Basic Science

AP19‐­00033

Cardiac arrhythmias during epilepsy linked to alterations of the adrenergic regulation of the cardiac sodium current

Robert Dumaine

Université de Sherbrooke, Canada

Introduction:

Evidence indicate that cardiac arrhythmias are involved in sudden death during epilepsy (SUDEP) and Dravet Syndrome and suggest that expression of non‐­cardiac sodium channels (neuronal) in the heart contributes to them. We will present an overview of the latest developments on the role of these channels to arrhythmia linked to epilepsy and non‐­cardiac diseases.

Methods:

We used a rat model of temporal lobe epilepsy (the most common form) to study how expression of neuronal sodium channels in the heart may alter the cardiac adrenergic response of the sodium current (INa) during epilepsy. We used the patch clamp technique to measure INa response to isoproterenol in acutely dissociated ventricular and atrial cells.

Result:

Epilepsy triggered expression of neuronal sodium channels in the heart. This overexpression of non‐­cardiac sodium channels increased the late sodium current by more than 50% and the sensitivity of the cardiac sodium current INa to isoproterenol. As a consequence, the duration of the ventricular action potential was prolonged by 40% during epilepsy. These changes are likely to alter cardiac conduction. Moreover, prolongation of the action potential duration is likely to result in increase the QT interval on the electrocardiogram at rest but also during an epileptic ictus. Long QT interval is a well known trigger of Torsade de pointes arrhythmias.

Conclusion:

Our results provide a basis to explain the QT prolongation and the conduction problems observed in epileptic patients and suggest that their heart may be prone to develop arrhythmia during adrenergic modulations commonly observed during seizures. Our data provide a potential link between alterations of INa, arrhythmia during epilepsy and SUDEP.

AP19‐­00047

In silico prediction of the effects of ethanol on cardiac cellular electrophysiology and reentrant arrhythmias

Henry Sutanto, Markéta Bébarová, Dobromir Dobrev, Paul Volders, Jordi Heijman

CARIM School for Cardiovascular Diseases, Maastricht University, Netherlands

Introduction:

Acute and chronic alcohol consumption alter cardiac electrophysiology and may promote arrhythmias, notably atrial fibrillation (AF). However, the underlying mechanisms and interaction between ethanol‐­induced and AF‐­related proarrhythmic remodeling remain incompletely understood. Here, we employ computational modeling to integrate recent experimental data about the acute effects of ethanol and study proarrhythmic consequences in the ventricles, and in the atria with and without AF‐­related remodeling.

Methods:

Multi‐­scale simulations were performed in Myokit using the Courtemanche human atrial and Passini human ventricular models. To simulate the effects of ethanol in long‐­standing persistent (‘chronic’) AF (cAF), a cAF version of the human atrial model with electrical remodeling of cardiac ion channels was implemented. Acute electrophysiological effects of ethanol were incorporated in all three models based on previously published experimental data: reduced INa, ICa, L, IKr and Ito, and dual effects on IK1 (inhibition at low concentrations, augmentation at high concentrations; Figure A). The potential proarrhythmic effect of ethanol was investigated at the cellular and tissue level. Reentry was simulated using an S1S2 induction protocol in homogeneous tissue of 8 × 8 cm (400 × 400 units).

Result:

Simulated application of 0.8, 80 and 400 mmol/L ethanol had distinct effects on action potential duration (APD) and resting membrane potential (RMP) in human atrial and ventricular cardiomyocyte models (Figure B). The lowest concentration of ethanol (0.8 mmol/L) prolonged APD by ˜5% in both control and cAF models and depolarized the RMP in the control atrial model, but had no effect on ventricular APD or RMP. However, 80 and 400 mmol/L ethanol significantly reduced atrial APD and hyperpolarized RMP, particularly in the control atrial model, while significantly prolonging ventricular APD (Figure B). At the tissue level, 0.8 mmol/L ethanol slightly increased conduction velocity (CV) while shifting the vulnerable window (WoV) to the right in the control atrial model (Figure C‐­E), but did not affect reentry in the ventricle (Figure F‐­H). By contrast, 80 mmol/L ethanol slightly reduced CV, shifted the vulnerable window to the left and prolonged the duration of reentry in the atria, but reduced the vulnerable window in the ventricle. The cAF model showed a large vulnerable window with unstable reentry and reentry duration was prolonged by ethanol (Figure I‐­K).

Conclusion:

Our simulations suggest that ethanol has concentration‐­dependent electrophysiological effects that differ between atria and ventricles, and in the absence or presence of AF‐­related remodeling. Low concentrations of ethanol could have anti‐­AF effects whereas moderate‐­ and high‐­concentrations may promote AF. These findings facilitate a better understanding of the complex effects of alcohol consumption on cardiac electrophysiology.

AP19‐­00057

A computational framework facilitating analyses of fundamental cellular electrophysiological features of clinically‐­used antiarrhythmic drugs

Henry Sutanto, Lian Laudy, Michael Clerx, Dobromir Dobrev, Harry Crijns, Jordi Heijman

CARIM School for Cardiovascular Diseases, Maastricht University, Netherlands

Introduction:

Cardiac arrhythmias remain a major cause of death and disability worldwide. Despite the improved understanding of arrhythmia mechanisms, progress in the development of new antiarrhythmic drugs (AADs) has been limited and clinical application of currently available AADs remains suboptimal, likely in large part due to the incomplete understanding of the complex mechanisms‐­ of‐­action of AADs. Here, we present a novel user‐­friendly computational framework to facilitate a better understanding of AADs (the Maastricht Antiarrhythmic Drug Evaluator; MANTA).

Methods:

MANTA integrates widely‐­used computational cardiomyocyte models of different species (mouse, guinea‐­pig, rabbit, dog, human), regions (atrial, ventricular, purkinje) and disease conditions (atrial fibrillation‐­ and heart failure‐­related remodeling; Figure A). It enables simulations of the effects of clinically available AADs (Vaughan‐­William Classes I, III, IV and multi‐­channel blockers) on action potential (AP) properties and the occurrence of proarrhythmic effects such as early‐­afterdepolarizations. AAD effects were simulated based on published IC50 values for each cardiac ion channel and by integrating state‐­dependent block of INa by Class I AADs using a Markov‐­model approach in all cardiomyocyte models (Figure B‐­C).

Result:

Markov model parameters were optimized to replicate published INa characteristics (voltage‐­ dependent activation, inactivation, recovery from inactivation) and AP upstroke velocity in all cardiomyocyte models and reproduced experimental use‐­dependent onset and recovery of INa inhibition by flecainide, lidocaine and vernakalant. MANTA provides a user‐­friendly graphical user interface (Figure C) allowing users to select and compare different AADs, concentrations, and experimental conditions (rate, electrolyte concentrations). Using MANTA, we characterized important species‐­, rate‐­, and condition‐­specific AAD effects, including 1) a stronger effect of Class III AADs in large mammals than in rodents (Figure D1); 2) a frequency‐­dependent decrease in upstroke velocity with Class I AADs and reverse use‐­dependence of Class III AADs (Figure D2); 3) ventricular‐­predominant effects of pure IKr blockers and preferential reduction in atrial AP upstroke velocity with vernakalant; and 4) excessive AP prolongation with Class III AADs during hypokalemia (Figure D3).

