Electrical Remodeling In Atrial Fibrillation Research Articles

Main Session: Molecular mechanisms in cardiomyopathies

# 95 High resolution multidimensional proteomics detects candidate arrhythmia biomarkers {#article-title-2} Purpose: Although biomarkers can predict prognosis, studies are limited to small panels, chosen a priori. Mass spectroscopy proteomic techniques powerfully and simultaneously demonstrate differential candidate proteins in an unbiased fashion but must overcome issues of resolving power caused by high abundant proteins. This study utilised a novel whole proteome technique to identify candidate biomarkers associated with arrhythmia. Methods: Consecutive device clinic patients were recruited. Baseline sera were later pooled according to death or arrhythmia occurrence. Samples underwent stable isotope labelling, fractionation and immunodepletion, followed by tandem mass spectroscopy. A separate validation set was tested using ELISA. Results: 243 patients (54% male, age 71±6) were included in the analysis. During follow up of 40 months, there were 10 cardiovascular deaths. 29 experienced VT>182bpm or VF, whilst 48 never experienced VT/VF (controls). Of 634 proteins identified, 94 were differentially expressed in those who died or experienced VT>182 or VF compared to controls. 9 proteins were selected for ELISA validation (table 1). Tropomyosin and VDAC1 were differentially expressed in both discovery and validation sets. Conclusion: This study provides proof of principle that proteomic techniques can identify candidate proteins for use as biomarkers of arrhythmia risk. Further investigation is needed to select proteins with potential for clinical application before testing in a prospective setting.[⇓][1] # 96 Regulation of the small conductance calcium-activated potassium channel 3 by microRNA-499 - potential role in atrial fibrillation {#article-title-3} Purpose: MicroRNAs have emerged as important regulators of gene expression, including those involving electrical remodeling in atrial fibrillation (AF). Recently, KCNN3, the gene that encodes the small conductance calcium-activated potassium channel 3 (SK3), was found to be strongly associated with AF. This study sought to evaluate the changes in atrial myocardial microRNAs in patients with permanent AF and to determine the role of microRNA on the regulation of cardiac SK3 expression. Methods: Atrial tissue obtained during cardiac surgery from patients (4 sinus rhythm and 4 permanent AF) was analyzed by microRNA arrays. Potential targets of microRNAs were predicted by software programs. The effects of specific microRNAs on target gene expression were evaluated in HL-1 cells from a continuously proliferating mouse hyperplastic atrial cardiomyocyte cell line. Interactions between microRNAs and targets were further evaluated by luciferase reporter assay using a vector expressing luciferase with the 3' UTR of SK3 mRNA subcloned into it. Expression of luciferase activity in cells transfected with the vector in the presence of specific microRNAs and microRNA negative controls were determined. The interaction between SK3 mRNA and specific microRNAs were also evaluated by Argonaute pull-down assay. Results: Twenty one microRNAs showed significant, greater than two-fold changes in AF. miR-499 was upregulated by 2.33 fold (p<0.01) in AF atria, whereas SK3 protein expression was down-regulated by 46% (n=3, p<0.05). Transfection of miR-499 mimic in HL-1 cells resulted in the downregulation of SK3 protein expression (reduction by 52%, n=3, p<0.05), while that of miR-499 inhibitor upregulated SK3 expression (54% increase, n=3, p<0.05). Binding of miR-499 to the 3' UTR of SK3 was confirmed by luciferase reporter assay. Exposure of miR-499 mimic to HL-1 cells transfected with luciferase reporter vector containing the SK3 3' UTR reduced luciferase activity by 53% (n=4, p<0.001) compared to cells exposed to microRNA negative control. Immunoprecipitates pull-downed by antibodies against Argonaute 1 and Argonaute 2 showed enhanced presence of SK3 mRNA in the microRNA-induced silencing complexes (RISC) after transfection with miR-499 mimic. Conclusion: Atrial microRNA expression profiles were different in patients with permanent AF compared to those with sinus rhythm. miRNA-499 binds to the 3' UTR of SK3 mRNA and effectively enriches SK3 mRNA in RISCs. Atrial miRNA-499 is significantly upregulated in AF, leading to SK3 downregulation and possibly contributing to the electrical remodeling in AF. [1]: #T1

