Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Loss of the Atrial Fibrillation-Related Gene, Zfhx3, Results in Atrial Dilation and Arrhythmias.

BACKGROUND: ZFHX3 (zinc finger homeobox 3), a gene that encodes a large transcription factor, is at the second-most significantly associated locus with atrial fibrillation (AF), but its function in the heart is unknown. This study aims to identify causative genetic variation related to AF at the ZFHX3 locus and examine the impact of Zfhx3 loss on cardiac function in mice. METHODS: CRISPR-Cas9 genome editing, chromatin immunoprecipitation, and luciferase assays in pluripotent stem cell-derived cardiomyocytes were used to identify causative genetic variation related to AF at the ZFHX3 locus. Cardiac function was assessed by echocardiography, magnetic resonance imaging, electrophysiology studies, calcium imaging, and RNA sequencing in mice with heterozygous and homozygous cardiomyocyte-restricted Zfhx3 loss (Zfhx3 Het and knockout, respectively). Human cardiac single-nucleus ATAC (assay for transposase-accessible chromatin)-sequencing data was analyzed to determine which genes in atrial cardiomyocytes are directly regulated by ZFHX3. RESULTS: We found single-nucleotide polymorphism (SNP) rs12931021 modulates an enhancer regulating ZFHX3 expression, and the AF risk allele is associated with decreased ZFHX3 transcription. We observed a gene-dose response in AF susceptibility with Zfhx3 knockout mice having higher incidence, frequency, and burden of AF than Zfhx3 Het and wild-type mice, with alterations in conduction velocity, atrial action potential duration, calcium handling and the development of atrial enlargement and thrombus, and dilated cardiomyopathy. Zfhx3 loss results in atrial-specific differential effects on genes and signaling pathways involved in cardiac pathophysiology and AF. CONCLUSIONS: Our findings implicate ZFHX3 as the causative gene at the 16q22 locus for AF, and cardiac abnormalities caused by loss of cardiac Zfhx3 are due to atrial-specific dysregulation of pathways involved in AF susceptibility. Together, these data reveal a novel and important role for Zfhx3 in the control of cardiac genes and signaling pathways essential for normal atrial function.

Atrial Fibrillation Risk Assessment after Embolic Stroke of Undetermined Source.

OBJECTIVE: Approximately 20% of strokes are embolic strokes of undetermined source (ESUS). Undetected atrial fibrillation (AF) remains an important cause. Yet, oral anticoagulation in unselected ESUS patients failed in secondary stroke prevention. Guidance on effective AF detection is lacking. Here, we introduce a novel, non-invasive AF risk assessment after ESUS. METHODS: Catch-Up ESUS is an investigator-initiated, observational cohort study conducted between 2018 and 2019 at the Munich University Hospital. Besides clinical characteristics, patients received ≥72 h digital electrocardiogram recordings to generate the rhythm irregularity burden. Uni- and multivariable regression models predicted the primary endpoint of incident AF, ascertained by standardized follow-up including implantable cardiac monitors. Predictors included the novel rhythm irregularity burden constructed from digital electrocardiogram recordings. We independently validated our model in ESUS patients from the University Hospital Tübingen, Germany. RESULTS: A total of 297 ESUS patients were followed for 15.6 ± 7.6 months. Incident AF (46 patients, 15.4%) occurred after a median of 105 days (25th to 75th percentile 31-33 days). Secondary outcomes were recurrent stroke in 7.7% and death in 6.1%. Multivariable-adjusted analyses identified the rhythm irregularity burden as the strongest AF-predictor (hazard ratio 3.12, 95% confidence interval 1.62-5.80, p < 0001) while accounting for the known risk factors age, CHA2 DS2 -VASc-Score, and NT-proBNP. Independent validation confirmed the rhythm irregularity burden as the most significant AF-predictor (hazard ratio 2.20, 95% confidence interval 1.45-3.33, p < 0001). INTERPRETATION: The novel, non-invasive, electrocardiogram-based rhythm irregularity burden may help adjudicating AF risk after ESUS, and subsequently guide AF-detection after ESUS. Clinical trials need to clarify if high-AF risk patients benefit from tailored secondary stroke prevention. ANN NEUROL 2023;93:479-488.

