The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is strongly associated with several comorbidities including heart failure (HF). AF in general, and specifically in the context of HF, is progressive in nature and associated with poor clinical outcomes. Current therapies for AF are limited in number and efficacy and do not target the underlying causes of atrial remodeling such as inflammation or fibrosis. We previously identified the calcium-activated SK4 K+ channels, which are preferentially expressed in the atria relative to the ventricles in both rat and human hearts, as attractive druggable target for AF treatment. Here, we examined the ability of BA6b9, a novel allosteric inhibitor of SK4 channels that targets the specific calmodulin-PIP2 binding domain, to alter AF susceptibility and atrial remodeling in a systolic HF rat postmyocardial infarction (post-MI) model. Daily BA6b9 injection (20 mg/kg/day) for 3 weeks starting 1-week post-MI prolonged the atrial effective refractory period, reduced AF induction and duration, and dramatically prevented atrial structural remodeling. In the post-MI left atrium (LA), pronounced upregulation of the SK4 K+ channel was observed, with corresponding increases in collagen deposition, α-SMA levels, and NLRP3 inflammasome expression. Strikingly, BA6b9 treatment reversed these changes while also significantly reducing the lateralization of the atrial connexin Cx43 in the LA of post-MI rats. Our findings indicate that the blockade of SK4 K+ channels using BA6b9 not only favors rhythm control but also remarkably reduces atrial structural remodeling, a property that is highly desirable for novel AF therapies, particularly in patients with comorbid HF.

An objective tool for quantifying atrial fibrillation substrate in rats.

The utility of rodents for research related to atrial fibrillation (AF) is growing exponentially. However, the obtained arrhythmic waveforms are often mixed with ventricular signals and the ability to analyze regularity and complexity of such events is limited. Recently, we introduced an implantable quadripolar electrode adapted for advanced atrial electrophysiology in ambulatory rats. Notably, we have found that the implantation itself leads to progressive atrial remodeling, presumably because of mechanical loading of the atria. In the present study, we developed an algorithm to clean the atrial signals from ventricular mixing and thereafter quantify the AF substrate in an objective manner based on waveform complexity. Rats were sequentially examined 1-, 4-, and 8-wk postelectrode implantation using a standard AF triggering protocol. Preburst ventricular mixing was sampled and automatically subtracted based on QRS detection in the ECG. Thereafter, the "pure" atrial signals were analyzed by Lempel-Ziv complexity algorithm and a complexity ratio (CR) was defined for each signal by normalizing the postburst to the preburst values. Receiver operating characteristic (ROC) curve analysis indicated an optimal CR cutoff of 1.236 that detected irregular arrhythmic events with high sensitivity (94.5%), specificity (93.1%), and area under the curve (AUC) (0.96, 95% confidence interval, 0.945-0.976). Automated and unbiased analysis indicated a gradual increase in signal complexity over time with augmentation of high frequencies in power spectrum analysis. Our findings indicate that CR algorithm detects irregularity in a highly efficient manner and can also detect the atrial remodeling induced by electrode implantation. Thus, CR analysis can strongly facilitate standardized AF research in rodents.NEW & NOTEWORTHY Rodents are increasingly used in AF research. However, because of technical difficulties including atrial waveform mixing by ventricular signals, most studies do not discriminate between irregular (i.e., AF) and regular atrial arrhythmias. Here, we develop an unbiased computerized tool to "pure" the atrial signals from ventricular mixing and thereafter analyze AF substrate based on the level of irregularity in an objective manner. This novel tool can facilitate standardized AF research in rodents.

Incorrectly corrected? QT interval analysis in rats and mice.