A novel adult human atrium slice technique for studying electrical remodeling in atrial fibrillation

A novel adult human atrium slice technique for studying electrical remodeling in atrial fibrillation

Effects of acetylcholine on electrical remodeling of human atrial fibers

Autonomic nervous system activation can result in significant changes of atrial electrophysiology and facilitate induction of atrial fibrillation. By recording influence of different concentrations of acetylcholine (ACh) on atrial fibers (AF), we investigated the role of the increased vagal tone in electrical remodeling in atrial fibrillation. Parameters of action potentials and force contraction (Fc) in atrial fibers were recorded by using standard intracellular microelectrode technique and force transducer. It was found that: (1) ACh at 0.1 μmol/L had no significant influence on spontaneous action potentials (SAPs) and Fc (n=6, P>0.05); ACh at both 1.0 and 10.0 μmol/L shortened action potential duration (APD) and Fc of human AF from right atrium (n=6, P<0.05); there was no significant difference in shortening APD between 10.0 and 1.0 μmol/L of ACh; (2) ACh at 0.1 μmol/L had no significant desensitization (n=6, P>0.05), but ACh at 1.0 and 10.0 μmol/L had desensitization (n=6, P<0.05) to SAPs and Fc. The desensitization of ACh on APD in AF was concentration- and time-dependent. It was shown that APD was longer than the control along with extending time of continuous Tyrode's solution perfusion after desensitization. It is concluded that ACh changes the electrophysiological characteristics of human AF, indicating that increased vagal tone plays a role in the development of a vulnerable substrate for atrial electrical remodeling in atrial fibrillation.

MicroRNA-328 Contributes to Adverse Electrical Remodeling in Atrial Fibrillation

A characteristic of both clinical and experimental atrial fibrillation (AF) is atrial electric remodeling associated with profound reduction of L-type Ca(2+) current and shortening of the action potential duration. The possibility that microRNAs (miRNAs) may be involved in this process has not been tested. Accordingly, we assessed the potential role of miRNAs in regulating experimental AF. The miRNA transcriptome was analyzed by microarray and verified by real-time reverse-transcription polymerase chain reaction with left atrial samples from dogs with AF established by right atrial tachypacing for 8 weeks and from human atrial samples from AF patients with rheumatic heart disease. miR-223, miR-328, and miR-664 were found to be upregulated by >2 fold, whereas miR-101, miR-320, and miR-499 were downregulated by at least 50%. In particular, miR-328 level was elevated by 3.9-fold in AF dogs and 3.5-fold in AF patients relative to non-AF subjects. Computational prediction identified CACNA1C and CACNB1, which encode cardiac L-type Ca(2+) channel α1c- and β1 subunits, respectively, as potential targets for miR-328. Forced expression of miR-328 through adenovirus infection in canine atrium and transgenic approach in mice recapitulated the phenotypes of AF, exemplified by enhanced AF vulnerability, diminished L-type Ca(2+) current, and shortened atrial action potential duration. Normalization of miR-328 level with antagomiR reversed the conditions, and genetic knockdown of endogenous miR-328 dampened AF vulnerability. CACNA1C and CACNB1 as the cognate target genes for miR-328 were confirmed by Western blot and luciferase activity assay showing the reciprocal relationship between the levels of miR-328 and L-type Ca(2+) channel protein subunits. miR-328 contributes to the adverse atrial electric remodeling in AF through targeting L-type Ca(2+) channel genes. The study therefore uncovered a novel molecular mechanism for AF and indicated miR-328 as a potential therapeutic target for AF.