Atrial fibrosis heterogeneity is a risk for atrial fibrillation in pigs with ischaemic heart failure.

BACKGROUND: Atrial fibrillation (AF) is the most common arrhythmia and is associated with considerable morbidity and mortality. Ischaemic heart failure (IHF) remains one of the most common causes of AF in clinical practice. However, ischaemia-mediated mechanisms leading to AF are still incompletely understood, and thus, current treatment approaches are limited. To improve our understanding of the pathophysiology, we studied a porcine IHF model. METHODS: In pigs, IHF was induced by balloon occlusion of the left anterior descending artery for 90 min. After 30 days of reperfusion, invasive haemodynamic measurements and electrophysiological studies were performed. Masson trichrome and immunofluorescence staining were conducted to assess interstitial fibrosis and myofibroblast activation in different heart regions. RESULTS: After 30 days of reperfusion, heart failure with significantly reduced ejection fraction (left anterior obique 30°, 34.78 ± 3.29% [IHF] vs. 62.03 ± 2.36% [control], p < .001; anterior-posterior 0°, 29.16 ± 3.61% vs. 59.54 ± 1.09%, p < .01) was observed. These pigs showed a significantly higher susceptibility to AF (33.90% [IHF] vs. 12.98% [control], p < .05). Histological assessment revealed aggravated fibrosis in atrial appendages but not in atrial free walls in IHF pigs (11.13 ± 1.44% vs. 5.99 ± .86%, p < .01 [LAA], 8.28 ± .56% vs. 6.01 ± .35%, p < .01 [RAA]), which was paralleled by enhanced myofibroblast activation (12.09 ± .65% vs. 9.00 ± .94%, p < .05 [LAA], 14.37 ± .60% vs. 10.30 ± 1.41%, p < .05 [RAA]). Correlation analysis indicated that not fibrosis per se but its cross-regional heterogeneous distribution across the left atrium was associated with AF susceptibility (r = .6344, p < .01). CONCLUSION: Our results suggest that left atrial cross-regional fibrosis difference rather than overall fibrosis level is associated with IHF-related AF susceptibility, presumably by establishing local conduction disturbances and heterogeneity.

Animal Models of Atrial Fibrillation.

Atrial fibrillation (AF) is the most common sustained arrhythmia encountered in humans and is a significant source of morbidity and mortality. Despite its prevalence, our mechanistic understanding is incomplete, the therapeutic options have limited efficacy, and are often fraught with risks. A better biological understanding of AF is needed to spearhead novel therapeutic avenues. Although "natural" AF is nearly nonexistent in most species, animal models have contributed significantly to our understanding of AF and some therapeutic options. However, the impediments of animal models are also apparent and stem largely from the differences in basic physiology as well as the complexities underlying human AF; these preclude the creation of a "perfect" animal model and have obviated the translation of animal findings. Herein, we review the vast array of AF models available, spanning the mouse heart (weighing 1/1000th of a human heart) to the horse heart (10× heavier than the human heart). We attempt to highlight the features of each model that bring value to our understanding of AF but also the shortcomings and pitfalls. Finally, we borrowed the concept of a SWOT analysis from the business community (which stands for strengths, weaknesses, opportunities, and threats) and applied this introspective type of analysis to animal models for AF. We identify unmet needs and stress that is in the context of rapidly advancing technologies, these present opportunities for the future use of animal models.

Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase.

BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.

Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls.

