavemapping studies in patients with different types of AF showed that longitudinal dissociation in conduction and epicardial breakthrough were the key elements of the substrate of longstanding persistent AF. A double layer of electrically dissociated waves propagating transmurally can explain persistence of AF (Double Layer Hypothesis) but the molecular mechanism is unknown. Derailment of proteasis -defined as the homeostasis in protein synthesis, folding, assembly, trafficking, guided by chaperones, and clearance by protein degradation systems - may play an important role in remodeling of the cardiomyocyte. As current therapies are not effective in attenuating AF progression, step-by-step analysis of this process, in order to identify potential targets for drug therapy, is essential. In addition, novel mapping approaches enabling assessment of the degree of electropathology in the individual patient are mandatory to develop patient-tailored therapies. The aims of this review are to 1) summarize current knowledge of the electrical and molecular mechanisms underlying AF 2) discuss the shortcomings of present diagnostic instruments and therapeutic options and 3) to present potential novel diagnostic tools and therapeutic targets.

Identification of MOAG-4/SERF as a regulator of age-related proteotoxicity.

Fibrillar protein aggregates are the major pathological hallmark of several incurable, age-related, neurodegenerative disorders. These aggregates typically contain aggregation-prone pathogenic proteins, such as amyloid-beta in Alzheimer's disease and alpha-synuclein in Parkinson's disease. It is, however, poorly understood how these aggregates are formed during cellular aging. Here we identify an evolutionarily highly conserved modifier of aggregation, MOAG-4, as a positive regulator of aggregate formation in C. elegans models for polyglutamine diseases. Inactivation of MOAG-4 suppresses the formation of compact polyglutamine aggregation intermediates that are required for aggregate formation. The role of MOAG-4 in driving aggregation extends to amyloid-beta and alpha-synuclein and is evolutionarily conserved in its human orthologs SERF1A and SERF2. MOAG-4/SERF appears to act independently from HSF-1-induced molecular chaperones, proteasomal degradation, and autophagy. Our results suggest that MOAG-4/SERF regulates age-related proteotoxicity through a previously unexplored pathway, which will open up new avenues for research on age-related, neurodegenerative diseases.