Arrhythmias In Mice Research Articles

A New target of ischemic ventricular arrhythmias-ITFG2.

ITFG2 is an intracellular protein known to modulate the immune response of T-cells. Our previous investigation revealed that ITFG2 specifically targets ATP5b to regulate ATP energy metabolism and maintain mitochondrial function, thereby protecting the heart from ischemic injury. However, the role of ITFG2 in ischemic ventricular arrhythmias and its underlying mechanisms have not been previously reported. In this study, we found ITFG2 overexpression, induced by an adeno-associated virus serotype 9 vector, partially reduced the incidence of ischemic ventricular arrhythmias and shortened the duration of ventricular arrhythmias in mice after myocardial infarction. Conversely, shRNA-mediated knockdown of endogenous ITFG2 aggravated ischemic ventricular arrhythmias. ITFG2 overexpression also shortened the prolonged QRS complex and increased the epicardial conduction velocity in MI mice. Additionally, the hearts from ITFG2 overexpression mice exhibited a higher maximal upstroke velocity at phase 0 of transmembrane action potential compared to MI mice. Patch-clamp analyses demonstrated a 50% increase in the peak current of voltage-dependent Na+ channel by ITFG2 overexpression in isolated ventricular cardiomyocytes post MI. In cultured neonatal mouse cardiomyocytes under hypoxic conditions, ITFG2 up-regulated Nav1.5 protein expression by inhibiting its ubiquitination. Co-immunoprecipitation experiments showed that ITFG2 reduces the binding affinity between NEDD4-2 and Nav1.5, thereby inhibiting Nav1.5 ubiquitination. Taken together, our data highlight the critical role of ITFG2 in reducing susceptibility to ischemic ventricular arrhythmias by down-regulating Nav1.5 ubiquitination. These findings suggest that ITFG2 may serve as a novel target for treating ischemic ventricular arrhythmias.

Inositol 1,4,5-Trisphosphate Receptor 1 Gain-of-Function Increases the Risk for Cardiac Arrhythmias in Mice and Humans.

Ca2+ mishandling in cardiac Purkinje cells is a well-known cause of cardiac arrhythmias. The Purkinje cell resident inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is believed to play an important role in Ca2+ handling, and ITPR1 gain-of-function (GOF) has been implicated in cardiac arrhythmias. However, nearly all known disease-associated ITPR1 variants are loss-of-function and are primarily linked to neurological disorders. Whether ITPR1 GOF has pathological consequences, such as cardiac arrhythmias, is unclear. This study aimed to identify human ITPR1 GOF variants and determine the impact of ITPR1 GOF on Ca2+ handling and arrhythmia susceptibility. There are a large number of rare ITPR1 missense variants reported in open data repositories. Based on their locations in the ITPR1 channel structure, we selected and characterized 33 human ITPR1 missense variants from open databases and identified 21 human ITPR1 GOF variants. We generated a mouse model carrying a human ITPR1 GOF variant, ITPR1-W1457G (W1447G in mice). We showed that the ITPR1-W1447G± and recently reported ITPR1-D2594K± GOF mutant mice were susceptible to stress-induced ventricular arrhythmias. Confocal Ca2+ and voltage imaging in situ in heart slices and Ca2+ imaging and patch-clamp recordings of isolated Purkinje cells showed that ITPR1-W1447G± and ITPR1-D2594K± variants increased the occurrence of stress-induced spontaneous Ca2+ release, delayed afterdepolarization, and triggered activity in Purkinje cells. To assess the potential role of ITPR1 variants in arrhythmia susceptibility in humans, we looked up a gene-based association study in the UK Biobank data set and identified 7 rare ITPR1 missense variants showing potential association with cardiac arrhythmias. Remarkably, in vitro functional characterization revealed that all these 7 ITPR1 variants resulted in GOF. Our studies in mice and humans reveal that enhanced function of ITPR1, a well-known movement disorder gene, increases the risk for cardiac arrhythmias.

Decreased ability to manage increases in reactive oxygen species may underlie susceptibility to arrhythmias in mice lacking Scn1b.