Role of Gap Junction in Atrial Fibrillation Pathophysiology

Initiation of atrial fibrillation (AF) occurred when there is a combination of triggering factors (mainly originated from thoracic veins) and arrhytmogenic substrate, such as : reduction of effective refractory period (ERP), increase in refractory spatial dispersion, or abnormal atrial impulse conduction. Atrial fibrillation has an ability to maintain its own progression, so called ‘AF begets AF’. Prologed AF episodes will lead to structural and electrical remodeling, making the patient prone to the recurrent and sustained AF. Structural remodeling, detected in the late phase, involve changes in mitochodrial size, disorder of sarcoplasmic reticulum in subcellular level, and myocardial cell hypertrophy, fiber disarray and elevated collagen deposition in the tissue level. Meanwhile, electrical remodeling of AF will cause delayed effective refractory period, promoting reentry mechanisms. Changes in gap junction regulation and distribution has been noted as part of this remodeling process. Mutation of connexin40 gene, a component protein of gap junction, also has a role in some cases of lone AF. (J Kardiol Indones. 2010; 31: 48-57.) Keywords: Gap Junction, atrial fibrillation

Electrical remodeling in atrial fibrillation

Electrical remodeling in atrial fibrillation

Electrical Remodeling in Atrial Fibrillation

Electrical remodeling refers to any change in electrical function that promotes atrial fibrillation (AF). A key advance in understanding the mechanisms of AF has been the identification of ion channels, gap junctions and regulators of intracellular Ca(2+) homeostasis as the molecular determinants of abnormal atrial electrical activity, and how these are modified in AF. This review focuses on the cellular and molecular basis of altered electrical properties of the atria in AF, with the goal of providing new insights into the potential molecular mechanisms and the identification of putative targets for antiarrhythmic therapy.

Long-Term Follow-up of Changes in Fibrillation Waves in Patients With Persistent Atrial Fibrillation Spectral Analysis of Surface ECG

Little is known about the shortening of atrial refractoriness as a result of electrical remodeling in atrial fibrillation (AF) in clinical cases, especially in terms of long-term follow-up, because of a lack of noninvasive testing methods. The present study population comprised 38 consecutive patients with persistent AF (PAF, >1 month). Before and after the follow-up period of 1-14 months, surface ECGs were recorded for analysis. In each case, the fibrillation wave was purified by subtracting the QRS-T complex template and then power spectral analysis was performed. The mean fibrillation cycle length (FCL) and FCL coefficient of variation (FCL-CV) were determined from peak power frequency in 20 epochs in each recording. The change in FCL (FCL) was calculated by subtracting the baseline FCL from the FCL after the follow-up period. To correct for the difference in the follow-up period, DeltaFCL was divided by the follow-up period in each case. In 38 cases, mean FCL decreased from 160+/-20 ms to 151+/-19 ms (p<0.05), and the FCL-CV also decreased from 15+/-9% to 12+/-5% (p<0.05). The corrected DeltaFCL was -2.4+/-7.6 (ms/month) and there was a significant negative correlation between corrected DeltaFCL and baseline FCL (p<0.01). Shortening of the FCL during a relatively long-term follow-up period was observed in patients with PAF.

Enalapril prevents electrical and structural remodeling in a canine model of atrial fibrillation: Molecular mechanisms

Recent animal models have shown that angiotensin II converting enzyme inhibitors (ACEI) prevent electrical remodeling in atrial fibrillation. The molecular mechanism of ACEI prevention of atrial remodeling still remain not well known.

Increased open probability of single cardiac L-type calcium channels in patients with chronic atrial fibrillation. role of phosphatase 2A.

