MicroRNAs in Atrial Fibrillation: from Expression Signatures to Functional Implications

Atrial fibrillation (AF) is the most common sustained arrhythmia and is associated with pronounced morbidity and mortality. Its prevalence, expected to further increase for the forthcoming years, and associated frequent hospitalizations turn AF into a major health problem. Structural and electrical atrial remodelling underlie the substrate for AF, but the exact mechanisms driving this remodelling remain incompletely understood. Recent studies have shown that microRNAs (miRNA), short non-coding RNAs that regulate gene expression, may be involved in the pathophysiology of AF. MiRNAs have been implicated in AF-induced ion channel remodelling and fibrosis. MiRNAs could therefore provide insight into AF pathophysiology or become novel targets for therapy with miRNA mimics or anti-miRNAs. Moreover, circulating miRNAs have been suggested as a new class of diagnostic and prognostic biomarkers of AF. However, the origin and function of miRNAs in tissue and plasma frequently remain unknown and studies investigating the role of miRNAs in AF vary in design and focus and even present contradicting results. Here, we provide a systematic review of the available clinical and functional studies investigating the tissue and plasma miRNAs in AF and will thereafter discuss the potential of miRNAs as biomarkers or novel therapeutic targets in A

Patch-Clamp Recordings of Action Potentials From Human Atrial Myocytes: Optimization Through Dynamic Clamp

Introduction: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Consequently, novel therapies are being developed. Ultimately, the impact of compounds on the action potential (AP) needs to be tested in freshly isolated human atrial myocytes. However, the frequent depolarized state of these cells upon isolation seriously hampers reliable AP recordings. Purpose: We assessed whether AP recordings from single human atrial myocytes could be improved by providing these cells with a proper inward rectifier K + current (I K1), and consequently with a regular, non-depolarized resting membrane potential (RMP), through “dynamic clamp”. Methods: Single myocytes were enzymatically isolated from left atrial appendage tissue obtained from patients with paroxysmal AF undergoing minimally invasive surgical ablation. APs were elicited at 1 Hz and measured using perforated patch-clamp methodology, injecting a synthetic I K1 to generate a regular RMP. The injected I K1 had strong or moderate rectification. For comparison, a regular RMP was forced through injection of a constant outward current. A wide variety of ion channel blockers was tested to assess their modulatory effects on AP characteristics. Results: Without any current injection, RMPs ranged from −9.6 to −86.2 mV in 58 cells. In depolarized cells (RMP positive to −60 mV), RMP could be set at −80 mV using I K1 or constant current injection and APs could be evoked upon stimulation. AP duration differed significantly between current injection methods (p < 0.05) and was shortest with constant current injection and longest with injection of I K1 with strong rectification. With moderate rectification, AP duration at 90% repolarization (APD 90) was similar to myocytes with regular non-depolarized RMP, suggesting that a synthetic I K1 with moderate rectification is the most appropriate for human atrial myocytes. Importantly, APs evoked using each injection method were still sensitive to all drugs tested (lidocaine, nifedipine, E-4031, low dose 4-aminopyridine, barium, and apamin), suggesting that the major ionic currents of the atrial cells remained functional. However, certain drug effects were quantitatively dependent on the current injection approach used. Conclusion: Injection of a synthetic I K1 with moderate rectification facilitates detailed AP measurements in human atrial myocytes. Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs

Differential sodium current remodeling identifies distinct cellular pro-arrhythmic mechanisms in paroxysmal versus persistent atrial fibrillation

BACKGROUND: The cellular mechanisms underlying progression from paroxysmal to persistent atrial fibrillation (AF) are not fully understood, but alterations in (late) sodium current (I Na) have been proposed. Human studies investigating electrophysiological changes at the paroxysmal stage of AF are sparse, with the majority employing right atrial appendage cardiomyocytes (CMs). We here investigated action potential (AP) characteristics and (late) I Na remodeling in left atrial appendage CMs (LAA-CMs) from patients with paroxysmal and persistent AF and patients in sinus rhythm (SR), as well as the potential contribution of the "neuronal" sodium channel SCN10A/Na V1.8. METHODS: Peak I Na, late I Na and AP properties were investigated through patch-clamp analysis on single LAA-CMs while qPCR was used to assess SCN5A/SCN10A expression levels in LAA tissue. RESULTS: In paroxysmal and persistent AF LAA-CMs, AP duration was shorter than in SR LAA-CMs. Compared to SR, peak I Na and SCN5A expression were significantly decreased in paroxysmal AF, while they were restored to SR levels in persistent AF. Conversely, while late I Na was unchanged in paroxysmal AF as compared to SR, it was significantly increased in persistent AF. Peak or late Na v1.8-based I Na was not detected in persistent AF LAA-CMs. Similarly, expression of SCN10A was not observed in LAAs at any stage. CONCLUSIONS: Our findings demonstrate differences in (late) I Na remodeling in LAA-CMs from patients with paroxysmal versus persistent AF, indicating distinct cellular pro-arrhythmic mechanisms in different AF forms. These observations are of particular relevance when considering potential pharmacological approaches targeting (late) I Na in AF

PREventive left atrial appenDage resection for the predICtion of fuTure atrial fibrillation: Design of the PREDICT AF study