Scn1b plays essential roles in the heart, where it encodes β1-subunits that serve as modifiers of gene expression, cell surface channel activity, and cardiac conductivity. Reduced β1 function is linked to electrical instability in various diseases with cardiac manifestations and increased susceptibility to arrhythmias. Recently, we demonstrated that loss of Scn1b in mice leads to compromised mitochondria energetics and reactive oxygen species (ROS) production. In this study, we examined the link between increased ROS and arrhythmia susceptibility in Scn1b-/- mice. In addition, ROS-scavenging capacity can be overwhelmed during prolonged oxidative stress, increasing arrhythmia susceptibility. Therefore, we isolated whole hearts and cardiomyocytes from Scn1b-/- and Scn1b+/+ mice and subjected them to an oxidative challenge with diamide, a glutathione oxidant. Next, we analyzed gene expression and activity of antioxidant enzymes in Scn1b-/- hearts. Cells isolated from Scn1b-/- hearts died faster and displayed higher rates of ROS accumulation preceding cell death compared with those from Scn1b+/+. Furthermore, Scn1b-/- hearts showed higher arrhythmia scores and spent less time free of arrhythmia. Lastly, we found that protein expression and enzymatic activity of glutathione peroxidase is increased in Scn1b-/- hearts compared with wild type. Our results indicate that Scn1b-/- mice have decreased capability to manage ROS during prolonged oxidative stress. ROS accumulation is elevated and appears to overwhelm ROS scavenging through the glutathione system. This imbalance creates the potential for altered cell energetics that may underlie increased susceptibility to arrhythmias or other adverse cardiac outcomes.NEW & NOTEWORTHY Using an oxidative challenge, we demonstrated that isolated cells from Scn1b-/- mice are more susceptible to cell death and surges in reactive oxygen species accumulation. At the whole organ level, they were also more susceptible to the formation of cardiac arrhythmias. This may in part be due to changes to the glutathione antioxidant system.

The Kv4 potassium channel modulator NS5806 attenuates cardiac hypertrophy in vivo and in vitro

The compound NS5806 is a Kv4 channel modulator. This study investigated the chronic effects of NS5806 on cardiac hypertrophy induced by transverse aortic constriction (TAC) in mice in vivo and on neonatal rat ventricular cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) in vitro. Four weeks after TAC, NS5806 was administered by gavage for 4 weeks. Echocardiograms revealed pronounced left ventricular (LV) hypertrophy in TAC-treated mice compared with sham mice. NS5806 attenuated LV hypertrophy, as manifested by the restoration of LV wall thickness and weight and the reversal of contractile dysfunction in TAC-treated mice. NS5806 also blunted the TAC-induced increases in the expression of cardiac hypertrophic and fibrotic genes, including ANP, BNP and TGF-β. Electrophysiological recordings revealed a significant prolongation of action potential duration and QT intervals, accompanied by an increase in susceptibility to ventricular arrhythmias in mice with cardiac hypertrophy. However, NS5806 restored these alterations in electrical parameters and thus reduced the incidence of mouse sudden death. Furthermore, NS5806 abrogated the downregulation of the Kv4 protein in the hypertrophic myocardium but did not influence the reduction in Kv4 mRNA expression. In addition, NS5806 suppressed in vitro cardiomyocyte hypertrophy. The results provide novel insight for further ion channel modulator development as a potential treatment option for cardiac hypertrophy.

Increased GIRK channel activity prevents arrhythmia in mice with heart failure by enhancing ventricular repolarization

Ventricular arrhythmia causing sudden cardiac death is the leading mode of death in patients with heart failure. Yet, the mechanisms that prevent ventricular arrhythmias in heart failure are not well characterized. Using a mouse model of heart failure created by transverse aorta constriction, we show that GIRK channel, an important regulator of cardiac action potentials, is constitutively active in failing ventricles in contrast to normal cells. Evidence is presented indicating that the tonic activation of M2 muscarinic acetylcholine receptors by endogenously released acetylcholine contributes to the constitutive GIRK activity. This constitutive GIRK activity prevents the action potential prolongation in heart failure ventricles. Consistently, GIRK channel blockade with tertiapin-Q induces QT interval prolongation and increases the incidence of arrhythmia in heart failure, but not in control mice. These results suggest that constitutive GIRK channels comprise a key mechanism to protect against arrhythmia by providing repolarizing currents in heart failure ventricles.

