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G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P 2 ). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP 2 , an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (I KACh ). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The I KACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the I KACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of I KACh . In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of I KACh , patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated I KACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active I KACh . Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.

The usage of flavored electronic nicotine delivery systems (ENDS) is popular, specifically in the teen and young adult age-groups. The possible cardiac toxicity of the flavoring aspect of ENDS is largely unknown. Vaping, a form of electronic nicotine delivery, uses “e-liquid” to generate “e-vapor,” an aerosolized mixture of nicotine and/or flavors. We report our investigation into the cardiotoxic effects of flavored e-liquids. E-vapors containing flavoring aldehydes such as vanillin and cinnamaldehyde, as indicated by mass spectrometry, were more toxic in HL-1 cardiomyocytes than fruit-flavored e-vapor. Exposure of human induced pluripotent stem cell-derived cardiomyocytes to cinnamaldehyde or vanillin-flavored e-vapor affected the beating frequency and prolonged the field potential duration of these cells more than fruit-flavored e-vapor. In addition, vanillin aldehyde-flavored e-vapor reduced the human ether-à-go-go-related gene (hERG)-encoded potassium current in transfected human embryonic kidney cells. In mice, inhalation exposure to vanillin aldehyde-flavored e-vapor for 10 wk caused increased sympathetic predominance in heart rate variability measurements. In vivo inducible ventricular tachycardia was significantly longer, and in optical mapping, the magnitude of ventricular action potential duration alternans was significantly larger in the vanillin aldehyde-flavored e-vapor-exposed mice than in controls. We conclude that the widely popular flavored ENDS are not harm free, and they have a potential for cardiac harm. More studies are needed to further assess their cardiac safety profile and long-term health effects. NEW & NOTEWORTHY The use of electronic nicotine delivery systems (ENDS) is not harm free. It is not known whether ENDS negatively affect cardiac electrophysiological function. Our study in cell lines and in mice shows that ENDS can compromise cardiac electrophysiology, leading to action potential instability and inducible ventricular arrhythmias. Further investigations are necessary to assess the long-term cardiac safety profile of ENDS products in humans and to better understand how individual components of ENDS affect cardiac toxicity.

G-protein–gated inwardly-rectifying K(+) (GIRK) channels are targets of G(i/o)-protein–signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with nonfunctional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report on a highly-specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel–phosphatidylinositol 4,5-bisphosphate interactions. Through brain-slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder. In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.

Atrial fibrillation (AF), the most common abnormal heart rhythm, is a major cause for stroke. Aging is a significant risk factor for AF; however, specific ionic pathways that can elucidate how aging leads to AF remain elusive. We used young and old wild-type and PKC epsilon- (PKCϵ) knockout mice, whole animal, and cellular electrophysiology, as well as whole heart, and cellular imaging to investigate how aging leads to the aberrant functioning of a potassium current, and consequently to AF facilitation. Our experiments showed that knocking out PKCϵ abrogates the effects of aging on AF by preventing the development of a constitutively active acetylcholine sensitive inward rectifier potassium current (I

Cigarette smoking (CS) is a major cause of cardiovascular diseases. Smokers are at a significantly higher risk for developing atrial fibrillation (AF), a dangerous and abnormal heart rhythm. In the US, 15.5% of adults are current smokers, and it is becoming clear that CS is an independent risk factor for AF, but a detailed mechanistic understanding of how CS contributes to the molecular patho-electrophysiology of AF remains elusive. We investigated if CS related AF is in part mediated through a mechanism that depends on the cardiac acetylcholine activated inward rectifier potassium current (I