Conclusion:

The effects of AADs are complex and highly dependent on the experimental or clinical conditions. MANTA is user‐­friendly, freely available framework able to reproduce a wide range of AAD characteristics that enables analyses of the underlying ionic mechanisms. Use of MANTA is expected to improve understanding of AAD effects on cellular electrophysiology under a wide range of conditions, which can provide clinically‐­relevant information on the safety and efficacy of AAD treatment.

AP19‐­00085

Angiotensin converting enzyme insertion/deletion gene polymorphism is associated with acquired sick sinus syndrome via linking a higher serum protein level

Jan‐Yow Chen, Ying‐Ming Liou, Kuan‐Cheng Chang

China Medical University Hospital, Taiwan

Introduction:

The replacement of nodal tissue with fibrotic tissue has been reported to play an essential role in the pathogenesis of acquired sick sinus syndrome (SSS). The activated atrial renin‐­angiotensin system (RAS) has been linked to atrial fibrosis and modification of ion channels, all of which can contribute to the abnormal pacemaker function of the sinus node. Angiotensin converting enzyme gene insertion/deletion (ACE I/D) polymorphism has been shown to modulate the expression of RAS genes. However, it remains to be determined whether ACE is involved in the pathogenesis of acquired SSS.

Methods:

The study included 110 SSS patients and 124 controls (matched for age and gender). Genotypes of the ACE I/D gene polymorphism were determined using the polymerase chain reaction‐­ fragment length polymorphism assay, followed by an association study. An enzyme‐­linked immunosorbent assay was used to determine the serum level of ACE.

Result:

The association study indicated that the ACE I/D polymorphism is linked to acquired SSS. Compared with the controls, the acquired SSS patients had a significantly higher frequency of the D dominant ID/DD genotypes than the controls (79/110 vs 62/124, OR 2.548, CI 1.478‐­4.393, P = .001). In addition, the D allele frequency was significantly higher in the SSS group than in the controls (87/220 vs 74/248, OR 1.538, CI 1.048‐­2.257, P = .028). Consistently, the ID/DD genotypes showed a higher ACE serum level than the II genotype (4.25 ± 2.50 vs 2.71 ± 1.76 ng/mL, P = .028) in controls.

Conclusion:

The ACE I/D gene polymorphism, correlated with a higher serum ACE level, is associated with the disease susceptibility to acquired SSS.

AP19‐­00093

Drug assessments using atrial cardiomyocytes generated from human induced pluripotent stem cells under chemically defined, albumin‐­free conditions

Chang Cui, Jiayi Huang, Yongping Lin, Minglong Chen

First Affiliated Hospital of Nanjing Medical University, China

Introduction:

Generation of homogeneous populations of subtype‐­specific cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) is crucial for studying the mechanisms underline atrial/ventricular cardiomyopathies and precisely developing chamber‐­specific drugs. The purpose of this study was to optimize a protocol to induce the differentiation of atrial cardiomyocytes with chemically defined and albumin‐­free conditions and test them in drug assessments.

Methods:

A novel chemically defined and albumin‐­free condition called 3F was applied for cardiac differentiation. Flow cytometry analyses, immunofluorescence, real time RT‐­PCR, and patch clamp were conducted to prove the phenotype of atrial‐­like and ventricular‐­like hiPSC‐­CMs generated though manipulating the RA pathway. The calcium transient waves and the force of engineered cardiac tissues (ECTs) were monitored to evaluate the drug responses to isoprenaline and nifedipine.

Result:

Molecular and electrophysiological characterizations of the derived cardiomyocytes revealed that RA‐­treated CMs and controlled CMs highly corresponded to the atrial (aCMs) and ventricular cardiomyocytes (vCMs), in concert with gene expressions. As golden standard, results from patch clamp verified that ˜85% CMs from RA group exhibited typical atrial‐­like action potentials. During drug assessments, hiPSC‐­aCMs and hiPSC‐­vCMs exhibited totally different patterns in both single‐­cell calcium transients and ECT levels at the baseline. Meanwhile, their responses to isoprenaline or nifedipine were highly parallel, supporting their suitability for drug tests.

Conclusion:

In the present study, we introduced a method to generate homogeneous atrial cardiomyocytes from hiPSCs under chemically defined and albumin‐­free conditions. This approach will be an important complement for drug screening in vitro.

AP19‐­00094

A missense mutation in PRKAG2 as a novel cause of familial atrial cardiomyopathy with features of glycogen deposition

Chang Cui, Shaojie Chen, Hailei Liu, Minglong Chen

First Affiliated Hospital of Nanjing Medical University, China

Introduction:

Atrial cardiomyopathy (ACM) is a novel subtype of cardiomyopathy characterized by atrial lesions which are associated with significant atrial arrhythmias. Rarely, heritable forms of ACM have been reported, and underlying mechanisms remain unknown. Herein, a 3‐­generation family affected by ACM with histories of syncope, atrial tachycardia, embolic events and pace maker implantations was enrolled.

Methods:

We carried out whole genome sequencing with linkage analysis in 3 affected members. The atrium samples were obtained from the proband via surgical intervention. Control atrium biopsies came from a patient with congenital heart disease. Comparative histology, transmission electron microscopy and western blot analyses were carried out to explore the pathogenesis in this ACM pedigree. Human induced pluripotent stem cell‐­derived atrial cardiomyocytes were transfected with adenovirus carrying the same mutation. Quantitative structural analyses were used to define the functional disturbances of the mutation.

Result:

Exome sequencing identified a missense mutation c.905G > A (R302Q) in the gene that encodes the gamma2 regulatory subunit of AMP‐­activated protein kinase (PRKAG2). Compared to control, PRKAG2‐­R302Q atria displayed disordered myofibrils, profound fibrosis and extensive glycogen deposition. Overexpressing PRKAG2‐­R302Q mutation in hiPSC‐­derived atrial cardiomyocytes resulted in significantly increased glycogen deposition and apoptosis ratio. Investigation of the protein expression levels revealed that PRKAG2‐­R302Q mutation led to increased AMPK activities.

Conclusion:

We have identified a variant in PRKAG2 as pathogenic variant for familial ACM. It demonstrated that PRKAG2‐­R302Q mutation could lead to glycogen deposition and further atrial lesions via AMPK pathway. These results improved our understanding of the molecular basis of ACM. Pedigrees considered to be relevant for ACM can be identified by the approach presented here.

AP19‐­00104

Therapeutic potentials of miRNA‐­21 regulation by peripheral blood derived‐­exosomes in myocardial infarction

Ji‐Young Kang, Hyoeun Kim, Dasom Mun, Nuri Yun, Boyoung Joung

Yonsei University College of Medicine, South Korea

Introduction:

Myocardial infarction (MI) is a public health problem and a leading cause of mortality globally. Due to insufficient current treatments, it is essential to develop effective alternatives for treating MI. Recently, microRNAs (miRNAs), a class of small non‐­coding RNAs that control gene expression of targeted mRNAs, have been explored in treating cardiac diseases. However, insufficient stability and poor cellular uptake in vivo restrict miRNAs’ clinical application. Lately, exosomes have emerged as a promising drug delivery system with high therapeutic efficacy, but quantity may constitute major issues. As human peripheral blood is easily obtained, the use of human peripheral blood‑derived exosomes as a miRNAs delivery system, has substantial potential as a therapeutic tool. Thus, this study investigated miRNA‐­21 regulation by human peripheral blood‐­derived exosomes may provide as an effective regulator for the treatment of MI.