Abstract 19102: E-Cigarettes Induce Persistent Cardiac Autonomic Dysfunction and Molecular Signatures of Dilated and Arrhythmogenic Cardiomyopathy

Introduction: Evidence is mounting that use of e-cigarettes (e-cigs) promotes cardiopulmonary injury and dysfunction, but the long-term consequences remain unclear. Consumption of menthol e-cigs has surged amid imminent bans on combustible menthol cigarettes and cigars. Recently, we found e-cig aerosols with menthol (but not tobacco) flavor acutely induced spontaneous ventricular arrhythmias in mice. Hypothesis: Menthol e-cigs induces persistent cardiac autonomic imbalance. Methods: Adult C57BL/6J male mice with electrocardiogram (ECG) and blood pressure (BP) telemeters were exposed for 4 weeks to menthol e-cig aerosols (JUUL, 180 puffs/day, 5 days/week; n=8) or filtered air (Air; n=7) and challenged with restraint stress to determine cardiovascular reactivity to stress. Hearts (n=5/group) were collected immediately after final exposure for phosphoproteomics. Results: Menthol e-cig exposures increased HR, decreased HR variability, and evoked ventricular premature beats only on exposure day 1, but consistently increased BP throughout the 20-day exposure (+16 mmHg systolic BP vs. Air). Additional exposures in females (n=4) recapitulated these acute effects. Pre-treatment with atenolol (n=4 males) abolished e-cig-induced arrhythmias, suggesting β 1 -adrenergic mediation of e-cig-induced arrhythmias. After 4-week JUUL Menthol exposure, mice had basal sinus bradycardia and impaired stress reactivity, characterized by reduced maximal HR and rapid recovery to baseline. All three effects persisted up to 3 weeks following cessation and were accompanied by prolonged ventricular repolarization (QT c ). Phosphoproteomic analysis revealed upregulation of protein kinase A signaling, and mitochondrial dysfunction in e-cig-exposed mice. Phosphoproteins associated with dilated & arrhythmogenic cardiomyopathy (ATP2A2, FN1, CACNB2, PFKM, ADCY6, and PRKAR1A) were differentially regulated in e-cig-exposed mice. Conclusions: We demonstrate that menthol e-cigs induce persistent cardiac autonomic imbalance associated with molecular signatures of dilated & arrhythmogenic cardiomyopathy. Autonomic imbalance and cardiac electrical dysfunction may contribute to the long-term health effects of repeated use of e-cigarettes.

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

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

An Innovative Toolkit to Investigate the Complex Mechanisms of Cardiac Arrhythmias

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

Maresin1 ameliorates ventricular remodelling and arrhythmia in mice models of myocardial infarction via NRF2/HO-1 and TLR4/NF-kB signalling.

Ventricular remodelling and arrhythmias are the main factors that affect the quality of life of patients with myocardial infarction (MI). Maresin 1 (Mar1) is associated with antioxidative and anti-inflammatory effects. However, the mechanisms underlying the effects of Mar1 in MI remain unclear. In this study, we aimed to explore the role and potential mechanisms of Mar1 in a mouse model of MI. The mice were divided into four groups: Sham, Sham+Mar1, MI, and MI+Mar1. In the MI groups, the left anterior descending coronary artery of the mice was ligated for 28days, while this ligation was not conducted in the Sham groups. Mar1 was injected into mice in the Sham+Mar1 and MI+Mar1 groups. H9c2 cells were cultured in vitro under hypoxic conditions for MI models, and then Mar1 was added to the medium for 24h. Our data demonstrated that Mar1 activated NRF2/HO-1 signalling and inhibited TLR4/NF-kB signalling in MI. These activities lead to inhibition of the release of inflammatory cytokines, reduction of myocardial apoptosis and interstitial fibrosis, decreased susceptibility to ventricular arrhythmias, and improved cardiac function. Similarly, our in vitro analyses showed that Mar1 inhibited inflammatory signalling by enhancing the antioxidative function of NRF2/HO-1 signalling. Furthermore, Mar1 inhibited hypoxia-activated apoptosis in cardiomyocytes. Taken together, our data demonstrate that Mar1 ameliorates ventricular remodelling and arrhythmias in mice post-MI via the activation of NRF2/HO-1 signalling and inhibition of the TLR4/NF-kB signalling pathways.