QT interval, a surrogate measure for ventricular action potential duration (APD) in the surface ECG, is widely used to identify cardiac abnormalities and drug safety. In humans, cardiac APD and QT interval are prominently affected by heart rate (HR), leading to widely accepted formulas to correct the QT interval for HR changes (QT corrected - QTc). While QTc is widely used in the clinic, the proper way to correct the QT interval in small mammals such as rats and mice is not clear. Over the years, empiric correction formulas were developed for rats and mice, which are widely used in the literature. Recent experimental findings obtained from pharmacological and direct pacing experiments in unanesthetized rodents show that the rate-adaptation properties are markedly different from those in humans and the use of existing QTc formulae can lead to major errors in data interpretation. In the present review, these experimental findings are summarized and discussed.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment.

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

Cannabinoid Receptor Agonist Inhibits Atrial Electrical Remodeling in a Tachypaced Ex Vivo Rat Model.

Introduction: Atrial fibrillation (AF) leads to rate-dependent atrial changes collectively defined as atrial remodelling (AR). Shortening of the atrial effective refractory period (AERP) and decreased conduction velocity are among the hallmarks of AR. Pharmacological strategies to inhibit AR, thereby reducing the self-perpetual nature of AF, are of great clinical value. Cannabinoid receptor (CBR) ligands may exert cardioprotective effects; CB13, a dual CBR agonist with limited brain penetration, protects cardiomyocytes from mitochondrial dysfunction induced by endothelin-1. Here, we examined the effects of CB13 on normal physiology of the rat heart and development of tachypacing-induced AR. Methods: Rat hearts were perfused in a Langendorff set-up with CB13 (1 µM) or vehicle. Hemodynamic properties of non-paced hearts were examined conventionally. In a different set of hearts, programmed stimulation protocol was performed before and after atrial tachypacing for 90 min using a mini-hook platinum quadrupole electrode inserted on the right atrium. Atrial samples were further assessed by western blot analysis. Results: CB13 had no effects on basal hemodynamic properties. However, the compound inhibited tachypacing-induced shortening of the AERP. Protein expression of PGC1α was significantly increased by CB13 compared to vehicle in paced and non-paced hearts. Phosphorylation of AMPKα at residue threonine 172 was increased suggesting upregulation of mitochondrial biogenesis. Connexin43 was downregulated by tachypacing. This effect was diminished in the presence of CB13. Conclusion: Our findings support the notion that peripheral activation of CBR may be a new treatment strategy to prevent AR in patients suffering from AF, and therefore warrants further study.

A new implantable tool for repeated assessment of supraventricular electrophysiology and atrial fibrillation susceptibility in freely moving rats.

The complex pathophysiology of atrial fibrillation (AF) is governed by multiple risk factors in ways that are still elusive. Basic electrophysiological properties, including atrial effective refractory period (AERP) and conduction velocity, are major factors determining the susceptibility of the atrial myocardium to AF. Although there is a great need for affordable animal models in this field of research, in vivo rodent studies are limited by technical challenges. Recently, we introduced an implantable system for long-term assessment of AF susceptibility in ambulatory rats. However, technical considerations did not allow us to perform concomitant supraventricular electrophysiology measurements. Here, we designed a novel quadripolar electrode specifically adapted for comprehensive atrial studies in ambulatory rats. Electrodes were fabricated from medical-grade silicone, four platinum-iridium poles, and stainless-steel fixating pins. Initial quality validation was performed ex vivo, followed by implantation in adult rats and repeated electrophysiological studies 1, 4, and 8 wk postimplantation. Capture threshold was stable. Baseline AERP values (38.1 ± 2.3 and 39.5 ± 2.0 using 70-ms and 120-ms S1-S1 cycle lengths, respectively) confirmed the expected absence of rate adaptation in the unanesthetized state and validated our prediction that markedly higher values reported under anesthesia are nonphysiological. Evaluation of AF substrate in parallel with electrophysiological parameters validated our recent finding of a gradual increase in AF susceptibility over time and demonstrated that this phenomenon is associated with an electrical remodeling process characterized by AERP shortening. Our findings indicate that the miniature quadripolar electrode is a potent new tool, which opens a window of opportunities for better utilization of rats in AF research.NEW & NOTEWORTHY Rodents are increasingly used in AF research. However, technical challenges restrict long-term supraventricular electrophysiology studies in these species. Here, we developed an implantable electrode adapted for such studies in the rat. Our findings indicate that this new tool is effective for long-term follow-up of critical parameters such as atrial refractoriness. Obtained data shed light on the normal electrophysiology and on the increased AF susceptibility that develops in rats with implanted atrial electrodes over time.