Methods:

We conducted a bioinformatics analysis and performed luciferase assay to confirm whether a direct target of miRNA‐­21 or not. To devise an effective strategy for clinical application with therapeutic miRNAs, we isolated exosomes from human peripheral blood using the Exoquick exosome precipitation kit (System Biosciences). Transmission electron microscopy (TEM), nanoparticle trafficking analysis (NTA) and western blotting were used to characterize the isolated exosomes. We then assessed the uptake and distribution of exosomes in vivo. For experimental mouse MI, the left anterior descending (LAD) artery ligation was done with a 6‐­0 silk suture.

Result:

The detection of morphology, size distribution and protein markers of typical exosomes indicated that the exosomes were successfully isolated from human peripheral blood. The PKH26 fluorescence became concentrated in the heart, observed using an in vivo imaging system (IVIS). In addition, PKH26‐­labeled exosomes co‐­localized with cTnI+ cells, suggesting an efficient in vivo uptake of the exosomes by cardiomyocytes. After validating our exosomes platform's potential utility in vivo, we evaluated whether miRNA‐­21 regulation by exosomes led to cardiac function recovery after MI. Mice treated with anti‐­miRNA‐­21‐­loaded exosomes significantly reduced infarct size (P < .001), and improved survival rate with the augmentation of cardiac function as measured by echocardiography. In contrast, mice treated with miRNA‐­21‐­loaded exosomes completely reversed the effect of anti‐­miRNA‐­21‐­loaded exosomes.

Conclusion:

In this study, we suggest that human peripheral blood‑derived exosomes function as efficient vehicles for the delivery of miRNAs, which in turn may potentially be used for the treatment of MI. Furthermore, our results show that anti‐­miRNA‐­21 exerts a cardioprotective effect through targeting novel genes, making them potential pharmacological candidates for MI treatment.

AP19‐­00182

The impact of irrigation flow rates on radiofrequency ablation lesions produced by Thermocool Smarttouch®SF & ThermoCool SmartTouch® catheters in horizontal & vertical orientation

Katsiarina Morgaenko, Syeda Noshin, William Stevenson, Nishaki Mehta

University of Virginia Health System, United States

Introduction:

The ThermoCool SmartTouch® catheter (6P) & ThermoCool SmartTouch SF® catheter (56P) are commonly used irrigated catheters with contact force (CF) sensing. Introduced in 2015, 56P catheter was designed to provide uniform surround flow cooling technology and now is the workhorse ablation catheter in several institutions. Prior studies with the 6P platform have suggested that irrigation flow rate (IFR) can impact radiofrequency ablation (RFA) lesion sizes. The comparison of the two catheters on lesion characteristics with interactions of varying IFRs, CFs & catheter orientation has not been reported.

Methods:

RFA lesions were created in strips of chicken muscle in a saline bath heated to 37°C with a 56P & 6P catheters using low flow rate (LFR) irrigation at 2 cc/min for 56P & 6P and standard flow rate (SFR) at 8 cc/min for 56P & 17 cc/min for 6P with catheter in horizontal (HO) & vertical (VO) orientations. Ablation power of 20 W was delivered for 30 seconds with a CF of 5, 10, 15 and 20 g. When coagulum or a steam‐­pop occurred, RFA was terminated & repeated at the different site under the same experimental settings.

Result:

A total of 176 lesions were analyzed using the 6P and 56P catheter. No lesions with coagulum or steam‐­pop were included in the analysis. For 56P & 6P catheters in HO & VO for power of 20W and studied CFs, lesion surface areas were significantly higher with LFR compared to SFR (P‐­values were 0.0001 & 0.0033, respectively). For 6P catheter, LFR resulted in a significant increase of lesion volume compared to SFR (P = .0078). With comparison of 56P and 6P under the same experimental settings, for studied IFRs, 6P created a deeper lesion with smaller surface areas (LFR: 0.0008 & 0.0001, SFR: 0.0015 & 0.0001, respectively). SFR resulted in lesions of a larger volume for 56P compared to 6P (P = .0008).

Conclusion:

For 56P and 6P catheters LFR created shallower with larger surface areas lesions likely from effective ionic shunting. For similar IFR & power setting among studied CFs, 56P irrigated catheter led to shallower lesions with greater surface area compared to 6P irrigated catheter. Increasing endocardial surface lesions without increasing depth is an attractive option in the thin walled posterior left atrium where collateral injury to the esophagus is a feared complication. However, deeper lesions would be advantageous in the ventricle to ensure transmurality. Therefore, the biophysics of lesion creation and the modulation of parameters are important to recognize for the different catheter platforms.

AP19‐­00186

Overexpression of the intermediate‐­conductance calcium‐­activated potassium channel (SK4) and the HCN2 channel to generate a biological pacemaker

Hongyi Zhao, Congxin Huang

Renmin Hospital of Wuhan University, China

Introduction:

Ion channels play important roles in the excitation‐­contraction coupling of cardiac myocytes. Previous studies have shown that the overexpression or activation of intermediate ‐­conductance calcium‐­activated potassium channel (SK4, encoded by KCNN4) in embryonic stem cell‐­ derived cardiomyocytes (ESC‐­CM) can significantly increase their automaticity. The mechanism underlying this effect is probably related to the activation of hyperpolarized cyclic nucleotide gated channel (HCN) channels.

Methods:

Ad‐­green fluorescent protein (GFP), Ad‐­KCNN4 and Ad‐­HCN2 recombinant adenoviruses were injected into the left ventricle of Sprague‐­Dawley (SD) rat hearts. The rats were divided into a GFP group (n = 10), a SK4 group (n = 10), a HCN2 group (n = 10) and a SK4 plus HCN2 (SK4/HCN2) group (n = 10). The isolated hearts were perfused after 5‐­7 days of gene expression, and a complete heart block model was established.

Result:

Compared with the GFP group, overexpressing SK4 alone did not increase the heart rate after establishment of a complete heart block model (98.1 ± 8.9 bpm, 96.7 ± 7.6 bpm, P > .05), The heart rates in the SK4/HCN2 (139.9 ± 21.9 bpm) and HCN2 groups (111.7 ± 5.5 bpm) were significantly increased compared with the GFP and SK4 groups, and the heart rates in the SK4/HCN2 group were significantly improved compared with the SK4 or HCN2 groups. In the HCN2 (n = 8) and the SK4/HCN2 (n = 7) groups, the shape of the spontaneous ventricular rhythm was the same as the ectopic rhythm after pacing the of the transgenically altered site. In contrast, these rhythms were different in the SK4 (n = 10) and GFP (n = 10) groups. There were no statistically significant differences in the APD alternans or ventricular arrhythmia inducibility between the four groups (all P > .05). Western blotting, PCR and immunohistochemistry showed that the expression levels of SK4 and HCN2 increased significantly at the transgene site.

Conclusion:

Biological pacemaker activity can be successfully generated by co‐­overexpression of SK4 and HCN2 without increasing the risk of ventricular arrhythmias. The overexpression of SK4 alone is insufficient to generate biological pacemaker activity. Our study provides evidence that SK4 and HCN2 combined could construct an ecotopic pacemaker, laying the groundwork for better construction biological pacing in the future.