Friedreich ataxia is an autosomal recessive congenital neurodegenerative disease caused by a deficiency in the frataxin protein and is often diagnosed in young adulthood. An expansion of guanine-adenine-adenine repeats in the first intron of the FXN gene leads to decreased frataxin expression. Frataxin plays an essential role in mitochondrial metabolism. Most Friedreich ataxia patients are diagnosed with left ventricular hypertrophic cardiomyopathy, and 60% of patients die with hypertrophic cardiomyopathy. However, the mitochondrial anatomy in Friedreich ataxia hypertrophic cardiomyopathy is still poorly understood. We investigated mitochondrial fission, fusion, and function using biochemical, microscopy, and computational stochastic analysis in human induced pluripotent stem cell derived cardiomyocytes from a patient with Friedreich ataxia hypertrophic cardiomyopathy and a healthy individual. We found a significantly higher mitochondrial footprint, decreased mitochondrial fission protein dynamin-related protein, and mitochondrial fission rate over fusion with more giant mitochondrial clusters in human induced pluripotent stem cell derived cardiomyocytes from a patient with Friedreich ataxia hypertrophic cardiomyopathy, compared to an unaffected individual. We also found significantly depolarized mitochondrial membrane potential and higher reactive oxygen species levels in Friedreich ataxia human induced pluripotent stem cell cardiomyocytes. Our results show that frataxin’s depletion may dampen the mitochondrial fission machinery by reducing dynamin-related protein1. The loss of mitochondrial fission might lead to elevated reactive oxygen species and depolarized mitochondrial membrane potential, which may cause oxidative damage in Friedreich ataxia hypertrophic cardiomyopathy. Further investigations are needed to identify the mechanism of downregulating dynamin-related protein1 due to the frataxin deficiency in Friedreich ataxia hypertrophic cardiomyopathy.

Loss-of-function (LOF) variants in SCN1B, encoding the voltage-gated sodium channel β1/β1B subunits, are linked to neurological and cardiovascular diseases. Scn1b-null mice have spontaneous seizures and ventricular arrhythmias and die by approximately 21 days after birth. β1/β1B Subunits play critical roles in regulating the excitability of ventricular cardiomyocytes and maintaining ventricular rhythmicity. However, whether they also regulate atrial excitability is unknown. We used neonatal Scn1b-null mice to model the effects of SCN1B LOF on atrial physiology in pediatric patients. Scn1b deletion resulted in altered expression of genes associated with atrial dysfunction. Scn1b-null hearts had a significant accumulation of atrial collagen, increased susceptibility to pacing induced atrial fibrillation (AF), sinoatrial node (SAN) dysfunction, and increased numbers of cholinergic neurons in ganglia that innervate the SAN. Atropine reduced the incidence of AF in null animals. Action potential duration was prolonged in null atrial myocytes, with increased late sodium current density and reduced L-type calcium current density. Scn1b LOF results in altered atrial structure and AF, demonstrating the critical role played by Scn1b in atrial physiology during early postnatal mouse development. Our results suggest that SCN1B LOF variants may significantly impact the developing pediatric heart.

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

[EN] BACKGROUND: The formation of recipient-to donor atrio-atrial connections (AAC) in patients after orthotopic heart transplantation (OHT) is poorly understood. We sought to investigate the mechanisms of atrial tachyarrhythmias after OHT, the role of AACs, and their relationship to the immunologic match. METHODS: In a large series of OHT patients, we performed a retrospective review of 42 patients who underwent catheter ablation for atrial arrhythmias. A realistic 3-dimensional computer model of human atria was used to study AAC conductivity. RESULTS: Patient age was 55 +/- 15 years (71% male). Biatrial anastomosis was present in 24/42 patients (57%). An AAC was found in 9/42 patients (21%, right-sided in 5 patients with biatrial anastomosis, left-sided in 4 patients). The AAC became apparent at the time of the electrophysiology study 10.1 +/- 7.6 years after OHT (range, 0.3-22.2 years). Donor-specific antibodies were present in no patient with AAC but were present in 69% of patients without AAC, P=0.002. In all patients with AAC, a recipient atrial tachycardia propagated via AAC to the donor atrium (4 patients presented with atrial fibrillation). Simulations showed AAC conduction requires an isthmus of >= 2 mm and is cycle length and location dependent. Patients without AAC (n=13) frequently presented with donor atrial arrhythmias, in 77% cavo-tricuspid isthmus flutter was ablated. The procedural success was high, although, 12 patients (29%) required reablation. CONCLUSIONS: AACs are found in 21% of OHT patients with atrial tachyarrhythmias and can manifest very early after OHT. Immune privilege characterized by the absence of donor-specific antibodies may facilitate AAC formation. Propagation across an AAC is width, cycle length, and location dependent. Patients with AAC present with focal atrial tachycardias or atrial fibrillation originating from the recipient atria; patients without most frequently present with cavo-tricuspid isthmus dependent atrial flutter. While multiple arrhythmias frequently require reablation, ablative therapy is highly effective., This study was supported in part by National Institutes of Health grants R21HL138064, and R01HL129136 to Dr Noujaim. This work was partially supported by the Direccion General de Politica Cientifica de la Generalitat Valenciana (PROMETEU2020/043).

Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects.

Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death. JV is a PhD fellow of the La Caixa Foundation International Fellowship Programme (La Caixa/CNB). This work was supported by the European Molecular Biology Organizat ion (STF-7649 to AF), the Spanish Ministry of Science, Innovation and Universities (MCIU), (BFU2011-26175 and BFU2014-57797-R to AN), and the network Ciber de Enfermedades Respiratorias (CIBERES) including the Improvement and Mobilit y Programme. The CNIC is a Severo Ochoa Center of Excellence (SEV-2015-0505). CNIC is supported by MCIU and the Pro CNIC Foundation. This study was supported by grants from Fondo Europeo de Desarrollo Regional (CB16/11/00458), grants SAF2015-65607-R and SAF2016-80324-R from MCIU (A.H. and D.F-R.) and fellowship SVP-2014-068595 to J.A.N-A. This study was supported by Frankel Cardiovascular Centre, Michigan Medicine (Grant 332475). JJ is supported in part by the National Heart, Lung, and Blood Institute (R01 Grant HL122352). S.F.N is supported in part by the National Heart, Lung, and Blood Institute grants R21HL138064 and R01HL129136. Sí

The acetylcholine-activated inward rectifier potassium current (I(KACh)) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that the blocking of I(KACh) with the small molecule chloroquine terminates persistent AF. We used a sheep model of tachypacing-induced, persistent AF, molecular modeling, electrophysiology, and structural biology approaches. The 50% inhibition/inhibitory concentration of I(KACh) block with chloroquine, measured by patch clamp, was 1 μM. In optical mapping of sheep hearts with persistent AF, 1 μM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of I(KACh)) by interacting with residues D260 and F255, in proximity to I228, Q227, and L299. (1)H (15)N heteronuclear single-quantum correlation of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical-shift perturbations upon drug binding. We then crystallized and solved a 2.5 Å X-ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drug’s binding to the pore becomes off centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A and D260A mutations significantly reduced I(KACh) block by chloroquine. With the use of numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel and exert antiarrhythmic effects. Chloroquine binds the I(KACh) channel at a site formed by specific amino acids in the ion-permeation pathway, leading to decreased I(KACh) and the subsequent termination of AF.—Takemoto, Y., Slough, D. P., Meinke, G., Katnik, C., Graziano, Z. A., Chidipi, B., Reiser, M., Alhadidy, M. M., Ramirez, R., Salvador-Montañés, O., Ennis, S., Guerrero-Serna, G., Haburcak, M., Diehl, C., Cuevas, J., Jalife, J., Bohm, A., Lin,Y.-S., Noujaim, S. F. Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

Background The incidence of sudden arrhythmic death is markedly increased in diabetics. Objective The purpose of this study was to develop a mouse model for postmyocardial infarction (post-MI) ventricular tachycardia (VT) in the diabetic heart and determine the mechanism of an antiarrhythmic effect of statins. Methods ECG transmitters were implanted in wild-type (WT), placebo, and pravastatin-treated type I diabetic Akita mice. MIs were induced by coronary ligation, and Ca 2+ transients were studied by optical mapping, and Ca 2+ transients and sparks in left ventricular myocytes (VM) by the Ionoptix system and confocal microscopy. Results Burst pacing of Akita mouse hearts resulted in rate-related QRS/T-wave alternans, which was attenuated in pravastatin-treated mice. Post-MI Akita mice developed QRS/T-wave alternans and VT at 2820 ± 879 beats per mouse, which decreased to 343 ± 115 in pravastatin-treated mice (n = 13, P 2+ alternans, both attenuated in hearts of statin-treated mice. Akita VM displayed Ca 2+ alternans, and triggered activity as well as increased Ca 2+ transient decay time (Tau), Ca 2+ sparks, and cytosolic Ca 2+ and decreased SR Ca 2+ stores all of which were in part reversed in cells from statin treated mice. Homogenates of Akita ventricles demonstrated decreased SERCA2a/PLB ratio and increased ratio of protein phosphatase (PP-1) to the PP-1 inhibitor PPI-1 which were reversed in homogenates of pravastatin-treated Akita mice. Conclusion Pravastatin decreased the incidence of post-MI VT and Ca 2+ alternans in Akita mouse hearts in part by revering abnormalities of Ca 2+ handling via the PP-1/PPI-1 pathway.