The L-type calcium channel (LCC) plays a crucial role in the electrical remodeling of atrial fibrillation (AF). AF is associated with reduction of L-type calcium current density, due to a transcriptional downregulation of the pore forming alpha(1c)-subunit of LCC. However, it is unclear, whether this current reduction is related to a decrease in channel number or to alterations in channel function. Hence, we performed a single LCC analysis to assess channel gating and function in human AF. We used the cell-attached patch-clamp technique in isolated atrial human cardiomyocytes of 25 patients with sinus rhythm (SR) and 15 patients with chronic AF. Protein expression of the pore-forming alpha(1c)-subunit of LCC was reduced by 40% in AF. Single channel peak average current was 1.7-fold higher in AF than in SR, due to a 3.1-fold higher open probability of LCC. Since phosphatase 2A (PP2A) is known to preferentially reduce LCC open probability via channel dephosphorylation, we assessed whether PP2A expression or activity is reduced in AF. Okadaic acid, an inhibitor of phosphatases, increased channel open probability in SR, but not in AF. However, Western blot analysis of atrial homogenates of the same patient population revealed unchanged expression of PP2A. Human AF is characterized by increased single LCC activity, due to an increase of channel open probability. The blunted effect of PP2A on LCC as shown by single channel analysis may be related to a reduction of cytosolic PP2A activity or impaired local interaction between PP2A and LCC in AF.

Remodeling in atrial fibrillation

Atrial fibrillation is associated with alterations in atrial electrophysiology that facilitate the initiation and persistence of the arrhythmia. This process was termed electrical remodeling in atrial fibrillation. The underlying cellular and molecular mechanisms have intensively been investigated over the past few years in patients with atrial fibrillation and in different experimental models. The results, that have substantially improved the understanding of the pathophysiology of atrial fibrillation, are reviewed. On the cellular level, atrial fibrillation leads to a strong shortening and an impaired rate adaptation of the action potential as well as to changes in action potential morphology. Atrial fibrillation is associated with an altered gene expression of the L-type calcium channel (ICa,L) and of potassium channels (Ito, IK1, IKACh). The molecular mechanisms of intraatrial conduction slowing are less well understood, changes in the expression or distribution of gap junction proteins or a decrease of the fast sodium inward channel (INa) have been reported in some studies. A trigger of initiation for electrical remodeling is an overload of the cytoplasm with Ca2+ and a consecutive decrease of the systolic calcium gradient, furthermore changes in calcium-handling proteins are detectable in atrial fibrillation. These changes in the cellular and molecular milieu importantly determine the clinical course and the efficacy of therapeutical interventions in atrial fibrillation. The clinical relevance and potential new therapeutic approaches are discussed in the last part.

The effects of atrial electrical remodeling on atrial defibrillation thresholds.

Electrical remodeling of atrial fibrillation may account for the increase in atrial defibrillation thresholds over time. The aim of this study was to examine the time course of electrical remodeling and the benefit of early defibrillation on the defibrillation threshold. Twenty-six mongrel dogs weighing 27.6 +/- 3.3 kg were induced into AF by repeated high output burst atrial pacing. Eight dogs were paced for multiple time periods of 5, 20, 40, and 60 minutes. Five dogs each had burst pacingfor 4 hours and 8 hours, and eight dogs were paced at a high rate (640 beats/min) for 48 hours. Biphasic atrial defibrillation shocks with a pulse width of 3/3 ms synchronized to the left apical electrogram were delivered to coil electrode catheters positioned in the lateral left and right atria. Defibrillation voltage was increased from 50 V in 20- to 30-V steps until defibrillation was successful. As the pacing period increased, a decrease in atrial fibrillation cycle lengths and atrial effective refractory period was not observed before 8 hours. Similarly, the defibrillation threshold did not change significantly until the 8-hour pacing period was reached. The defibrillation thresholds were 69 +/- 28 V for 5 minutes, 64 +/- 20 V for 20 minutes, 99 +/- 85 V for 40 minutes, 78 +/- 51 V for 60 minutes, 78 +/- 38 V for 4 hours, 124 +/- 33 V for 8 hours, and 133 +/- 32 V for 48 hours (mean +/- SD) (P < 0.05). Atrial electrical remodeling in a rapid atrial pacing canine model is not observed until after 4 hours of burst atrial pacing. The atrial defibrillation threshold increases with increasing duration of burst atrial pacing, and follows a similar time course to other parameters of electrical remodeling.