Key words: SK4; HCN2; heart rate; pacemaker

AP19‐­00254

Acute and chronic anti‐­arrhythmogenic effect of liposome‐­encapsulated hemoglobin (HbV) on the Myocardium through improving myocardial electrical remodeling and the arrhythmogenic substrate in hemorrhagic shock‐­induced heart

Bonpei Takase, Yuko Higashimura

National Defense Medical College, Japan

Introduction:

Prolonged blood pressure <40 mm Hg in hemorrhagic shock (HS) causes irreversible heart dysfunction, “Shock Heart Syndrome” (SHS), which is associated with lethal arrhythmias (VT/VF).

Methods:

To investigate whether liposome‐­encapsulated human hemoglobin (HbV) is comparable to washed red blood cells (wRBCs) for improving arrhythmogenesis in SHS in either acute or chronic phase, optical mapping analysis (OMP), electrophysiological study (EPS) and pathological examinations were performed in Sprague‐­Dawley rat hearts, being obtained from both acute and chronic phase when each rat survived. The first, they were subjected to acute HS by withdrawing 30% of total blood volume. After HS, rats were immediately resuscitated by transfusing exactly same amount of 5% albumin (5% ALB, n = 13), HbV (n = 13), or wRBCs (n = 13). All rats in chronic phase survived at least several weeks. After excising heart, OMP and EPS were performed in Langendorff‐­perfused hearts.

Result:

In both acute and chronic phase, OMP showed tendency for abnormal conduction and significantly impaired action potential duration dispersion (APDd) in left ventricle with 5%ALB (25 ± 9/

24 ± 10 ms, Acute / Chronic phase). In contrast, myocardial conduction and APDd were substantially preserved with HbV (14 ± 3/

13 ± 5 ms) and wRBCs (14 ± 3 / 15 ± 3 ms) as shown in Figure. Sustained VT/VF was easily provoked by burst pacing stimulus to left ventricle with 5%ALB. No VT/VF was induced with HbV and wRBCs. Pathology showed myocardial structural damage characterized by worse myocardial cell damage and Connexin43 with 5%ALB, whereas it was attenuated with HbV and wRBCs in both acute and chronic phase.

Conclusion:

Ventricular structural remodeling after HS causes VT/VF in the presence of APDd. Transfusion of HbV prevents acutely and chronically VT/VF, similarly to transfusion of wRBCs, by preventing chronic electrical remodeling of APDd and preserving myocardial structures in HS‐­induced SHS.

AP19‐­00259

Transcription factor TBX18 reprograms vascular smooth muscle cells of ascending aorta into pacemaker‐­like cells

Fengyuan Wang, Congxin Wang

Renmin Hospital of Wuhan University, China

Introduction:

Biological pacemaker is aimed to find a better replacement to treat bradycardia. Transcription factor TBX18 has been successfully applied for constructing biological pacemaker. And vascular smooth muscle cells (VSMCs) of the ascending aorta and SAN originated from the second heart field. The study explored whether ascending aortic smooth muscle cells in vitro could be reprogrammed into pacemaker‐­like cells with human TBX18.

Methods:

The vascular smooth muscle cells (VSMCs) of ascending aorta were cultured by tissue block adherence. After 4‐­7 days, the cell morphology was observed under light microscope. After passaging, the cells were randomly divided into TBX18 group, GFP group and Null group. TBX18 group was transfected with adenovirus carrying TBX18 transcription factor and green fluorescent protein (GFP), and GFP group was transfected with equal amount of GFP adenovirus as empty virus. And blank group was not transfected with virus as control group (Null group). When transfected for 4 days, and then VSMCs were cocultured with neonatal rat ventricular cardiomyocytes (NRVMs) for 5 days in vitro. Three groups of transcription factors TBX3, human dwarf homeobox gene SHOX2, insulin gene enhancer binding protein 1 (Isl1), hyperpolarization‐­activated cyclic nucleotide‑gated channel 4 (HCN4), NKx2.5, Connexin 43 (Cx43) and cardiomyocyte specificity cardiac troponin I (cTnI) firstly were detected by RT‐­qPCR and Western blot after 4 days. And the expression of HCN4 protein in TBX18 group and GFP group was detected by immunofluorescence. When VSMCs were cocultured for 5 days, funny current (If current) and action potentials were detected by the whole cell patch clamp and current clamp, respectively.

Result:

The purity of vascular smooth muscle cells reached above 90% with α‐­SMA and MHC antibody. By overexpressing TBX18, the transfected VSMCs expressed high levels of TBX3, Shox2, Isl1, HCN4 and cTnI and low level of Cx 43 and NKx2.5 in both RT‐­qPCR and Western blot. The result of immunofluorescence showed that HCN4 protein (red fluorescence) in the TBX18 group was expressed and almost consistent with green fluorescent protein and cell nucleus (blue fluorescence), while the GFP group showed barely red fluorescence. In co‐­culture conditions, If current that recorded by patch clamp appeared the time and voltage dependence in TBX18 group, which the amplitude of If density was from −5.164 ± 0.662 to −0.765 ± 0.358 pA/pF (n = 14). Furthermore, the beating rate of TBX18‐­overexpressing VMSCs was faster than in other groups in co‐­culture systems (178.00 ± 7.55 bpm, P < .05), and most importantly, the transfected cells by TBX18 exhibited sinoatrial‐­like APs which do not exist in the remaining groups.

Conclusion:

Transcription factor TBX18 could reprogram vascular smooth muscle cells of ascending aorta into pacemaker‐­like cells in vitro.

AP19‐­00264

C1q/TNF‐­related protein‐­9 attenuates diabetic microangiopathy in db/db mice

Wei Li, Mingxin Liu, Yanhong Tang

武汉大学人民医院, China

Introduction:

This study was to investigate the protective effects of the adiponectin paralog CTRP9 on the diabetic microangiopathy and the underlying molecular mechanisms in db/db mice.

Methods:

Our study contained diabetic nephropathy and diabetic retinopathy. The db/db mice (12 weeks old) ,a diabetic animal model, were randomly divided into DM group (n = 20), DM+Ad‐­GFP group (n = 20), DM+Ad‐­CTRP9 group (n = 20), the non‐­diabetic db/m mice serve as control group. Adenovirus (Ad) vectors involving the green fluorescence protein (Ad‐­GFP) or full‐­length mice CTRP9 (Ad‐­CTRP9) at 3*108 (pfu) was respectively injected into db/db mice of DM+Ad‐­CTRP9 group and DM+Ad‐­GFP group via tail vein, at day 15 post injection, and the process was repeated, the db/m mice were treated with equivalent amounts of saline. We detected the biomarker of inflammation, apoptosis, oxidative stress, fibrosis in kidney and retina tissues, determined the breakdown of blood retina barrier using evens blue.

Result:

The result showed that CTRP9 was reduced in kidney and retina tissues of diabetic mice. CTRP9 suppresses the expression of interleukin‐­1 beta, tumor necrosis factor‐­alpha, monocyte chemotactic protein‐­1 and adhesion molecules in the retina of db/db mice. CTRP9 can balance the expression of pigment epithelium‐­derived factor and vascular endothelial growth factor. CTRP9 can also inhibit the activation of nuclear factor Kappa B in the retina of db/db mouse. In addition, CTRP9 can prevent the breakdown of BRB and downregulation of tight‐­junction proteins in the retina of db/db mice. In the kidney tissues ,CTRP9 ameliorate renal dysfunction and injury at the structural and functional level in diabetic mice, inhibited glomerular and tubular glycogen accumulation and fibrosis, prevented the upregulation of TGF‐­β, α‐­SMA, fibronectin, collagen IV, laminin, promoted the expression of nephrin.Moreover, CTRP9 relived hyperglycemia‐­mediated oxidative stress, reactive oxygen species level and apoptosis, inhibited the activation of MAPK/ERK/JNK signaling pathway.