Variations in body mass impose constraints on the structure and function of mammalian species, including those of the cardiovascular system. Numerous biological processes, including cardiovascular parameters, have been shown to scale with body mass (BM) according to the law of allometric scaling: Y=Y =a·BMb (Y, biological process; a, normalization constant; b, scaling exponent, which in many instances is a multiple of ¼). These parameters include heart and breathing rates, intervals and subintervals of the electrocardiogram (ECG), action potential duration (APD), metabolic rate, and temporal properties of ventricular fibrillation. For instance, the hierarchical branching networks of the vascular system, and of the specialized conduction system in the heart have been proposed to be important determinants of allometric scaling. A global and unifying molecular mechanism of allometric scaling has not been put forth, but changes in gene expression have been proposed to play an important role. Even though it is accepted that differences in body size have cardiovascular effects, the use of scaling in the clinical setting is limited. An increase in the clinical utilization of scaling is thought to lead to improved cardiovascular disease diagnosis and management in patients.

[EN] In clinical practice, reducing the burden of persistent atrial fibrillation by pharmacological means is challenging. We explored if blocking the background and the acetylcholine-activated inward rectifier potassium currents (I-K1 and I-KACh) could be antiarrhythmic in persistent atrial fibrillation. We thus tested the hypothesis that blocking I-K1 and I-KACh with chloroquine decreases the burden of persistent atrial fibrillation. We used patch clamp to determine the IC50 of I-K1 and I-KACh block by chloroquine and molecular modeling to simulate the interaction between chloroquine and Kir2.1 and Kir3.1, the molecular correlates of I-K1 and I-KACh. We then tested, as a proof of concept, if oral chloroquine administration to a patient with persistent atrial fibrillation can decrease the arrhythmia burden. We also simulated the effects of chloroquine in a 3D model of human atria with persistent atrial fibrillation. In patch clamp the IC50 of I-K1 block by chloroquine was similar to that of I-KACh. A 14-day regimen of oral chloroquine significantly decreased the burden of persistent atrial fibrillation in a patient. Mathematical simulations of persistent atrial fibrillation in a 3D model of human atria suggested that chloroquine prolonged the action potential duration, leading to failure of reentrant excitation, and the subsequent termination of the arrhythmia. The combined block of I-K1 and I-KACh can be a targeted therapeutic strategy for persistent atrial fibrillation., This work was supported in part by National Institutes of Health grants R21HL138064, R01HL129136, by the Direccion General de Politica Cientifica de la Generalitat Valenciana (PROMETEO 2016/088), and by the ACM SIGHPC/Intel Computational & Data Science fellowship.

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3β, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3β, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3β might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3β. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3β. Using this mouse, we demonstrate that attenuation of GSK3β expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3β deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3β activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression.

Potassium channels play a critical role in modulating cardiac excitability and action potential repolarization. They include the voltage-gated, inwardly rectifying (Kir), small conductance calcium-activated, two-pore domain K+ channels. In this chapter, we will focus on Kir channels, in light of advancements made in the structural biology of these proteins and our increased understanding of their regulation by pharmacological agents.

The sympathetic and parasympathetic branches of the autonomic nervous system exert fine-tuned reciprocal influences on the heart. Sympathetic stimulation increases heart rate, electrical conductivity, and contractility, whereas in general, parasympathetic stimulation produces opposing inhibitory effects. However, the cardiac regulation driven by the autonomic nervous system is complex. In fact, the electromechanical function of the heart is subject to the influence of two separate autonomic nervous systems: the extrinsic cardiac nervous system (ECNS) and the intrinsic cardiac nervous system (ICNS). Here, we will explore the influence of the intrinsic nervous system on the regulation of heart rate.