Effect of sotagliflozin on ventricular arrhythmias in mice with myocardial infraction

BackgroundVentricular arrhythmias (VAs) after myocardial infarction (MI) are an important cause of death in patients. Inhibition of sodium-glucose ligand transporter type 2 (SGLT-2) has been shown to reduce cardiovascular mortality in myocardial infarction independent of glycemic sham and prevent ventricular arrhythmias, and it is unclear whether these effects can be further enhanced by additional SGLT-1 inhibition. We investigated the effects of chronic treatment with the dual SGLT-1 and 2 inhibitor sotagliflozin (Sto) on VAs and their underlying mechanisms after MI. MethodsMI was induced by ligation of the left anterior coronary artery in 6–8 weeks-old male mice, which were fed Sto at a dose of 30 mg/kg/day for 4 weeks after surgery. The effects were assessed by echocardiography, histopathology, Langendorff Heart Perfusion System, ambulatory electrocardiography, and Western blot. In vitro experiments were performed to assess the effect of Sto on cardiomyocytes subjected to hypoxic. ResultsCompared to the MI group, Sto treatment significantly improved cardiac function and reduced infarct size and fibrosis levels. Sto also decreased action potential duration (APD) and APD alternation thresholds. Ambulatory ECG and BURST stimulation showed that Sto reduced the incidence of VAs. In addition, Sto significantly improved the expression levels of ion channels and CX43 and ameliorated the abnormal expression of Ca2+ handling proteins after MI. Mechanistically, in vivo and in vitro experiments confirmed that TLR4/CaMKII activation was enhanced after MI, while Sto significantly inhibited the activation of the TLR4/CaMKII signaling pathway. ConclusionSto improved LV remodeling and reduced the incidence of VAs after MI, which was regulated by modulating the TLR4/CaMKII signaling pathway.

Stimulation of the mitochondrial calcium uniporter mitigates chronic heart failure-associated ventricular arrhythmia in mice.

An aberrant increase in the diastolic calcium concentration ([Ca2+]i) level is a hallmark of heart failure (HF) and the cause of delayed afterdepolarization and ventricular arrhythmia (VA). Although mitochondria play a role in regulating [Ca2+]i, whether they can compensate for the [Ca2+]i abnormality in ventricular myocytes is unknown. The purpose of this study was to investigate whether enhanced Ca2+ uptake of mitochondria may compensate for an abnormal increase in the [Ca2+]i of ventricular myocytes in HF to effectively mitigate VA. We used a HF mouse model in which myocardial infarction was induced by permanent left anterior descending coronary artery ligation. The mitochondrial Ca2+ uniporter was stimulated by kaempferol. Ca2+ dynamics and membrane potential were measured using an epifluorescence microscope, a confocal microscope, and the perforated patch-clamp technique. VA was induced in Langendorff-perfused hearts, and hemodynamic parameters were measured using a microtip transducer catheter. Protein expression of the mitochondrial Ca2+ uniporter, as assessed by its subunit expression, did not change between HF and sham mice. Treatment of cardiomyocytes with kaempferol, isolated from HF mice 28 days after coronary ligation, reduced the appearance of aberrant diastolic [Ca2+]i waves and sparks and spontaneous action potentials. Kaempferol effectively reduced VA occurring in Langendorff-perfused hearts. Intravenous administration of kaempferol did not markedly affect left ventricular hemodynamic parameters. The effects of kaempferol in HF of mice implied that mitochondria may have the potential to compensate for abnormal [Ca2+]i. Mechanisms involved in mitochondrial Ca2+ uptake may provide novel targets for treatment of HF-associated VA.