VDAC1 in the diseased myocardium and the effect of VDAC1-interacting compound on atrial fibrosis induced by hyperaldosteronism.

The voltage-dependent anion channel 1 (VDAC1) is a key player in mitochondrial function. VDAC1 serves as a gatekeeper mediating the fluxes of ions, nucleotides, and other metabolites across the outer mitochondrial membrane, as well as the release of apoptogenic proteins initiating apoptotic cell death. VBIT-4, a VDAC1 oligomerization inhibitor, was recently shown to prevent mitochondrial dysfunction and apoptosis, as validated in mouse models of lupus and type-2 diabetes. In the present study, we explored the expression of VDAC1 in the diseased myocardium of humans and rats. In addition, we evaluated the effect of VBIT-4 treatment on the atrial structural and electrical remodeling of rats exposed to excessive aldosterone levels. Immunohistochemical analysis of commercially available human cardiac tissues revealed marked overexpression of VDAC1 in post-myocardial infarction patients, as well as in patients with chronic ventricular dilatation\dysfunction. In agreement, rats exposed to myocardial infarction or to excessive aldosterone had a marked increase of VDAC1 in both ventricular and atrial tissues. Immunofluorescence staining indicated a punctuated appearance typical for mitochondrial-localized VDAC1. Finally, VBIT-4 treatment attenuated the atrial fibrotic load of rats exposed to excessive aldosterone without a notable effect on the susceptibility to atrial fibrillation episodes induced by burst pacing. Our results indicate that VDAC1 overexpression is associated with myocardial abnormalities in common pathological settings. Our data also indicate that inhibition of the VDAC1 can reduce excessive fibrosis in the atrial myocardium, a finding which may have important therapeutic implications. The exact mechanism\s of this beneficial effect need further studies.

An implantable system for long-term assessment of atrial fibrillation substrate in unanesthetized rats exposed to underlying pathological conditions.

Atrial fibrillation (AF) is a progressive arrhythmia with underlying mechanisms that are not fully elucidated, partially due to lack of reliable and affordable animal models. Here, we introduce a system for long-term assessment of AF susceptibility (substrate) in ambulatory rats implanted with miniature electrodes on the atrium. Rats were subjected to excessive aldosterone (Aldo) or solvent only (Sham). An additional group was exposed to myocardial infarction (MI). AF substrate was tested two- and four-weeks post implantation and was also compared with implanted rats early post-implantation (Base). Aldo and MI increased the AF substrate and atrial fibrosis. In the MI group only, AF duration was correlated with the level of atrial fibrosis and was inversely correlated with systolic function. Unexpectedly, Shams also developed progressive AF substrate relative to Base individuals. Further studies indicated that serum inflammatory markers (IL-6, TNF-alpha) were not elevated in the shams. In addition, we excluded anxiety\depression due to social-isolation as an AF promoting factor. Finally, enhanced biocompatibility of the atrial electrode did not inhibit the gradual development of AF substrate over a testing period of up to 8 weeks. Overall, we successfully validated the first system for long-term AF substrate testing in ambulatory rats.

Rapid Atrial Pacing Promotes Atrial Fibrillation Substrate in Unanesthetized Instrumented Rats.