PURPOSE: Stringent variant interpretation guidelines can lead to high rates of variants of uncertain significance (VUS) for genetically heterogeneous disease like long QT syndrome (LQTS) and Brugada syndrome (BrS). Quantitative and disease-specific customization of American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines can address this false negative rate. METHODS: We compared rare variant frequencies from 1847 LQTS (KCNQ1/KCNH2/SCN5A) and 3335 BrS (SCN5A) cases from the International LQTS/BrS Genetics Consortia to population-specific gnomAD data and developed disease-specific criteria for ACMG/AMP evidence classes-rarity (PM2/BS1 rules) and case enrichment of individual (PS4) and domain-specific (PM1) variants. RESULTS: Rare SCN5A variant prevalence differed between European (20.8%) and Japanese (8.9%) BrS patients (p = 5.7 × 10-18) and diagnosis with spontaneous (28.7%) versus induced (15.8%) Brugada type 1 electrocardiogram (ECG) (p = 1.3 × 10-13). Ion channel transmembrane regions and specific N-terminus (KCNH2) and C-terminus (KCNQ1/KCNH2) domains were characterized by high enrichment of case variants and >95% probability of pathogenicity. Applying the customized rules, 17.4% of European BrS and 74.8% of European LQTS cases had (likely) pathogenic variants, compared with estimated diagnostic yields (case excess over gnomAD) of 19.2%/82.1%, reducing VUS prevalence to close to background rare variant frequency. CONCLUSION: Large case-control data sets enable quantitative implementation of ACMG/AMP guidelines and increased sensitivity for inherited arrhythmia genetic testing.

Porcine models for studying complications and organ crosstalk in diabetes mellitus.

The worldwide prevalence of diabetes mellitus and obesity is rapidly increasing not only in adults but also in children and adolescents. Diabetes is associated with macrovascular complications increasing the risk for cardiovascular disease and stroke, as well as microvascular complications leading to diabetic nephropathy, retinopathy and neuropathy. Animal models are essential for studying disease mechanisms and for developing and testing diagnostic procedures and therapeutic strategies. Rodent models are most widely used but have limitations in translational research. Porcine models have the potential to bridge the gap between basic studies and clinical trials in human patients. This article provides an overview of concepts for the development of porcine models for diabetes and obesity research, with a focus on genetically engineered models. Diabetes-associated ocular, cardiovascular and renal alterations observed in diabetic pig models are summarized and their similarities with complications in diabetic patients are discussed. Systematic multi-organ biobanking of porcine models of diabetes and obesity and molecular profiling of representative tissue samples on different levels, e.g., on the transcriptome, proteome, or metabolome level, is proposed as a strategy for discovering tissue-specific pathomechanisms and their molecular key drivers using systems biology tools. This is exemplified by a recent study providing multi-omics insights into functional changes of the liver in a transgenic pig model for insulin-deficient diabetes mellitus. Collectively, these approaches will provide a better understanding of organ crosstalk in diabetes mellitus and eventually reveal new molecular targets for the prevention, early diagnosis and treatment of diabetes mellitus and its associated complications.

Non-coding RNA and Cardiac Electrophysiological Disorders.

Cardiac arrhythmias are common diseases affecting millions of people worldwide. A broad and diverse array of arrhythmias exists, ranging from harmless ones such as sinus arrhythmia to fatal disorders such as ventricular fibrillation. The underlying pathophysiology of arrhythmogenesis is complex and still not fully understood. Since their discovery, non-coding RNAs (ncRNAs) and especially microRNAs (miRNAs) came into the spotlight of arrhythmia research as it has been shown that they play an important role in regulating normal development of the cardiac conduction system and are involved in remodeling processes leading to arrhythmias. This chapter will give a brief overview on basic electrophysiologic concepts and will summarize the current knowledge on ncRNAs and their role in arrhythmogenesis.

A Functional Variant Associated with Atrial Fibrillation Regulates PITX2c Expression through TFAP2a.