Abstract P1084: Mature Ipsc-derived Atrial Cardiomyocytes Show That Saturated Fatty Acids Differentially Modulate Atrial Remodeling In Obesity-induced Atrial Fibrillation Compared To Mono And Poly-unsaturated Fatty Acids.

Background: Increased free fatty acids (FFA) and FA infiltration, both major hallmarks of obesity can individually modulate changes leading to AF. For example, mass spectrometry on obese mouse heart tissues have shown that saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) are all significantly increased compared to controls. The highest increase was shown to be in linoleic acid (LA), a PUFA followed by palmitic acid (PA, SFA) and oleic acid (OA, MUFA). Although reports suggest that some FAs induce ventricular arrhythmias in mice, the differential electrophysiological effects of SFAs, MUFAs, and PUFAs on the atria remain unclear. Objective: To determine the individual roles of SFAs, MUFAs, and PUFAs in mediating atrial remodeling in obesity-induced AF using mature human induced pluripotent stem-derived atrial cardiomyocytes (iPSC-aCMs). Methods: We compared iPSC-aCMs and HL-1 cells treated with bovine serum albumin (BSA) with palmitic acid (PA), oleic acid (OA) and LA cells (600 uM each) for 24 hours and 1 week. Ion channel function were assessed using optical voltage mapping and whole-cell patch clamping, and we assessed atrial remodeling using qPCR, and western blotting. Results: iPSC-aCMs treated with PA, OA, and LA displayed higher expression of FABP3 and CPT1A suggesting increased FA metabolism (Figure A-C). In HL1 cells, protein expression of Cpt1a was increased in all groups after FA treatment (Figure D). PA differentially modulated expression of Kv7.1 and NCX1 in HL1 cells (Figure E-F) and ion channel mRNA expression in iPSC-aCMs (Figure G-L). After 1 week PA and LA treatment b-adrenergic signaling was significantly increased in iPSC-aCMs while OA decreased it compared to controls (Figure M-O). Lastly, 24-hour PA, OA, LA treatment differentially modulated the atrial action potential (AP). While PA and LA significantly shorten the action potential, there was no significant changes to the AP after OA treatment. Conclusions: Using mature iPSC-aCMs, we showed for the first time that SFA, MUFA, and PUFA not only increased FA metabolism but differentially affected b-adrenergic signaling and ion channel remodeling leading to differential effects on the atrial AP. Our findings may have important implications for the management of obesity-induced AF in patients.

Pharmacological activation of exchange protein directly activated by cAMP 1 (EPAC1) increases action potential duration in mouse atrial myocytes

Atrial Fibrillation (AF) is the most common sustained cardiac arrhythmia worldwide. Several mechanisms are known to be involved in AF development and sustainability, such as abnormal Ca2+ cycling and action potential (AP) duration. Molecules involved in these mechanisms are potential relevant therapeutic candidates. One of these candidates is the Exchange Protein directly Activated by cAMP 1 (EPAC1), which has been recently shown to increase arrhythmogenic activity in ventricular cardiomyocytes and to be related to AF in an EPAC1-/- mouse model. Although strong evidence suggests that EPAC1 is involved in cardiac arrhythmia in mice, its role in AF has not been fully investigated. To investigate the effect of EPAC1 pharmacological activation in the development of atrial arrhythmogenic activity. Atrial myocytes from EPAC1-/- and Wild Type (WT) mice were dissociated by enzymatic digestion. AP were recorded using the whole cell configuration of the patch clamp technique in the current clamp mode. To assess the selective role of EPAC1, EPAC proteins were acutely activated by superfusion of 8-CPT-AM, a preferential EPAC1 activator (10 μM) and inhibited by superfusion of AM-001 compound, an EPAC1 non-competitive selective inhibitor (20 μM). Activation of EPAC1 by 8-CPT-AM drastically increased AP duration at 90% of repolarization (APD90) by 106.6 ± 14.3% (vs control; p < 0.0001) in WT atrial myocytes. This effect was attenuated by AM-001 perfusion (APD90 decreased by 38.9 ± 8.3% vs 8-CPT-AM; P < 0.05). Interestingly, EPAC1 deleted cardiomyocytes displayed a lower APD90 increase (+42.3 ± 15.1% vs control; P < 0.01) after treatment by 8-CPT-AM than that of WT atrial myocytes ( P < 0.05). Concomitant treatment of EPAC-/- cells with 8-CPT-AM and AM-001 failed to influence the effect on APD90 compared to 8-CPT-AM alone ( P = 0.85). Our results show that EPAC1 activation is involved in the modulation of AP duration in mouse atrial myocytes. In addition, our data suggest that EPAC1 could participate in AF initiation and/or sustainability by promoting an arrhythmogenic ectopic activity. Therefore, EPAC1 may constitute a therapeutic target in AF management.