Conclusion:

In conclusion, our study, for the first time, suggests that CTRP9 has a protective therapeutic effect on diabetic nephropathy and diabetic retinopathy, providing a potentially effective clinical method for the treatment of patients with diabetic microangiopathy.

AP19‐­00269

C1q/TNF‐­related protein‐­9 improves diabetes‐­induced cardiac perivascular fibrosis by inhibiting endothelial‐­to‐­mesenchymal transition

Hongyao Hu, Mingxin Liu, Wei Li, Yanhong Tang

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China, China

Introduction:

C1q/TNF‐­Related Protein‐­9 (CTRP9) attenuates adverse cardiac remodeling partially by inhibiting interstitial fibrosis, endothelial cells respond by endothelial‐­to‐­mesenchymal transition (EndMT) plays a significant role in CF of diabetes. However, little is known about CTRP9‐­associated EndMT.

Methods:

We investigated the potential role and mechanism of CTRP9 in diabetes‐­induced EndMT. After treated with Ad‐­GFP or Ad‐­CTRP9 for 4 weeks of 16‐­week‐­old db/db mice, untreated db/db mice and age‐­matched controls were sacrificed in 20 weeks. EndMT related markers, histopathological staining were detected. In vitro, high glucose (HG)‐­induced human umbilical vein endothelial cells (HUVECs) were treated with gCTRP9/short‐­hairpin CTRP9 to explore the mechanism of CTRP9 in the function of EndMT.

Result:

We observed that diabetes decreased CTRP9 expression, and promoted the progression of EndMT and aggravated collagen deposition in vivo. Whereas CTRP9 overexpression improved the process without affecting the cardiac function of diabetic mice. In vitro, HG induced EndMT and production of collagen, TGF‐­β1, CTGF, gCTRP9 ameliorated HG–induced EndMT in HUVECs Conversely, silencing CTRP9 further exacerbated HG–induced EndMT. These protective effects of CTRP9 on EndMT were likely mediated by inactivating Smad2/Smad3 and inhibiting Snail signaling pathways.

Conclusion:

Taken together, our results showed that CTRP9 alleviates diabetes‐­induced EndMT partially via Smad2/3 and Snail pathways.

AP19‐­00310

Transcription factor prrx1 promotes brown adipose‐­derived stem cells differentiation to sinus node‐­like cells

Lin Yin, Cong‐Xin Huang

Department of Cardiology, Renmin Hospital of Wuhan University, PR China, China

Introduction:

Biological pacing is the production of a specific gene or cell‐­induced cell‐­like cell by genetic engineering to construct a pacing site similar to the atrial junction to replace the original damaged pacing cell, and to obtain the desired heart rate for the patient to be able to meet the normal physiological activity. Transfecting the adenovirus overexpressing prrx1 into BADSCs, our study aimed to investigate whether overexpression of prrx1 can successfully induce the differentiation of BADSCs into sinus nodel cells and construct biological pacing.

Methods:

BADSCs of SD rats were isolated and cultured, and the cells were identified by flow cytometry when they were passaged to passages 3‐­5. The experimental groups were divided into two groups: BADSCs were transfected with empty adenovirus GFP and adenovirus prrx1 (ie, Ad‐­GFP group, Ad‐­prrx1 group). Cell morphology and fluorescence intensity were observed under fluorescence microscope. After 5‐­7 days of virus transfection, sinus node cell‐­associated pacing protein (HCN4) and ion channel (Cacnalg, encoding T‐­type calcium channel) as well as the expression levels of transcription factors (TBX18, ISL‐­1, pitx2, shox2, etc.) were detected by Western blot and RT‐­qPCR. Then immunofluorescence assay to detect whether cell co‐­expressed prrx1 with HCN4, TBX18 and ISL‐­1. Finally, whole‐­cell patch clamp technique records pacing current If.

Result:

The newly isolated cells were round, and after being attached to the wall, they were long fusiform and spirally growing. After identification by flow cytological cell surface molecules, the isolated cells showed CD90 positive and almost no CD45, indicating that BADSCs were successfully isolated from rats. Repeated experiments confirmed that the optimal MOI for adenovirus transfection of BADSCs was 100. After 5‐­7 days of transfection of adenovirus into cells, the biochemical tests showed that the mRNA levels and protein expressions of pacing‐­related factors (TBX18, ISL‐­1, HCN4, shox2, Cacnalg) in Ad‐­prrx1 group were significantly higher than those in Ad‐­GFP group. However, the expression level of pitx2 was decreased, and there was a statistical difference between the two groups (P 0.05). Immunofluorescence showed that prrx1 co‐­expressed with TBX18, ISL‐­1 and HCN4 in Ad‐­ prrx1 group, but no expression of pacing‐­related protein was found in Ad‐­GFP group. Whole cell patch clamps were able to record the If current in the experimental group and this current was blocked by 4 mmol/L CsCl.

Conclusion:

Overexpression of prrx1 can successfully induce the differentiation of BADSCs into sinus node‐­like cells with biochemical characteristics and electrophysiological characteristics.

AP19‐­00338

Cardiac‐­targeted exosome‐­mediated delivery of RAGE siRNA for the effective treatment of myocarditis

Hyoeun Kim, Dasom Mun, Jiyoung Kang, Nuri Yun, Boyoung Joung

Yonsei University College of Medicine, South Korea

Introduction:

Receptor for advanced glycation end‐­products (RAGE) is participation in proinflammatory/proapoptotic processes. Blocked the expression of RAGE via RNAi mechanism avoid deleterious effects of overwhelming inflammation. Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by blood, urine, and cultured medium of cell cultures. Recently, the organ‐­specific delivery of exosomes was improved by expressing target peptides with Lamp2 on the surface of exosomes. However, siRNA therapy using cardiac target exosomes has not been studied yet. This study evaluated whether RAGE siRNA delivery using cardiac‐­targeted exosomes can relieve myocarditis.

Methods:

We use vectors encoding LAMP2B (CTL‐­Exo) or CTP‐­LAMP2B (CTP‐­Exo) into HEK 293 cells expressing mCherry‐­CD81, an exosome marker. Exosomes were purified from culture media of HEK 293 cells by serial centrifugation followed by tangential flow filtration (TFF) system. CTL‐­Exo and CTP‐­Exo were loaded with siRNAs by Exo‐­Fect™ exosome transfection reagent, and were treated into H9C2 rat cardiomyocyte. Lipopolysaccharides (LPS) were then added to the cells to induce inflammation. And exosomes were intravenously injected into myocarditis rat. Inflammation factors of in vitro and in vivo inflammation model were identified by western blot. echocardiographic examination was also performed in rat.