Prevention and reversal of electrical remodeling in atrial fibrillation by TH1 177, a novel calcium channel antagonist Joseph G. Akar, Thomas H. Everett, IV, Lai Chow Kok, Timothy Macdonald, Gabor Szabo, David E. Haines. University of Virginia Health Sciences Center, Charlottesville, VA

Prevention and reversal of electrical remodeling in atrial fibrillation by TH1 177, a novel calcium channel antagonist Joseph G. Akar, Thomas H. Everett, IV, Lai Chow Kok, Timothy Macdonald, Gabor Szabo, David E. Haines. University of Virginia Health Sciences Center, Charlottesville, VA

Molecular Basis of Electrical Remodeling in Atrial Fibrillation

D. R. Van Wagoner and J. M. Nerbonne. Molecular Basis of Electrical Remodeling in Atrial Fibrillation.Journal of Molecular and Cellular Cardiology (2000) 32, 1101–1117. Atrial fibrillation (AF) is the most common cardiac arrhythmia, and is often associated with other cardiovascular disorders and diseases. AF can lead to thromboembolism, reduced left ventricular function and stroke, and, importantly, it is independently associated with increased mortality. AF is a progressive disease; numerous lines of evidence suggest that disease progression results from cumulative electrophysiological and structural remodeling of the atria. There is considerable interest in delineating the molecular mechanisms involved in the remodeling that occurs in the atria of patients with AF. Cellular electrophysiological studies have revealed marked reductions in the densities of the L-type voltage-gated Ca2+current, ICa,L, the transient outward K+current, ITO, and the ultrarapid delayed rectifier K+current,IKur , in atrial myocytes from patients in chronic AF. Similar (but not identical) changes in currents are evident in myocytes isolated from a canine model of AF and, in this case, the changes in currents are correlated with reduced expression of the underlying channel forming subunits. In both human and canine AF, the reduction inICa,L appears to be sufficient to explain the observed decreases in action potential duration and effective refractory period that are characteristic features of the remodeled atria. In addition, expression of the sarcoplasmic reticulum Ca2+ATPase is reduced, suggesting that calcium cycling is affected in AF. These recent studies suggest that calcium overload and perturbations in calcium handling play prominent roles in AF-induced atrial remodeling. Although considerable progress has been made, further studies focused on defining the detailed structural, cellular and molecular changes that accompany the different stages of AF in humans, as well as in animal models of AF, are clearly warranted. It is anticipated that molecular insights gleaned from these studies will facilitate the development of improved therapeutic approaches to treat AF and to prevent the progression of the arrhythmia.

Ionic mechanisms of electrical remodeling in human atrial fibrillation

Atrial fibrillation (AF) is associated with a decrease in atrial ERP and ERP adaptation to rate as well as changes in atrial conduction velocity. The cellular changes in repolarization and the underlying ionic mechanisms in human AF are only poorly understood. Action potentials (AP) and ionic currents were studied with the patch clamp technique in single atrial myocytes from patients in chronic AF and compared to those from patients in stable sinus rhythm (SR). The presence of AF was associated with a marked shortening of the AP duration and a decreased rate response of atrial repolarization. L-type calcium current (ICa,L) and the transient outward current (Ito) were both reduced about 70% in AF, whereas an increased steady-state outward current was detectable at test potentials between -30 and 0 mV. The inward rectifier potassium current (IKI) and the acetylcholine-activated potassium current (IKACh) were increased in AF at hyperpolarizing potentials. Voltage-dependent inactivation of the fast sodium current (INa) was shifted to more positive voltages in AF. AF in humans leads to important changes in atrial potassium and calcium currents that likely contribute to the decrease in APD and APD rate adaptation. These changes contribute to electrical remodeling in AF and are therefore important factors for the perpetuation of the arrhythmia.