The most significantly associated genetic locus for atrial fibrillation (AF) is in chromosomal region 4q25, where four independent association signals have been identified. Although model-system studies suggest that altered PITX2c expression might underlie the association, the link between specific variants and the direction of effect on gene expression remains unknown for all four signals. In the present study, we analyzed the AF-associated region most proximal to PITX2 at 4q25. First, we identified candidate regulatory variants that might confer AF risk through a combination of mammalian conservation, DNase hypersensitivity, and histone modification from ENCODE and the Roadmap Epigenomics Project, as well as through in vivo analysis of enhancer activity in embryonic zebrafish. Within candidate regions, we then identified a single associated SNP, rs2595104, which displayed dramatically reduced enhancer activity with the AF risk allele. CRISPR-Cas9-mediated deletion of the rs2595104 region and editing of the rs2595104 risk allele in human stem-cell-derived cardiomyocytes resulted in diminished PITX2c expression in comparison to that of the non-risk allele. This differential activity was mediated by activating enhancer binding protein 2 alpha (TFAP2a), which bound robustly to the non-risk allele at rs2595104, but not to the risk allele, in cardiomyocytes. In sum, we found that the AF-associated SNP rs2595104 altered PITX2c expression via interaction with TFAP2a. Such a pathway could ultimately contribute to AF susceptibility at the PITX2 locus associated with AF.

Coding and non-coding variants in the SHOX2 gene in patients with early-onset atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia with a strong genetic component. Molecular pathways involving the homeodomain transcription factor Shox2 control the development and function of the cardiac conduction system in mouse and zebrafish. Here we report the analysis of human SHOX2 as a potential susceptibility gene for early-onset AF. To identify causal variants and define the underlying mechanisms, results from 378 patients with early-onset AF before the age of 60 years were analyzed and compared to 1870 controls or reference datasets. We identified two missense mutations (p.G81E, p.H283Q), that were predicted as damaging. Transactivation studies using SHOX2 targets and phenotypic rescue experiments in zebrafish demonstrated that the p.H283Q mutation severely affects SHOX2 pacemaker function. We also demonstrate an association between a 3'UTR variant c.*28T>C of SHOX2 and AF (p = 0.00515). Patients carrying this variant present significantly longer PR intervals. Mechanistically, this variant creates a functional binding site for hsa-miR-92b-5p. Circulating hsa-miR-92b-5p plasma levels were significantly altered in AF patients carrying the 3'UTR variant (p = 0.0095). Finally, we demonstrate significantly reduced SHOX2 expression levels in right atrial appendages of AF patients compared to patients with sinus rhythm. Together, these results suggest a genetic contribution of SHOX2 in early-onset AF.

Mutation of a common amino acid in NKX2.5 results in dilated cardiomyopathy in two large families.

BACKGROUND: The genetic basis for dilated cardiomyopathy (DCM) can be difficult to determine, particularly in familial cases with complex phenotypes. Next generation sequencing may be useful in the management of such cases. METHODS: We report two large families with pleiotropic inherited cardiomyopathy. In addition to DCM, the phenotypes included atrial and ventricular septal defects, cardiac arrhythmia and sudden death. Probands underwent whole exome sequencing to identify potentially causative variants. RESULTS: Each whole exome sequence yielded over 18,000 variants. We identified distinct mutations affecting a common amino acid in NKX2.5. Segregation analysis of the families support the pathogenic role of these variants. CONCLUSION: Our study emphasizes the utility of next generation sequencing in identifying causative mutations in complex inherited cardiac disease. We also report a novel pathogenic NKX2.5 mutation.

One-year clinical outcome after ablation with a novel multipolar irrigated ablation catheter for treatment of atrial fibrillation: potential implications for clinical use.