Result:

The RAGE siRNA loaded exosome was still targeted to the heart. As a result, the silencing of RAGE was more efficiently achieved by CTP‐­Exo than CTL‐­Exo loaded with RAGE siRNA. In LPS induced H9C2 cell, CTP‐­Exo decreased inflammatory factors such as TNF‐­alpha and IL‐­6. Also, in myocarditis group, inflammation factors were increased, but CTP‐­Exo loaded with RAGE siRNA treated myocarditis group were relieved inflammatory response and echocardiography confirmed recovery of LVEDD, LVESD, and LVEF in the CTP‐­Exo‐­treated group. Histological examination also confirmed that inflammation was reduced in the CTP‐­Exo‐­treated group.

Conclusion:

Inflammation‐­inducing gene targeted siRNAs delivery via CTP‐­exosomes was successful. In addition, RAGE siRNA loaded CTP‐­Exo decreased inflammation level both in vitro and in vivo disease model. The results suggested that CTP‐­exosome might be used as a therapeutic tool for heart disease.

AP19‐­00342

Pericardium‐­myocardium interaction in lipopolysaccharide‐­induced left atrium arrhythmogenesis

Lin Fong‐Jhih, Lu Yen‐Yu, Li Shao‐Jung, Chen Yao‐Chang, Chen Shih‐Ann, Chen Yi‐Jen

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Taiwan

Introduction:

Atrial fibrillation (AF) is the commonest sustained arrhythmia, and the pathogenesis of AF is multifactorial. Pericarditis may be associates with occurrence and recurrence of post‐­operation AF. Toll‐­like receptor 4 (TLR4) expression significantly increases in atrial fibrillation. TLR4 binds to lipopolysaccharide (LPS) to induce pro‐­inflammatory cytokines. Therefore, the purpose of this study will to evaluate the role of TLR in LPS‐­induced left atrial (LA) arrhythmogenesis.

Methods:

We use LPS (1‐­2 mg/kg) injected into pericardium of the New Zealand rabbits to induce pericarditis. Conventional microelectrodes were used to record the action potentials (APs) and spontaneous activity of LA posterior wall (LAPW) and LA appendage (LAA) covered with pericardium with or without treatment of LPS or TLR4 inhibitor (TAK‐­242, 100 ng/mL).

Result:

1. LPS can increase spontaneous activity rate in LAPW (P < .05) (Figure 1A), but not in LAA. Rapid atrial pacing did not induce sustained spontaneous activity in control LAPW, but induced burst firing in LPS‐­treated LAPW (Figure 1B). Rapid atrial pacing did not induce sustained spontaneous activity in control

LAA, but induced burst firing in LPS treated LAA (Figure 1C). 2. All LPS‐­treated LAPW with burst firing, the rate increased after covering with LPS‐­treated pericardium (Figure 2A), but subsided after covering with control pericardium (Figure 2 B). 3. Under rapid atrial pacing, sustained spontaneous activity was found in 9 of 9 control LAPW covered with LPS‐­treated pericardium (Figure 2C), and 5 of 9 control LAPW covered with control pericardium (Figure 2D). 4. Among LPS‐­treated LAA with burst firing, the rate increased after covering with LPS‐­treated pericardium (Figure 3A), but subsided after covering with control pericardium (Figure 3B). 5. Control LAA did not show spontaneous activity under rapid atrial pacing, and control pericardium did not induced triggered activity (Figure 3C) but LPS‐­treated pericardium induced sustained spontaneous activity in control LAA (Figure 3D). 6. Moreover, all burst firing induced by LPS‐­treated pericardium can be subsided by TAK‐­242 in LPAW with or without LPS treatment (Figure 4), and in LAA with or without LPS treatment (Figure 5).

Conclusion:

LPS‐­induced pericarditis model can show that pericarditis may modulate electrophysiological characteristics of LAPW and LAA, and increase arrhythmogenesis. TLR4 inhibition can inhibit the occurrence of arrhythmia that can be used as a reference for AF treatment in the future.

AP19‐­00391

Effect of angiotensin receptor neprilysin inhibition in atrial and ventricular arrhythmogenesis following left atrial appendage closure in animal model

Isaiah Lugtu, Li‐Wei Lo

Taipei Veterans General Hospital, Taiwan

Introduction:

LAA closure decreases ANP level which indirectly causes increased risk of atrial and ventricular arrhythmogenesis especially in heart failure. The study aims to determine the effect of ARNi in atrial and ventricular arrhythmogenesis following LAA closure in animal models.

Methods:

Twenty‐­four rabbits were randomized to four groups: (a) control, (b) with LAA closure, (c) heart failure with LAA closure, and (d) HF‐­LAA closure with Sacubitril/Valsartan. Heart failure models were developed in groups 3 and 4 by RV pacing. Epicardial LAA closure was performed in groups 2, 3 and 4. ANP levels were measured at baseline and after LAA closure. Electrophysiologic study were performed individually in each group. Atrial and ventricular myocardia were harvested for Western blot and Trichrome stain.

Result:

Right and left atrial effective refractory periods (ERPs) were prolonged in group 3 following LAA closure, while ERPs were restored to baseline in group 4 after neprilysin inhibition (Figure A). Left ventricular ERPs were longest in group 3, while no difference was noted between groups 1 and 4 (Figure B). AF window of vulnerability was significantly elevated in groups 2 and 4, but group 4 is lower when compared to that of group 3 (Figure C). The VF inducibility was highest in Gr 3 (51 ± 5%, P < .001) followed by Gr 2 (30 ± 4%, P = .006) while groups 1 and 4 had no significant difference in VF inducibility (25 ± 5%, P = .11 vs 18 ± 4%, respectively). Atrial ANP was decreased in group 2 (785 ± 103 pg/mL, P = .03), and failed to increase in group 3 (917 ± 172 pg/mL, P = .3), increased in group 4 (1524 ± 126 pg/mL, P < .01) when compared to group 1 (1014 ± 56 pg/mL). Ventricular ANP level was not elevated in group 3 (781 ± 191, P = .54), but elevated in group 4 (1524 ± 126, P < .01) when compared to group 1 (932 ± 102 pg/mL). Western blot showed extensively decreased expressions of atrial calcium handle proteins (CaV1.2, RyR, SERCA and NCX) in groups 2 & 3, moderately decreased in group 4, compared group 1 (Figure D). Changes of ventricular Ca handling proteins (CaV1.2, SERCA and NCX) were observed in groups 2 & 3, when compared to group 1, while it restored to baseline in group 4 (Figure E). Advanced fibrosis was noted in groups 2, 3 & 4 in both ventricles, when compared to group 1 (Fig F, H).

Conclusion:

LAA closure causes neurohormonal remodeling with decreasing ANP, which in turn increases AF and VF inducibility. Atrial and ventricular arrhythmogenesis were both suppressed by ARNi.

AP19‐­00446

Transition of lesion characterization in late gadolinium enhancement following cryo ablation – serial mr imaging

Wataru Igawa, Kennosuke Yamashita, Morio Ono, Junpei Saito, Masahiko Ochiai

Showa University Northern Yokohama Hospital, Japan

Introduction:

RF ablation has been shown to mature over the span of 8‐­12 weeks but the extent and time course of cryo lesion maturation is still unknown. Our goal was to assess cryo lesion maturation using serial magnetic resonance imaging (MRI).