PO-691-04 SATURATED FATTY ACIDS DIFFERENTIALLY MODULATE ION CHANNEL AND STRUCTURAL REMODELING IN MATURE IPSC-DERIVED ATRIAL CARDIOMYOCYTES LEADING TO OBESITY-INDUCED ATRIAL FIBRILLATION

Obesity-induced atrial fibrillation (AF) is modulated increased fatty infiltration in the atrial myocardium leading to increased exposure to free fatty acids (FFA). Mass spectrometry studies of obese mice have shown increased saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) compared to lean controls with the highest increase in the PUFA, linoleic acid. While some reports suggest that some SFAs and MUFAs induce ventricular arrhythmias in mice, the differential electrophysiological effects of SFAs, MUFAs, and PUFAs on the atria remain unclear.

Abstract 11606: Mature Ipsc-Derived Atrial Cardiomyocytes Show That Saturated Fatty Acids Differentially Modulate Ion Channel and Structural Remodeling in Obesity-Induced Atrial Fibrillation

Background: Obesity-induced atrial fibrillation (AF) is modulated in part by increased serum free fatty acids (FFA) and FA infiltration of the heart. Mass spectrometry data on obese mice have shown increased saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) compared to lean controls with the largest increase in the PUFA, linoleic acid (LA). Although reports suggest that some FAs induce ventricular arrhythmias in mice, the differential electrophysiological (EP) effects of SFAs, MUFAs, and PUFAs on the atria remain unclear. Objective: To determine if SFAs, MUFAs, and PUFAs differentially modulate atrial remodeling using mature human induced pluripotent stem-derived atrial cardiomyocytes (iPSC-aCMs) and HL1 cells. . Methods: We compared iPSC-aCMs and HL-1 cells treated with bovine serum albumin (BSA) with palmitic acid (PA), oleic acid (OA) and LA (600 uM each) for 1 week. Ion channel function was assessed using whole-cell patch clamping and we assessed β-oxidation and atrial remodeling with using ELISA, qPCR, and Western blotting. Results: iPSC-aCMs treated with PA, OA, and LA displayed higher expression of FABP3 , ANGPTL4 , and CPT1A suggesting increased FA metabolism and β-oxidation (Figure A-C). In HL1 cells, protein expression of Cpt1a was increased in all groups after FA treatment (Figure D). PA differentially modulated expression of Kv7.1 and NCX1 in HL1 cells (Figure E-F) and ion channel mRNA expression in iPSC-aCMs (Figure G-L). After 1 week PA and LA treatment β-adrenergic signaling was significantly increased in iPSC-aCMs while OA decreased it compared to controls (Figure M-O). Conclusions: Using mature iPSC-aCMs, we showed for the first time that SFA, MUFA, and PUFA not only increased FA metabolism but also differentially modulated ion channel and structural remodeling and β-adrenergic signaling in the atria. Our findings may have important implications for the management of obesity-induced AF in patients.