AIMS: Pulmonary vein isolation (PVI) is an established therapy for atrial fibrillation (AF). However, PVI remains a time-consuming procedure. A novel multipolar irrigated radiofrequency (RF) ablation catheter (nMARQ™) is aiming to improve PVI. We investigated the influence on procedural parameters and assessed clinical outcomes after PVI using this novel catheter. METHODS AND RESULTS: Fifty-eight consecutive patients with paroxysmal AF were equally allocated (n = 29/group) to PVI treatment with (i) the novel multipolar ablation catheter (nMARQ™) and (ii) a standard single-tip ablation catheter (SAC). Study endpoints included procedure time, fluoroscopy time, radiation dose, RF time, number of energy applications, and clinical outcome defined as freedom from AF after a single procedure. Successful PVI was confirmed by a separate circular, multipolar mapping catheter in all patients treated with the nMARQ™. Pulmonary vein isolation was achieved in 100% in the SAC group. In the nMARQ™ group, PVI was suggested in all patients. However, confirmatory mapping revealed persistent pulmonary vein (PV) conduction in 19 out of 29 nMARQ™ patients. These patients underwent further ablation, which still failed to achieve PVI in 5 of the 29 nMARQ™ patients, mainly due to significant temperature rise in the oesophagus and device-related limitations reaching the right inferior PV. Mean fluoroscopy time (31 ± 12 vs. 23 ± 10 min, P < 0.05) and (132 ± 37 vs. 109 ± 30 min, P < 0.05) were longer in nMARQ™ vs. SAC patients. Radiofrequency time was shorter in nMARQ™ vs. SAC group (21 ± 9 vs. 35 ± 12 min, P < 0.001). Radiation dose and the number of energy applications did not differ between both groups. Clinical outcome analysis revealed no significant differences (nMARQ™: 72 vs. SAC: 72%) after a mean follow-up of 373 ± 278 days. CONCLUSION: The use of the nMARQ™ catheter is associated with important device-related limitations to achieve successful PVI. However, clinical outcomes were equivalent in nMARQ™- and SAC-treated patients.

Detailed characterization of microRNA changes in a canine heart failure model: Relationship to arrhythmogenic structural remodeling.

Heart failure (HF) causes left-atrial (LA) and left-ventricular (LV) remodeling, with particularly-prominent changes in LA that create a substrate for atrial fibrillation (AF). MicroRNAs (miRs) are potential regulators in cardiac remodeling. This study evaluated time-dependent miR expression-changes in LA and LV tissue, fibroblasts and cardiomyocytes in experimental HF. HF was induced in dogs by ventricular tachypacing (varying periods, up to 2weeks). Following screening-microarray, 15 miRs were selected for detailed real-time qPCR assay. Extracellular matrix mRNA-expression was assessed by qPCR. Tachypacing time-dependently reduced LV ejection-fraction, increased LV-volume and AF-duration, and caused tissue-fibrosis with LA changes greater than LV. Tissue miR-expression significantly changed in LA for 10 miRs; in LV for none. Cell-selective analysis showed significant time-dependent changes in LA-fibroblasts for 10/15 miRs, LV-fibroblasts 8/15, LA-cardiomyocytes in 6/15 and LV-cardiomyocytes 3/15. Cell-expression specificity did not predict cell-specificity of VTP-induced expression-changes, e.g. 4/6 cardiomyocyte-selective miRs changed almost exclusively in fibroblasts (miR-1, miR-208b, miR133a/b). Thirteen miRs directly implicated in fibrosis/extracellular-matrix regulation were prominently changed: 9/13 showed fibroblast-selective alterations and 5/13 LA-selective. Multiple miRs changed in relation to associated extracellular-matrix targets. Experimental HF causes tissue and cell-type selective, time-dependent changes in cardiac miR-expression. Expression-changes are greater in LA versus LV, and greater in fibroblasts than cardiomyocytes, even for most cardiomyocyte-enriched miRs. This study, the first to examine time, chamber and cell-type selective changes in an experimental model of HF, suggests that multiple miR-changes underlie the atrial-selective fibrotic response and emphasize the importance of considering cell-specificity of miR expression-changes in cardiac remodeling paradigms.

MicroRNA29: a mechanistic contributor and potential biomarker in atrial fibrillation.