Methods:

Ventricular ablation was performed in a canine model using a cryo catheter (Freezor® MAX, Medtronic) with freeze times of 120 and 240 seconds. T2‐­weighted edema imaging and Late Gadolinium Enhancement (LGE) MRI were done immediately after ablation and repeated after 1, 4, and 12 weeks. After the final MRI, hearts were excised for pathological and histological evaluation. Edema, microvascular obstruction (MVO), and hyperenhancement (HE) volumes were calculated (Panel A) and normalized to chronic pathological lesion volume.

Result:

Thirty‐­three ventricular lesions were evaluated. Immediately after contrast administration, the MVO volume was 1.9 ± 0.5 times and the HE volume was 6.9 ± 3.7 times larger than the chronic pathological lesion volume. Fifty minutes after contrast administration, the normalized HE volume was 4.8 ± 0.5 and the MVO volume was 0.5 (Panel B). The logarithmic curve fitted to the normalized MVO showed that the MVO volume at 19.1 minutes (95% prediction interval, 17.2–21.1 minutes) after contrast injection (Panel B) was the best predictor of chronic lesion volume. When analyzing ablation lesions with 120 and 240 seconds freeze times the predictive values were 18.2 and 20.2 minutes, respectively.

Conclusion:

In cryo ablation, the MVO volume measured 19.1 minutes after contrast injection on acute LGE‐­MRI predicts the chronic lesion volume. This time was not strongly affected by the freeze time.

AP19‐­00558

A case of permanent pace maker implantation ‐­ woes during and after…

Abhilash Sreevilasam Pushpangadhan, Mukund A. Prabhu, Krishna Kumar Mohanan Nair, K. K. Narayanan Namboodiri, V. K. Ajit Kumar

Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India, India

Introduction:

Patient was a 6 years old girl who had a small ASD and a small VSD. Both were kept for medical follow up from infancy. During the course of follow up, she was found to have complete heart block. (See Figure 1). She had one episode of syncope and her echo showed mild LV dysfunction. She was planned for a permanent pacemaker at our hospital. We opted for a rate responsive single chamber pace maker (considering small kid) ‐­ a VVI‐­R pacemaker.

FIGURE 1 ECG showing complete heart block (CHB)

Methods:

Patient being a kid, we programmed the pulse generator to 80 bpm in VVI‐­R mode prior to implantation of pace maker. Lead was screwed in at the RV apical septum and threshold of lead was being checked. The trouble started then. The R wave and impedance were good and EGM showed more than 50% injury. But the lead was not capturing 1:1 at even high out puts. It showed 2:1 capture. (See Figure 2) We were planning to change lead position to another location assuming threshold was not good at the implanted site. At this point we could make out that, apart from CHB, there was significant QT prolongation in the basal ECG. We realised that alternate pacing beats were not captured because they fell on the refractory period of the myocardium due to prolonged QT interval.

FIGURE 2 Alternate pacing beats were not capturing since they fell on the refractory period of myocardium when paced at 100 bpm. Please note the prolonged QT.

Result:

We started pacing at a slower 70 bpm rate. (See Figure 3). At 70 bpm, same lead position showed consistent 1:1 capture. Threshold at the same lead position was 0.8V and hence we did not have to change the lead position. The issues did not settle there. We had programmed the pacemaker pulse generator to 80 bpm in VVI‐­R mode prior to implantation. This pre‐­programmed pulse generator was connected and at 80 bpm per minute capture was inconsistent. And as the child woke up from general anaesthesia, rate responsive pacemaker was increasing heart rate to 100 bpm, further affecting the capture. So we had to re‐­programme the pace maker to 70 bpm VVI mode. (Rate responsiveness was switched off as well).

FIGURE 3 Showing 1:1 capture at 70 bpm. Note each pacing artefact falling beyond the prolonged QT segment

Conclusion:

The QT prolongation in this kid is due to long duration of bradycardia and this is potentially reversible with correction of heart rate. We observed the child for 3 more days in ICU. Beta blockers were started to reduce the QT dispersion. See the surface ECG on the next day of implantation (Figure 4) and pre discharge interrogation of pace maker. (Figure 5) We could observe that the myocardium which was not capturing 1:1 at 100 bpm 3 days back, once QT got corrected, capturing at 120 bpm now. We programmed pacemaker again to VVI‐­R at 80 bpm at discharge. Our plan with beta blocker was to continue it for 1 more month.

FIGURE 4 Surface ECG on the next day of pace maker implantation. Upper panel shows consistent capture at 70 bpm with reduction in QT interval. Lower panel shows 1:1 capture at 85 bpm(at magnet rate)

FIGURE 5 Pre discharge pace maker interrogation showing 1:1 ventricular capture at 120 bpm

AP19‐­00574

Catestatin protects oxidative‐­stress‐­induced apoptosis by activating the β2 adrenergic receptor and PKB/Akt pathway in ischemic‐­reperfused myocardium

Song‐Yun Chu, Fen Peng, Jie Wang, Lin Liu, Lei Meng, Jing Zhao, Xiao‐Ning Han, Wen‐Hui Ding

Peking University First Hospital, China

Introduction:

Apoptosis induced by oxidative stress is one of the most important cardiomyocytes losses during ischemia‐­reperfusion (I/R). Catestatin (CST) has been demonstrated to have the anti‐­ oxidative capacity in vitro. We hypothesized that CST could reduce apoptosis of cardiomyocytes induced by oxidative stress in I/R.

Methods:

In Langendorff‐­perfused rat heart global I/R model, cardiac function including LV developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), and maximal time derivative of left ventricular pressure (±dp/dtmax) were recorded. After I/R, MDA, SOD, and GSH‐­PX content in myocardial tissue were measured. TTC staining and TUNEL staining were used to evaluated the infarction area and apoptosis. Heart homogenates were used for LDH assay and Western blots. Primary cultured neonatal rat cardiomyocytes were exposed to H2O2. Apoptosis of the cardiomyocytes was assessed by Hoechst 33342 staining and DNA laddering.

Result:

Global I/R induced infarction. Average infarct size of (33.66 ± 3.61)% was found in I/R group. Treatment with CST led to reduced infarct size (20.25 ± 3.23)% (P = .011). LDH content in myocardium decreased significantly after reperfusion. CST intervention alleviated the LDH loss [(8994.4 ± 963.8) vs (6843.5 ± 1136.0), P < .001)]. After I/R, LVDP and ±dp/dtmax decreased while LVEDP increased significantly. CST improved LVDP and ±dp/dtmax (P = .005), and reduced LVEDP (P = .008). Reperfusion significantly increased apoptosis of cardiomyocytes and caspase‐­3 cleavage. CST intervention showed a significant decrease of apoptotic nuclei and caspase‐­3 cleavage. Ischemia‐­ reperfusion induced elevation of MDA and depletion of anti‐­oxidative enzymes. CST intervention led to decrease of MDA level and restored activities of SOD and GSH‐­PX [SOD (222.8 ± 26.6) vs (175.3 ± 20.6) in I/R group (U/mg) and GSH‐­Px (53.3 ± 15.9) vs (37.3 ± 11.0) in I/R group (U/mg), all P < .05]. Ischemia‐­ reperfusion led to an increased expression of total ERK, phosphorylation activation of AMPK, and Akt. CST contributed to reduced AMPK activation but further Akt phosphorylation. CST did not influence the phosphorylation of ERK. In primary cultured neonatal cardiomyocytes, H2O2 stimulation induced apoptosis of the cells, activation of caspase‐­3 and phosphorylation of Akt. CST intervention further stimulated phosphorylation of Akt and alleviated the effect of H2O2 by decreasing apoptosis and inhibiting cleavage of caspase‐­3. PI3K inhibitor LY294002 partly abolished the protective effect of CST. Moreover, β2 inhibitor ICI118551 pretreatment suppressed the protective effect of CST toward Akt activation and caspase‐­3 cleavage.