Transcription factor Meis1 act as a new regulator of ischemic arrhythmias in mice

IntroductionThe principal voltage-gated Na+ channel, NaV1.5 governs heart excitability and conduction. NaV1.5 dysregulation is responsible for ventricular arrhythmias and subsequent sudden cardiac death (SCD) in post-infarct hearts. The transcription factor Meis1 performs a significant role in determining differentiation fate and regenerative capability of cardiomyocytes. However, the functions of Meis1 in ischemic arrhythmias following myocardial infarction (MI) are still largely undefined. ObjectivesHere we aimed to study whether Meis1 could act as a key regulator to mediate cardiac Na+ channel and its underlying mechanisms. MethodsHeart-specific Meis1 overexpression was established by AAV9 virus injection in C57BL/6 mice. The QRS duration, the incidence of ventricular arrhythmias and cardiac conduction velocity were evaluated by ECG, programmed electrical stimulation and optical mapping techniques respectively. The conventional patch clamp technique was performed to explore the INa characteristics of isolated mouse ventricular myocytes. In vitro, Meis1 was also overexpressed in hypoxic-treated neonatal cardiomyocytes. The analysis of immunoblotting and immunofluorescence were used to detect the changes in the expression of NaV1.5 in each group. ResultsWe found that forced expression of Meis1 rescued the prolongation of QRS complex, produced anti-arrhythmic activity and improved epicardial conduction velocity in infarcted mouse hearts. In terms of mechanisms, cardiac electrophysiological changes of MI mice can be ameliorated by the recovery of Meis1, which is characterized by the restoration of INa current density and NaV1.5 expression level of cardiomyocytes in the marginal zone of MI mouse hearts. Furthermore, in vitro studies showed that Meis1 was also able to rescue hypoxia-induced decreased expression and dysfunction of NaV1.5 in ventricular myocytes. We further revealed that E3 ubiquitin ligase CDC20 led to the ubiquitination and degradation of Meis1, which blocked the transcriptional regulation of SCN5A by Meis1 and ultimately led to the electrophysiological remodeling in ischemic-hypoxic cardiomyocytes. ConclusionCDC20 mediates ubiquitination of Meis1 to govern the transcription of SCN5A and cardiac electrical conduction in mouse cardiomyocytes. This finding uncovers a new mechanism of NaV1.5 dysregulation in infarcted heart, and provides new therapeutic strategies for malignant arrhythmias and sudden cardiac death following MI.

Molecular Remodeling of Cardiac Sinus Node Associated with Acute Chagas Disease Myocarditis.

Chagas disease principally affects Latin-American people, but it currently has worldwide distribution due to migration. Death among those with Chagas disease can occur suddenly and without warning, even in those who may not have evidence of clinical or structural cardiac disease and who are younger than 60 years old. HCN4 channels, one of the principal elements responsible for pacemaker currents, are associated with cardiac fetal reprogramming and supraventricular and ventricular arrhythmias, but their role in chagasic arrhythmias is not clear. We found that a single-dose administration of ivabradine, which blocks HCN4, caused QTc and QRS enlargement and an increase in P-wave amplitude and was associated with ventricular and supraventricular arrhythmias in mice challenged with isoproterenol, a chronotropic/ionotropic positive agent. Continuous treatment with ivabradine did not alter the QTc interval, but P-wave morphology was deeply modified, generating supraventricular arrhythmias. In addition, we found that repolarization parameters improved with ivabradine treatment. These effects could have been caused by the high HCN4 expression observed in auricular and ventricular tissue in infected mice. Thus, we suggest, for the first time, that molecular remodeling by overexpression of HCN4 channels may be related to supraventricular arrhythmias in acute Chagas disease, causing ivabradine over-response. Thus, ivabradine treatment should be administered with caution, while HCN4 overexpression may be an indicator of heart failure and/or sudden death risk.

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice.