BACKGROUND: Congestive heart failure (CHF) causes atrial fibrotic remodeling, a substrate for atrial fibrillation (AF) maintenance. MicroRNA29 (miR29) targets extracellular matrix proteins. In the present study, we examined miR29b changes in patients with AF and/or CHF and in a CHF-related AF animal model and assessed its potential role in controlling atrial fibrous tissue production. METHODS AND RESULTS: Control dogs were compared with dogs subjected to ventricular tachypacing for 24 hours, 1 week, or 2 weeks to induce CHF. Atrial miR29b expression decreased within 24 hours in both whole atrial tissue and atrial fibroblasts (-87% and -92% versus control, respectively; p<0.001 for both) and remained decreased throughout the time course. Expression of miR29b extracellular matrix target genes collagen-1A1 (COL1A1), collagen-3A1 (COL3A1), and fibrillin increased significantly in CHF fibroblasts. Lentivirus-mediated miR29b knockdown in canine atrial fibroblasts (-68%; p<0.01) enhanced COL1A1, COL3A1, and fibrillin mRNA expression by 28% (p<0.01), 19% (p<0.05), and 20% (p<0.05), respectively, versus empty virus-infected fibroblasts and increased COL1A1 protein expression by 90% (p<0.05). In contrast, 3-fold overexpression of miR29b decreased COL1A1, COL3A1, and fibrillin mRNA by 65%, 62%, and 61% (all p<0.001), respectively, versus scrambled control and decreased COL1A1 protein by 60% (p<0.05). MiR29b plasma levels were decreased in patients with CHF or AF (by 53% and 54%, respectively; both p<0.001) and were further decreased in patients with both AF and CHF (by 84%; p<0.001). MiR29b expression was also reduced in the atria of chronic AF patients (by 54% versus sinus rhythm; p<0.05). Adenoassociated viral-mediated knockdown of miR29b in mice significantly increased atrial COL1A1 mRNA expression and cardiac tissue collagen content. CONCLUSIONS: MiR29 likely plays a role in atrial fibrotic remodeling and may have value as a biomarker and/or therapeutic target.

Detection of anti-ß1-AR autoantibodies in heart failure by a cell-based competition ELISA.

RATIONALE: Autoantibodies directed against the second extracellular loop of the cardiac ß1-adrenergic receptor (ß1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. OBJECTIVE: We analyzed sera from 167 DCM patients (ejection fraction<45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell-based ELISA, using cells expressing the full transgene for the human ß1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. METHODS AND RESULTS: The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease ("third-generation assay") and also detects the target antibodies by competition with a specific monoclonal anti-ß1-AR antibody (ß1-AR MAb) directed against the functionally relevant ß1-AR epitope. Anti-ß1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-ß1-AR antibody titers (defined as inhibition of ß1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular ß1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. CONCLUSIONS: We established a simple and efficient screening assay detecting disease-relevant ß1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to "good laboratory practice" and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.

Biomarker Periodic Repolarization Dynamics Indicates Enhanced Risk for Arrhythmias and Sudden Cardiac Death in Myocardial Infarction in Pigs.

BACKGROUND: Periodic repolarization dynamics (PRD) is an electrocardiographic biomarker that captures repolarization instability in the low frequency spectrum and is believed to estimate the sympathetic effect on the ventricular myocardium. High PRD indicates an increased risk for postischemic sudden cardiac death (SCD). However, a direct link between PRD and proarrhythmogenic autonomic remodeling has not yet been shown. METHODS AND RESULTS: We investigated autonomic remodeling in pigs with myocardial infarction (MI)-related ischemic heart failure induced by balloon occlusion of the left anterior descending artery (n=17) compared with pigs without MI (n=11). Thirty days after MI, pigs demonstrated enhanced sympathetic innervation in the infarct area, border zone, and remote left ventricle paralleled by altered expression of autonomic marker genes/proteins. PRD was enhanced 30 days after MI compared with baseline (pre-MI versus post-MI: 1.75±0.30 deg2 versus 3.29±0.79 deg2, P<0.05) reflecting pronounced autonomic alterations on the level of the ventricular myocardium. Pigs with MI-related ventricular fibrillation and SCD had significantly higher pre-MI PRD than pigs without tachyarrhythmias, suggesting a potential role for PRD as a predictive biomarker for ischemia-related arrhythmias (no ventricular fibrillation versus ventricular fibrillation: 1.50±0.39 deg2 versus 3.18±0.53 deg2 [P<0.05]; no SCD versus SCD: 1.67±0.32 deg2 versus 3.91±0.63 deg2 [P<0.01]). CONCLUSIONS: We demonstrate that ischemic heart failure leads to significant proarrhythmogenic autonomic remodeling. The concomitant elevation of PRD levels in pigs with ischemic heart failure and pigs with MI-related ventricular fibrillation/SCD suggests PRD as a biomarker for autonomic remodeling and as a potential predictive biomarker for ventricular arrhythmias/survival in the context of MI.