Although a reduction in the high-frequency (HF) component of heart rate variability (HRV) is a major complication of diabetes and a risk factor for sudden death, its relationship to ventricular tachycardia (VT) is unknown. We developed a mouse model for the study of VT and its relationship to changes in HRV in the Akita type 1 diabetic mouse. Programmed ventricular stimulation of anesthetized mice demonstrated that Akita mice were more inducible for VT compared with wild-type mice: 78.6% versus 28.6% ( P = 0.007). Optical mapping of perfused hearts demonstrated multifocal breakthroughs that occasionally gave rise to short-lived rotors consistent with focal initiation and maintenance of VT. Treatment of Akita mice with pravastatin, which had been previously shown clinically to decrease ventricular ectopy and to increase HRV, decreased the inducibility of VT: 36.8% compared with 75.0% with placebo treatment ( P = 0.022). The HF fraction of HRV was reduced in Akita mice (48.6 ± 5.2% vs. 70.9 ± 4.8% in wild-type mice, P = 0.005) and was increased compared with placebo treatment in pravastatin-treated mice. Pretreatment of Akita mice with the muscarinic agonist carbamylcholine or the β-adrenergic receptor blocker propranolol decreased the inducibility of VT ( P = 0.001). In conclusion, the increased inducibility of focally initiated VT and reduced HF fraction in Akita mice were partially reversed by both pravastatin treatment and pharmacologic reversal of parasympathetic dysfunction. In this new animal model for the study of the pathogenesis of VT in type 1 diabetes, pravastatin may play a role in the prevention of VT by attenuating parasympathetic dysfunction.

Rationale: Autonomic nerves from sinoatrial node (SAN) ganglia are known to regulate SAN function. However, it is unclear whether remote pulmonary vein ganglia (PVG) also modulate SAN pacemaker rhythm. Objective: To investigate whether in the mouse heart PVG modulate SAN function. Methods and Results: In hearts from 45 C57BL and 7 Connexin40+/GFP mice, we used tyrosine-hydroxylase (TH) and choline-acetyltransferase (ChAT) immunofluorescence labeling to characterize adrenergic and cholinergic elements, repectively, within the PVG and SAN. PVG project postganglionic nerves to the SAN. TH and ChAT stained nerves, enter the SAN as an extensive, dense mesh-like neural network. Neurons in PVG are biphenotypic, containing ChAT and TH positive neurons. In Langendorff-perfused hearts, we compared effects of electrical stimulation of PVG, posterior (PRCVG) and anterior right vena cava ganglia (ARCVG) using 200-2000 ms trains of pulses (300μs, 0.2-0.6mA, 200Hz). Sympathetic and/or parasympathetic blockade was achieved using 0.5μM propranolol and 1μM atropine, respectively. Epicardial optical mapping of SAN activation was performed before, during and after ganglion stimulation. PVG stimulation increased the P-P interval by 36±9%; PRCVG stimulation increased the P-P interval by 42±11%. ARCVG stimulation produced no change. Propranolol perfusion increased the PVG stimulation effect to 43±13%. Atropine caused a 5±6% decrease. In optical mapping experiments of whole hearts and isolated atrial preparations, PVG stimulation shifted the origin of SAN discharges to varying locations. Conclusions: PVG contain cholinergic, adrenergic and biphenotipic neurons whose axons project across the right atrium to richly innervate the SAN region and contribute significantly to regulation of SAN function.

We describe a mutation (E299V) in KCNJ2 , the gene that encodes the strong inward rectifier K + channel protein (Kir2.1), in an 11-y-old boy. The unique short QT syndrome type-3 phenotype is associated with an extremely abbreviated QT interval (200 ms) on ECG and paroxysmal atrial fibrillation. Genetic screening identified an A896T substitution in a highly conserved region of KCNJ2 that resulted in a de novo mutation E299V. Whole-cell patch-clamp experiments showed that E299V presents an abnormally large outward I K1 at potentials above −55 mV ( P < 0.001 versus wild type) due to a lack of inward rectification. Coexpression of wild-type and mutant channels to mimic the heterozygous condition still resulted in a large outward current. Coimmunoprecipitation and kinetic analysis showed that E299V and wild-type isoforms may heteromerize and that their interaction impairs function. The homomeric assembly of E299V mutant proteins actually results in gain of function. Computer simulations of ventricular excitation and propagation using both the homozygous and heterozygous conditions at three different levels of integration (single cell, 2D, and 3D) accurately reproduced the electrocardiographic phenotype of the proband, including an exceedingly short QT interval with merging of the QRS and the T wave, absence of ST segment, and peaked T waves. Numerical experiments predict that, in addition to the short QT interval, absence of inward rectification in the E299V mutation should result in atrial fibrillation. In addition, as predicted by simulations using a geometrically accurate three-dimensional ventricular model that included the His–Purkinje network, a slight reduction in ventricular excitability via 20% reduction of the sodium current should increase vulnerability to life-threatening ventricular tachyarrhythmia.