Arrhythmias are common, affecting millions of patients worldwide. Current treatment strategies are associated with significant side effects and remain ineffective in many patients. To improve patient care, novel and innovative therapeutic concepts causally targeting arrhythmia mechanisms are needed. To study the complex pathophysiology of arrhythmias, suitable animal models are necessary, and mice have been proven to be ideal model species to evaluate the genetic impact on arrhythmias, to investigate fundamental molecular and cellular mechanisms, and to identify potential therapeutic targets. Implantable telemetry devices are among the most powerful tools available to study electrophysiology in mice, allowing continuous ECG recording over a period of several months in freely moving, awake mice. However, due to the huge number of data points (>1 million QRS complexes per day), analysis of telemetry data remains challenging. This article describes a step-by-step approach to analyze ECGs and to detect arrhythmias in long-term telemetry recordings using the software, Ponemah, with its analysis modules, ECG Pro and Data Insights, developed by Data Sciences International (DSI). To analyze basic ECG parameters, such as heart rate, P wave duration, PR interval, QRS interval, or QT duration, an automated attribute analysis was performed using Ponemah to identify P, Q, and T waves within individually adjusted windows around detected R waves. Results were then manually reviewed, allowing adjustment of individual annotations. The output from the attribute-based analysis and the pattern recognition analysis was then used by the Data Insights module to detect arrhythmias. This module allows an automatic screening for individually defined arrhythmias within the recording, followed by a manual review of suspected arrhythmia episodes. The article briefly discusses challenges in recording and detecting ECG signals, suggests strategies to improve data quality, and provides representative recordings of arrhythmias detected in mice using the approach described above.

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Analyzing Long-Term Electrocardiography Recordings to Detect Arrhythmias in Mice

Early onset epilepsy and sudden unexpected death in epilepsy with cardiac arrhythmia in mice carrying the early infantile epileptic encephalopathy 47 gain-of-function FHF1(FGF12) missense mutation.

Fibroblast growth factor homologous factors (FHFs) are brain and cardiac sodium channel-binding proteins that modulate channel density and inactivation gating. A recurrent de novo gain-of-function missense mutation in the FHF1(FGF12) gene (p.Arg52His) is associated with early infantile epileptic encephalopathy 47 (EIEE47; Online Mendelian Inheritance in Man database 617166). To determine whether the FHF1 missense mutation is sufficient to cause EIEE and to establish an animal model for EIEE47, we sought to engineer this mutation into mice. The Arg52His mutation was introduced into fertilized eggs by CRISPR (clustered regularly interspaced short palindromic repeats) editing to generate Fhf1R52H/F+ mice. Spontaneous epileptiform events in Fhf1R52H/+ mice were assessed by cortical electroencephalography (EEG) and video monitoring. Basal heart rhythm and seizure-induced arrhythmia were recorded by electrocardiography. Modulation of cardiac sodium channel inactivation by FHF1BR52H protein was assayed by voltage-clamp recordings of FHF-deficient mouse cardiomyocytes infected with adenoviruses expressing wild-type FHF1B or FHF1BR52H protein. All Fhf1R52H/+ mice experienced seizure or seizurelike episodes with lethal ending between 12 and 26days of age. EEG recordings in 19-20-day-old mice confirmed sudden unexpected death in epilepsy (SUDEP) as severe tonic seizures immediately preceding loss of brain activity and death. Within 2-53s after lethal seizure onset, heart rate abruptly declined from 572±16bpm to 108±15bpm, suggesting a parasympathetic surge accompanying seizures that may have contributed to SUDEP. Although ectopic overexpression of FHF1BR52H in cardiomyocytes induced a 15-mV depolarizing shift in voltage of steady-state sodium channel inactivation and slowed the rate of channel inactivation, heart rhythm was normal in Fhf1R52H/+ mice prior to seizure. The Fhf1 missense mutation p.Arg52His induces epileptic encephalopathy with full penetrance in mice. Both Fhf1 (p.Arg52His) and Scn8a (p.Asn1768Asp) missense mutations enhance sodium channel Nav 1.6 currents and induce SUDEP with bradycardia in mice, suggesting an FHF1/Nav 1.6 functional axis underlying altered brain sodium channel gating in epileptic encephalopathy.