Atrial fibrillation is the most common cardiac arrhythmia, posing a heavy burden on patients'wellbeing and healthcare budgets. Patients undergoing cardiac surgery are at risk of developing postoperative atrial fibrillation (POAF), new-onset atrial fibrillation and subsequent atrial fibrillationrelated complications, including stroke. Sufficient clinical identification of patients at risk fails while the pathological substrate changes that precede atrial fibrillation remain unknown. Here, we describe the PREDICT AF study design, which will be the first study to associate tissue pathophysiology and blood biomarkers with clinical profiling and follow-up of cardiothoracic surgery patients for the prediction of future atrial fibrillation. Methods PREDICT AF will include 150 patients without atrial fibrillation and a CHA2DS2-VASc score of at least 2 undergoing cardiac surgery. The left atrial appendage will be excised during surgery and blood samples will be collected before surgery and at 6 and 12 months' follow-up. Tissue and blood analysis will be used for the discovery of biomarkers including microRNAs and protein biomarkers. The primary study endpoint is atrial fibrillation, which will be objectified by 24 h Holters and ECGs after 30 days for POAF and after 6, 12 and 24 months for new-onset atrial fibrillation. Secondary endpoints include the dynamic changes of blood biomarkers over time and other atrial arrhythmias. PREDICT AF participants may benefit from extensive postoperative care with clinical phenotyping, rhythm monitoring and primary prevention of stroke. Conclusion We here describe the PREDICT AF trial design, which will enable the discovery of biomarkers that truly predict POAF and new-onset atrial fibrillation by combining tissue and plasma-derived biomarkers with comprehensive clinical follow-up data

Absence of Functional Nav1.8 Channels in Non-diseased Atrial and Ventricular Cardiomyocytes

Purpose: Several studies have indicated a potential role for SCN10A/NaV1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/NaV1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of NaV1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of NaV1.8 to the peak and late sodium current (INa) under normal conditions in different species. Methods: The effects of the NaV1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs). Results: A-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, INa density was unchanged after exposure to A-803467 and NaV1.8-based late INa was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs. Conclusion: We here demonstrate the absence of functional NaV1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or NaV1.8 activity in cell types other than CMs

Neutrophil degranulation interconnects over-represented biological processes in atrial fibrillation

Despite our expanding knowledge about the mechanism underlying atrial fibrillation (AF), the interplay between the biological events underlying AF remains incompletely understood. This study aimed to identify the functionally enriched gene-sets in AF and capture their interconnection via pivotal factors, that may drive or be driven by AF. Global abundance of the proteins in the left atrium of AF patients compared to control patients (n = 3/group), and the functionally enriched biological processes in AF were determined by mass-spectrometry and gene set enrichment analysis, respectively. The data were validated in an independent cohort (n = 19–20/group). In AF, the gene-sets of innate immune system, metabolic process, cellular component disassembly and ion homeostasis were up-regulated, while the gene-set of ciliogenesis was down-regulated. The innate immune system was over-represented by neutrophil degranulation, the components of which were extensively shared by other gene-sets altered in AF. In the independent cohort, an activated form of neutrophils was more present in the left atrium of AF patients with the increased gene expression of neutrophil granules. MYH10, required for ciliogenesis, was decreased in the atrial fibroblasts of AF patients. We report the increased neutrophil degranulation appears to play a pivotal role, and affects multiple biological processes altered in AF

Epicardial and endothelial cell activation concurs with extracellular matrix remodeling in atrial fibrillation

BACKGROUND: Improved understanding of the interconnectedness of structural remodeling processes in atrial fibrillation (AF) in patients could identify targets for future therapies. METHODS: We present transcriptome sequencing of atrial tissues of patients without AF, with paroxysmal AF, and persistent AF (total n = 64). RNA expression levels were validated in the same and an independent cohort with qPCR. Biological processes were assessed with histological and immunohistochemical analyses. RESULTS: In AF patients, epicardial cell gene expression decreased, contrasting with an upregulation of epithelial-to-mesenchymal transition (EMT) and mesenchymal cell gene expression. Immunohistochemistry demonstrated thickening of the epicardium and an increased proportion of (myo)fibroblast-like cells in the myocardium, supporting enhanced EMT in AF. We furthermore report an upregulation of endothelial cell proliferation, angiogenesis, and endothelial signaling. EMT and endothelial cell proliferation concurred with increased interstitial (myo)fibroblast-like cells and extracellular matrix gene expression including enhanced tenascin-C, thrombospondins, biglycan, and versican. Morphological analyses discovered increased and redistributed glycosaminoglycans and collagens in the atria of AF patients. Signaling pathways, including cell-matrix interactions, PI3K-AKT, and Notch signaling that could regulate mesenchymal cell activation, were upregulated. CONCLUSION: Our results suggest that EMT and endothelial cell proliferation work in concert and characterize the (myo)fibroblast recruitment and ECM remodeling of AF. These processes could guide future research toward the discovery of targets for AF therapy

Neutrophil degranulation interconnects over-represented biological processes in atrial fibrillation

Despite our expanding knowledge about the mechanism underlying atrial fibrillation (AF), the interplay between the biological events underlying AF remains incompletely understood. This study aimed to identify the functionally enriched gene-sets in AF and capture their interconnection via pivotal factors, that may drive or be driven by AF. Global abundance of the proteins in the left atrium of AF patients compared to control patients (n = 3/group), and the functionally enriched biological processes in AF were determined by mass-spectrometry and gene set enrichment analysis, respectively. The data were validated in an independent cohort (n = 19-20/group). In AF, the gene-sets of innate immune system, metabolic process, cellular component disassembly and ion homeostasis were up-regulated, while the gene-set of ciliogenesis was down-regulated. The innate immune system was over-represented by neutrophil degranulation, the components of which were extensively shared by other gene-sets altered in AF. In the independent cohort, an activated form of neutrophils was more present in the left atrium of AF patients with the increased gene expression of neutrophil granules. MYH10, required for ciliogenesis, was decreased in the atrial fibroblasts of AF patients. We report the increased neutrophil degranulation appears to play a pivotal role, and affects multiple biological processes altered in AF