An Innovative Toolkit to Investigate the Complex Mechanisms of Cardiac Arrhythmias.

ARTICLES DISCUSSED: Tomsits, P., Schuttler, D., Kaab, S., Clauss, S., Voigt, N. Isolation of high quality murine atrial and ventricular myocytes for simultaneous measurements of Ca2+ transients and L-Type calcium current. Journal of Visualized Experiments. (165), 10.3791/61964 (2020). Seibertz, F., Reynolds, M., Voigt, N. Single-Cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes. Journal of Visualized Experiments. (166), 10.3791/61890 (2020). Xia, R. et al. Isolation and culture of resident cardiac macrophages from the murine sinoatrial and atrioventricular node. Journal of Visualized Experiments. (171), 10.3791/62236 (2021). Xia, R. et al. Whole-Mount immunofluorescence staining, confocal imaging and 3D reconstruction of the sinoatrial and atrioventricular node in the mouse. Journal of Visualized Experiments. (166), 10.3791/62058 (2020). Kumar, P., Si, M., Paulhus, K., Glasscock, E. Microelectrode array recording of sinoatrial node firing rate to identify intrinsic cardiac pacemaking defects in mice. Journal of Visualized Experiments. (173), 10.3791/62735 (2021). Scherschel, K. et al. Location, dissection, and analysis of the murine stellate ganglion. Journal of Visualized Experiments. (166), 10.3791/62026 (2020). Shimura, D., Hunter, J., Katsumata, M., Shaw, R. M. Removal of an internal translational start site from mRNA while retaining expression of the full-length protein. Journal of Visualized Experiments. (181), 10.3791/63405 (2022). Rotzer, R. D. et al. Implantation of combined telemetric ECG and blood pressure transmitters to determine spontaneous baroreflex sensitivity in conscious mice. Journal of Visualized Experiments. (168), 10.3791/62101 (2021). Tomsits, P. et al. Analyzing long-term electrocardiography recordings to detect arrhythmias in mice. Journal of Visualized Experiments. (171), 10.3791/62386 (2021). Grab, M. et al. Development and evaluation of 3D-printed cardiovascular phantoms for interventional planning and training. Journal of Visualized Experiments. (167), 10.3791/62063 (2021).

Medetomidine/midazolam/fentanyl narcosis alters cardiac autonomic tone leading to conduction disorders and arrhythmias in mice.

Arrhythmias are critical contributors to cardiovascular morbidity and mortality. Therapies are mainly symptomatic and often insufficient, emphasizing the need for basic research to unveil the mechanisms underlying arrhythmias and to enable better and ideally causal therapies. In translational approaches, mice are commonly used to study arrhythmia mechanisms in vivo. Experimental electrophysiology studies in mice are performed under anesthesia with medetomidine/midazolam/fentanyl (MMF) and isoflurane/fentanyl (IF) as commonly used regimens. Despite evidence of adverse effects of individual components on cardiac function, few data are available regarding the specific effects of these regimens on cardiac electrophysiology in mice. Here we present a study investigating the effects of MMF and IF narcosis on cardiac electrophysiology in vivo in C57BL/6N wild-type mice. Telemetry transmitters were implanted in a group of mice, which served as controls for baseline parameters without narcosis. In two other groups of mice, electrocardiogram and invasive electrophysiology studies were performed under narcosis (with either MMF or IF). Basic electrocardiogram parameters, heart rate variability parameters, sinus node and atrioventricular node function, and susceptibility to arrhythmias were assessed. Experimental data suggest a remarkable influence of MMF on cardiac electrophysiology compared with IF and awake animals. While IF only moderately reduced heart rate, MMF led to significant bradycardia, spontaneous arrhythmias, heart rate variability alterations as well as sinus and AV node dysfunction, and increased inducibility of ventricular arrhythmias. On the basis of these observed effects, we suggest avoiding MMF in mice, specifically when studying cardiac electrophysiology, but also whenever a regular heartbeat is required for reliable results, such as in heart failure or imaging research.