Histone H3 lysine 4 (H3K4me) methyltransferases and their cofactors are essential for embryonic development and the establishment of gene expression patterns in a cell-specific and heritable manner. However, the importance of such epigenetic marks in maintaining gene expression in adults and in initiating human disease is unclear. Here, we addressed this question using a mouse model in which we could inducibly ablate PAX interacting (with transcription-activation domain) protein 1 (PTIP), a key component of the H3K4me complex, in cardiac cells. Reducing H3K4me3 marks in differentiated cardiomyocytes was sufficient to alter gene expression profiles. One gene regulated by H3K4me3 was Kv channel-interacting protein 2 (Kcnip2), which regulates a cardiac repolarization current that is downregulated in heart failure and functions in arrhythmogenesis. This regulation led to a decreased sodium current and action potential upstroke velocity and significantly prolonged action potential duration (APD). The prolonged APD augmented intracellular calcium and in vivo systolic heart function. Treatment with isoproterenol and caffeine in this mouse model resulted in the generation of premature ventricular beats, a harbinger of lethal ventricular arrhythmias. These results suggest that the maintenance of H3K4me3 marks is necessary for the stability of a transcriptional program in differentiated cells and point to an essential function for H3K4me3 epigenetic marks in cellular homeostasis.

Background Complex fractionated atrial electrograms (CFAEs) are thought to identify high-frequency electrical sources and have become an important target for radiofrequency ablation of atrial fibrillation (AF). Methods used to identify CFAEs and locate suitable ablation sites usually depend on subjective analysis of the electrograms but may also involve objective, computer-based paradigms through either time- or frequency-domain approaches. Methods We generated a set of simulated test electrograms, which were defined by a combination of a basic cycle length, phase-resetting noise, and phase-preserving noise, accounting for far-field effects. The simulated electrograms were analyzed separately by well-known basic time-domain (complex fractionated electrogram [CFE]) and frequency-domain (dominant frequency [DF]) methods, and the results were compared with each other to determine objectively the potential reliability of either method to accurately estimate the cycle length of the atrial electrogram. Results The behavior of the time-domain method depends on the assumed amplitude-sensitivity threshold and can be tuned to its optimal performance but only for signals having stable (and known a priori ) amplitude. When the signal amplitude varies randomly (with ±20% standard deviation range), the time-domain method loses performance. By contrast, the performance of the frequency-domain method remains stable. Conclusion Despite potentially good performance of time-domain methods to estimate the cycle length during AF and localize ablation sites, their performance is easily prone to degradation. The frequency-domain method seems to be much more robust.

Many cardiac electrophysiological abnormalities are accompanied by autonomic nervous system dysfunction. Here, we review mechanisms by which the cardiac nervous system controls normal and abnormal excitability and may contribute to atrial and ventricular tachyarrhythmias. Moreover, we explore the potential antiarrhythmic and/or arrhythmogenic effects of modulating the autonomic nervous system by several strategies, including ganglionated plexi ablation, vagal and spinal cord stimulations, and renal sympathetic denervation as therapies for atrial and ventricular arrhythmias.

Background: The acetylcholine activated inward rectifier potassium current (IKACh) was shown to be constitutively active in chronic atrial fibrillation. Its blockade has been proposed to be a possible antifibrillatory pharmacotherapy. We therefore tested the hypothesis that blocking IKACh with the bee venom peptide tertiapin, or the small molecule chloroquine, terminates chronic AF in the sheep heart. Methods and Results: We tested our hypothesis using biochemistry, electrophysiology and molecular modeling. In patch clamp, the IC50 of IKACh block by tertiapin, and chloroquine was 60 nM, and 710 nM respectively, while dofetilde, a currently used class III antiarrhythmic, did not block the current. On the other hand, dofetilide and chloroquine blocked the rapid delayed rectifier potassium current (IKr) with an IC50 of 50nM, and 2.3 microM respectively, while tertiapine had no effects. Furthermore, molecular modeling indicated that 1 chloroquine molecule blocks the intracellular ion permeation vestibule of the tetrameric Kir3.1, a molecular correlate of IKACh, by interacting with amino acids important for the channel’s rectification. Dofetilide did not interact with Kir3.1. In vitro fluorescence measurements of chloroquine titration into the purified Kir3.1 intracellular domain protein confirmed the computational results and indicated that chloroquine directly binds Kir3.1 with a stoichiometry of 1 chloroquine molecule per Kir3.1 tetramer. Finally, optical mapping of the chronically fibrillating sheep atria showed that while tertiapine, chloroquine, and dofetilide slowed down chronic AF and prolonged the action potential, only tertiapine and chloroquine (the IKACh blockers) restored normal sinus rhythm. Conclusions: IKACh could be a more powerful anti atrial fibrillation target compared to IKr. Pharmacological blockade of IKACh with a peptide, or a small molecule similar to chloroquine could terminate chronic AF and restore normal sinus rhythm.

