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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.

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.

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.

SARS-CoV-2 is a novel coronavirus that mainly affects the upper airways. Approximately one third of all detected cases is asymptomatic. We report an asymptomatic individual who tested positive for SARS-CoV-2 over a period of nine months. Of this individual, whole mouth saliva was tested by a novel fluorescence in situ hybridization-based assay which detects only the ‘active’ form of the virus. During the observation period of nine months, there was a possible co-infection with a second SARS-CoV-2 variant accompanied by none or very low antibody production until the possible co-infection. We suspect that the SARS-CoV-2 infection in this individual is limited to the salivary glands and does not spread (much) throughout the systemic compartment(s) of the body.

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.

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.

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.

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.

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.

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.

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.

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.