Down-regulation of L-type calcium channel and sarcoplasmic reticular Ca2+-ATPase mRNA in human atrial fibrillation without significant change in the mRNA of ryanodine receptor, calsequestrin and phospholamban: An insight into the mechanism of atrial electrical remodeling

OBJECTIVESWe investigated the gene expression of calcium-handling genes including L-type calcium channel, sarcoplasmic reticular calcium adenosine triphosphatase (Ca2+-ATPase), ryanodine receptor, calsequestrin and phospholamban in human atrial fibrillation.BACKGROUNDRecent studies have demonstrated that atrial electrical remodeling in atrial fibrillation is associated with intracellular calcium overload. However, the changes of calcium-handling proteins remain unclear.METHODSA total of 34 patients undergoing open heart surgery were included. Atrial tissue was obtained from the right atrial free wall, right atrial appendage, left atrial free wall and left atrial appendage, respectively. The messenger ribonucleic acid (mRNA) amount of the genes was measured by reverse transcription–polymerase chain reaction and normalized to the mRNA levels of glyceraldehyde 3-phosphate dehydrogenase.RESULTSThe mRNA of L-type calcium channel and of Ca2+-ATPase was significantly decreased in patients with persistent atrial fibrillation for more than 3 months (0.36 ± 0.26 vs. 0.90 ± 0.88 for L-type calcium channel; 0.69 ± 0.42 vs. 1.21 ± 0.68 for Ca2+-ATPase; both p < 0.05, all data in arbitrary unit). We further demonstrated that there was no spatial dispersion of the gene expression among the four atrial tissue sampling sites. Age, gender and underlying cardiac disease had no significant effects on the gene expression. In contrast, the mRNA levels of ryanodine receptor, calsequestrin and phospholamban showed no significant change in atrial fibrillation.CONCLUSIONSL-type calcium channel and the sarcoplasmic reticular Ca2+-ATPase gene were down-regulated in atrial fibrillation. These changes may be a consequence of, as well as a contributory factor for, atrial fibrillation.

Electrical remodeling in atrial fibrillation. Time course and mechanisms.

Atrial fibrillation is self-perpetuating, suggesting that the tachyarrhythmia causes electrophysiological changes that contribute to the progressive nature of the disease. In animal models, pacing-induced rapid atrial rates result in sustained atrial fibrillation. This is mediated by shortening of refractory periods termed electrical remodeling. The purpose of the present study was to characterize the time course of electrical remodeling and to define mechanisms of the phenomenon. Closed-chest dogs were anesthetized, pretreated with atropine and propranolol, and subjected to 7 hours of atrial pacing at 800 bpm. The effective and absolute refractory periods (ARP and ERP) were measured during and after rapid pacing, and transvenous endocardial biopsy specimens were examined using electron microscopy. Despite autonomic blockade and the absence of change in right atrial pressure, persistent atrial tachycardia caused ARP and ERP to fall by > 10%. Electrical remodeling developed quickly, with more than half of the phenomenon occurring during the first 30 minutes of high-rate pacing. Pretreatment with glibenclamide in doses sufficient to block the ATP-sensitive potassium current had no effect. Atrial electrical remodeling was blocked by verapamil and accentuated by hypercalcemia. Biopsy specimens from controls subjected to rapid pacing showed mitochondrial swelling consistent with calcium overload. Biopsies from verapamil-treated animals were normal. Atrial electrical remodeling develops quickly, is progressive, and may be persistent. Shifts in autonomic tone, atrial stretch, or depletion of high-energy phosphates do not contribute significantly to the phenomenon. Results of the study suggest that atrial electrical remodeling is mediated by rate-induced intracellular calcium overload.