Previous studies have suggested an important role for the inward rectifier K+ current (IK1) in stabilizing rotors responsible for ventricular tachycardia (VT) and fibrillation (VF). To test this hypothesis, we used a line of transgenic mice (TG) overexpressing Kir 2.1–green fluorescent protein (GFP) fusion protein in a cardiac-specific manner. Optical mapping of the epicardial surface in ventricles showed that the Langendorff-perfused TG hearts were able to sustain stable VT/VF for 350 ± 1181 s at a very high dominant frequency (DF) of 44.6 ± 4.3 Hz. In contrast, tachyarrhythmias in wild-type hearts (WT) were short-lived (3 ± 9 s), and the DF was 26.3 ± 5.2 Hz. The stable, high frequency, reentrant activity in TG hearts slowed down, and eventually terminated in the presence of 10 μm Ba2+, suggesting an important role for IK1. Moreover, by increasing IK1 density in a two-dimensional computer model having realistic mouse ionic and action potential properties, a highly stable, fast rotor (≈45 Hz) could be induced. Simulations suggested that the TG hearts allowed such a fast and stable rotor because of both greater outward conductance at the core and shortened action potential duration in the core vicinity, as well as increased excitability, in part due to faster recovery of Na+ current. The latter resulted in a larger rate of increase in the local conduction velocity as a function of the distance from the core in TG compared to WT hearts, in both simulations and experiments. Finally, simulations showed that rotor frequencies were more sensitive to changes (doubling) in IK1, compared to other K+ currents. In combination, these results provide the first direct evidence that IK1 up-regulation in the mouse heart is a substrate for stable and very fast rotors.

Rationale: Most cardiac ryanodine receptor (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) are postulated to cause a distinctive form of Ca 2+ release dysfunction. Considering the spread distribution of CPVT mutations, we hypothesized that dysfunctional heterogeneity also was feasible. Objective: To determine the molecular and cellular mechanisms by which a novel RyR2-V2475F mutation associated with CPVT in humans triggers Ca 2+ -dependent arrhythmias in whole hearts and intact mice. Methods and Results: Recombinant channels harboring CPVT-linked RyR2 mutations were functionally characterized using tritiated ryanodine binding and single-channel recordings. Homologous recombination was used to generate a knock-in mouse bearing the RyR2-V2475F mutation. Ventricular myocytes from mice heterozygous for the mutation (RyR2-V2475F +/− ) and their wild-type littermates were Ca 2+ -imaged by confocal microscopy under conditions that mimic stress. The propensity of wild-type and RyR2-V2475F +/− mice to have development of arrhythmias was tested at the whole heart level and in intact animals. Recombinant RyR2-V2475F channels displayed increased cytosolic Ca 2+ activation, abnormal protein kinase A phosphorylation, and increased activation by luminal Ca 2+ . The RyR2-V2475F mutation appears embryonic-lethal in homozygous mice, but heterozygous mice have no alterations at baseline. Spontaneous Ca 2+ release events were more frequent and had shorter latency in isoproterenol-stimulated cardiomyocytes from RyR2-V2475F +/− hearts, but their threshold was unchanged with respect to wild-type. Adrenergically triggered tachyarrhythmias were more frequent in RyR2-V2475F +/− mice. Conclusions: The mutation RyR2-V2475F is phenotypically strong among other CPVT mutations and produces heterogeneous mechanisms of RyR2 dysfunction. In living mice, this mutation appears too severe to be harbored in all RyR2 channels but remains undetected under basal conditions if expressed at relatively low levels. β-adrenergic stimulation breaks the delicate Ca 2+ equilibrium of RyR2-V2475F +/− hearts and triggers life-threatening arrhythmias.