AIM: The self-perpetuating nature of atrial fibrillation (AF) has been a subject of intense research in large mammalian models exposed to rapid atrial pacing (RAP). Recently, rodents are increasingly used to gain insight into the pathophysiology of AF. However, little is known regarding the effects of RAP on the atria of rats and mice. Using an implantable device for electrophysiological studies in rodents, we examined on a daily basis, the effects of continuous RAP on the developed AF substrate of unanesthetized rats and mice. METHODS AND RESULTS: Aggressive burst pacing did not induce AF at baseline in the large majority of rodents, but repeatedly induced AF episodes in rats exposed to RAP for more than 2 days. A microarray study of left atrial tissue from rats exposed to RAP for 2 days vs. control pacing identified 304 differentially expressed genes. Enrichment analysis and comparison with a dataset of atrial tissue from AF patients revealed indications of increased carbohydrate metabolism and changes in pathways that are thought to play critical roles in human AF, including TGF-beta and IL-6 signaling. Among 19 commonly affected genes in comparison with human AF, downregulation of FOXP1 and upregulation of the KCNK2 gene encoding the Kir2.1 potassium channel were conspicuous findings, suggesting NFAT activation. Further results included reduced expression of MIR-26 and MIR-101, which is in line with NFAT activation. CONCLUSION: Our results demonstrate electrophysiological evidence for AF promoting effects of RAP in rats and several molecular similarities between the effects of RAP in large and small mammalian models.

Unanesthetized Rodents Demonstrate Insensitivity of QT Interval and Ventricular Refractory Period to Pacing Cycle Length.

Aim: The cardiac electrophysiology of mice and rats has been analyzed extensively, often in the context of pathological manipulations. However, the effects of beating rate on the basic electrical properties of the rodent heart remain unclear. Due to technical challenges, reported electrophysiological studies in rodents are mainly from ex vivo preparations or under deep anesthesia, conditions that might be quite far from the normal physiological state. The aim of the current study was to characterize the ventricular rate-adaptation properties of unanesthetized rats and mice. Methods: An implanted device was chronically implanted in rodents for atrial or ventricular pacing studies. Following recovery from surgery, QT interval was evaluated in rodents exposed to atrial pacing at various frequencies. In addition, the frequency dependence of ventricular refractoriness was tested by conventional ventricular programmed stimulation protocols. Results: Our findings indicate total absence of conventional rate-adaptation properties for both QT interval and ventricular refractoriness. Using monophasic action potential recordings in isolated mice hearts we could confirm the previously reported shortening of the action potential duration at fast pacing rates. However, we found that this mild shortening did not result in similar decrease of ventricular refractory period. Conclusion: Our findings indicate that unanesthetized rodents exhibit flat QT interval and ventricular refractory period rate-dependence. This data argue against empirical use of QT interval correction methods in rodent studies. Our new methodology allowing atrial and ventricular pacing of unanesthetized freely moving rodents may facilitate more appropriate utility of these important animal models in the context of cardiac electrophysiology studies.

Prominent differences in left ventricular performance and myocardial properties between right ventricular and left ventricular-based pacing modes in rats.

Biventricular pacing is an important modality to improve left ventricular (LV) synchronization and long-term function. However, the biological effects of this treatment are far from being elucidated and existing animal models are limited and demanding. Recently, we introduced an implanted device for double-site epicardial pacing in rats and echocardiographically demonstrated favorable effects of LV and biventricular (LV-based) pacing modes typically observed in humans. Here, this new animal model was further characterized. Electrodes were implanted either on the right atria (RA) and right ventricle (RV) or on the RV and LV. Following recovery, rats were either used for invasive hemodynamic measurements (pressure-volume analysis) or exposed to sustained RV vs. biventricular tachypacing for 3 days. RV pacing compromised, while LV-based pacing modes markedly enhanced cardiac performance. Changes in LV performance were associated with prominent compensatory changes in arterial resistance. Sustained RV tachypacing increased the electrocardiogram QTc interval by 7.9 ± 3.1 ms (n = 6, p < 0.05), dispersed refractoriness between the right and left pacing sites and induced important molecular changes mainly in the early-activated septal tissue. These effects were not observed during biventricular tachypacing (n = 6). Our results demonstrate that the rat is an attractive new model to study the biological consequences of LV dyssynchrony and resynchronization.