The congenital long QT syndrome (LQTS) is an inherited disease that causes cardiac arrhythmias and sudden cardiac death in the absence of evident structural heart defects. It is mainly attributed to genetic mutations that affect the function of ion channels that regulate the action potential waveform. LQTS is characterized by an abnormal prolongation of cardiac repolarization, leading to an increased QT interval, notched or biphasic T waves, and T-wave alternans. Affected patients are at a higher risk for developing arrhythmias, including polymorphic ventricular tachycardia, torsades de pointes, and ventricular fibrillation. The estimated prevalence of LQTS (based on electrocardiographic-guided identification of disease-causing mutations among whites) is approximately 1:2500 in apparently healthy live births. LQTS was initially described in the Jervell and Lange-Nielsen syndrome, where QT prolongation and deafness were present. A few years later, an autosomal dominant form of LQTS, in the absence of deafness, was reported. During the last decade, 13 LQTS variants have been described. Mutations in KCNQ1, KCNH2, and SCN5A genes account for approximately 90% of the positivegenotype cases of LQTS and underlie LQTS1, LQTS2, and LQTS3. Those genes encode Kv7.1, Kv11.1, and Nav1.5, respectively. Genetic analyses have identified more than 200 LQTS2causing mutations in KCNH2. Missense mutations leading to single amino acid substitutions in hERG (human ethera-gogo-related gene) protein—the alpha subunit of the rapid delayed rectifier current (IKr)—are the predominant abnormality. At the functional level, these mutations result in a decreased IKr. 11 It has been shown that LQTS2 mutations reduce IKr through a variety of mechanisms including aberrant hERG protein folding, trafficking, or ion-channel gating. Although not all identified mutations have been investigated, the majority of the mutations produce failure of the hERG subunits to exit the endoplasmic

Background— Blockade of inward-rectifier K + channels by chloroquine terminates reentry in cholinergic atrial fibrillation (AF). However, it is unknown whether inward-rectifier K + channels and reentry are also important in maintaining stretch-induced AF (SAF). We surmised that reentry underlies SAF, and that abolishing reentry with chloroquine terminates SAF more effectively than traditional Na + -channel blockade by flecainide. Methods and Results— Thirty Langendorff-perfused sheep hearts were exposed to acute and continuous atrial stretch, and mapped optically and electrically. AF dynamics were studied under control and during perfusion of either chloroquine (4 µmol/L, n=7) or flecainide (2–4 µmol/L, n=5). Chloroquine increased rotor core size and decreased reentry frequency from 10.6 ± 0.7 Hz in control to 6.3 ± 0.7 Hz ( P Kr blockade by E-4031 (n=7) did not terminate AF when frequency values were >8 Hz. During pacing (n=11), flecainide reversibly reduced conduction velocity (≈30% at cycle length 300, 250, and 200 ms; P P P Conclusions— Chloroquine is more effective than flecainide in terminating SAF in isolated sheep hearts by significantly increasing core size and decreasing reentry frequency. Chloroquine’s effectiveness may be explained by its inward-rectifier K + channel blockade profile and suggest that reentry is important to maintain acute SAF.

This Document is Protected by copyright and was first published by Frontiers. All rights reserved. it is reproduced with permission, Atrial fibrillation (AF) is the most common sustained arrhythmia encountered in clinical practice with growing prevalence in developed countries. Several medical and interventional therapies, such as atrial specific drugs and pulmonary vein isolation, have demonstrated prevention of recurrences. However, their suboptimal long-term success and significant rate of secondary effects have led to intensive research in the last decade focused on novel alternative and supplemental therapies. One such candidate is polyunsaturated fatty acids (PUFAs). Because of their biological properties, safety, simplicity, and relatively cheap cost, there is a special clinical interest in omega-3 PUFAs as a possible antiarrhythmic agent. Obtained from diets rich in fish, they represent one of the current supplemental therapies. At the cellular level, an increasing body of evidence has shown that n-3 PUFAs exert a variety of effects on cardiac ion channels, membrane dynamic properties, inflammatory cascade, and other targets related to AF prevention. In this article, we review the current basic and clinical evidence pertinent to n-3 PUFAs in AF treatment and prevention.We also discuss controversial outcomes among clinical studies and propose specific subsets of AF patients who will benefit most from n-3 PUFAs, NHLBI Grant K99-HL105574 to SFN and the Alfonso Martín Escudero Foundation Grant to DFR