Conclusion:

Administration of CST might protect myocardial I/R injury by activationβ2 receptor and reperfusion injury salvage kinase (RISK) pathway to reduce apoptosis of cardiomyocytes induced by oxidative stress.

AP19‐­00626

A Mendelian randomization analysis: The causal association between serum uric acid and atrial fibrillation

Myunghee Hong, Pil‐Sung Yang, Inseok Hwang, Tae‐Hoon Kim, Hee‐Tae Yu, Jae‐Sun Uhm, Boyoung Joung, Moon‐Hyoung Lee, Hui‐Nam Pak

Yonsei University, South Korea

Introduction:

Observational studies were shown that high levels of serum uric acid (UA) were associated with the atrial fibrillation (AF). However, the causal effect of urate on risk of AF is still unknown. To clarify the potential causal association between uric acid and atrial fibrillation, we performed the Mendelian randomization analysis using genetic instrumental variables (IVs).

Methods:

In a Korean GWAS dataset consisting of 672 patients with AF (mean age 50.5 ± 7.8 years, 81.0% male, Yonsei AF Ablation cohort) who underwent radiofrequency catheter ablation and 3700 controls (Korea Genome Epidemiology Study), we selected the 10 SNPs with P < .05 associated with the increasing serum level of UA. Additionally, we also calculated the weighted genetic risk score (wGRS) using selected 10 SNPs in order to use as an instrumental variable. Mendelian randomization analysis was calculated by the 2‐­stage least square (2SLS) method.

Result:

The conventional association between serum uric acid and AF was shown the significance level (P = .004) after adjusting the potential confounding factors. The SNP rs1165196 on SLC17A1 (F‐­ statistics = 221.82, 0.18 mg/mL per allele change, P < .001) and wGRS (F‐­statistics = 237.27; 0.20 mg/mL per 1SD change, P < .001) were significantly associated with increase the UA level. Mendelian randomization analysis was shown the causally associated with rs1165196 only (estimated odds ratio (OR) = 0.25, 95% CI = 0.08–0.77, P = .016), but not wGRS (estimated OR = 0.98, 95% CI = 0.54–1.76, P = .944).

Conclusion:

In contrast to the observational studies, the increasing levels of serum UA are causally associated with decreasing AF risk at SLC17A1 gene. It is warranted to test the reproducibility in the higher number of population.

FIGURE 1 Observational association between serum urate and atrial fibrillation

FIGURE 2 Association of genetic variants with serum uric acid (exposure) and atrial fibrillation (outcome)

FIGURE 3 The causal effect of uric acid levels on atrial fibrillation using Mendelian randomization analysis

AP19‐­00630

Association of obesity on obstructive sleep apnea in patients with atrial fibrillation: a Mendelian randomization analysis

Myunghee Hong, Inseok Hwang, Tae‐Hoon Kim, Hee‐Tae Yu, Jae‐Sun Uhm, Boyoung Joung, Moon‐Hyoung Lee, Hui‐Nam Pak

Yonsei University, South Korea

Introduction:

Obesity and obstructive sleep apnea (OSA) are associated with the atrial fibrillation (AF) risk. However, the study of causal relationship of obesity on risk of OSA in patients with AF is limited. The aim of study was to examine the causal effect between obesity and OSA in patients with AF using a Mendelian randomization study design.

Methods:

546 patients with AF (mean age 59.9 ± 10.5 years, 74.2% male) who underwent radiofrequency catheter ablation were used for Mendelian randomization analysis from GWAS dataset. To use the genetic instrumental variables (IVs), we choose the 6 SNPs (P < .05) associated with the increasing level of body mass index (BMI; mg/kg). In addition, we also constructed the weighted genetic risk score (wGRS) by using selected 6 SNPs as an IV.

Result:

We found the significant association (P < .001) of BMI on OSA after adjusting age and gender. Additionally, a BMI levels in the upper quartile associated with a 4.84 fold increased risk of apnea/hyponea index compare to lower quartile (95% CI = 2.62–8.93, P < .001). The top SNP rs2535633 on ITIH4 (0.65 mg/kg per allele change, P < .001) and wGRS (0.86 mg/kg per 1SD change, P < .001) were significantly associated with increase the BMI level. In Mendelian randomization analysis, the estimated the causal effect (beta coefficient) of OSA risk was 4.97 (95% CI = 1.75‐­8.18, P = .003) and 5.06 (95% CI = 2.25‐­7.88, P < .001) using ITIH4 and wGRS, respectively.

FIGURE 1 Observational association between BMI and OSA‐­AHI in patients with AF after RFCA. (A) BMI according to Apnea/hypopnea index groups. (B) Risk of Apnea/hypopnea index on BMI quartiles.

FIGURE 2 Association between BMI or OSA‐­AHI and genetic instrumental variables using BMI related SNPs and weighted genetic risk score.

FIGURE 3 Mendelian randomization analysis of BMI on OSA‐­AHI risk in patients with AF.

Conclusion:

Our finding suggests that increasing OSA risk in patients with AF is causally associated obesity at the genetic levels. It may valuable finding in order to reduce the AF risk.

AP19‐­00659

Effects of renal denervation to cardiac autonomics during sleep in rats with myocardial infarction

Wei‐Lun Lin, Chun‐Ting Lai, Yu‐Hui Chou, Shin‐Huei Liu, Wen‐Han Cheng, Tsung‐Ying Tsai, Cheryl C. H Yang, Terry B. J. Kuo, Li‐Wei Lo, Shih‐Ann Chen

Taipei Veterans General Hospital, Taiwan

Introduction:

Sympathetic hyperactivity and poor sleep quality are reported in MI patients. Sleep is an important modulator of cardiovascular function, both in physiological conditions and in disease states. Objective: We aimed to evaluate the effects of renal denervation (RDN) on cardiac autonomic activity and disordered sleep pattern.

Methods:

Wireless transmission of polysomnographic recording was performed in sham (n = 6) and left coronary artery (LCA) ligation (n = 7) male rats during normal daytime sleep before and after RDN treatment. Spectral analyses of the electroencephalogram (EEG) and electromyogram (EMG) were evaluated to define active waking (AW), quiet and paradoxical sleeps (QS, PS). Cardiac autonomic activities were measured by analyzing the power spectrum of heart rate variability (HRV).

Result:

In LCA ligation group, there was a higher LF/HF ratio during sleep, and LF/HF ratio was reduced significantly after RDN treatment in all sleep stages, when compared to that before RDN treatment (Fig A). The frequency of sleep interruption was increased before RDN treatment in LCA ligation rats compared to sham, and that change was ameliorated and restored to baseline after RDN treatment in LCA ligation group (Fig E). Percentages of LVEF and FS were significant decreased in LCA ligation rats compared to sham (Fig F).

Conclusion:

Our results demonstrate a significant sleep fragmentation with sympathetic hyperactivity after MI, and RDN restores the autonomic dysfunction and sleep disorder. The findings suggest that RDN improve sleep‐­related arrhythmia and sudden cardiac death after MI by restoring autonomic homeostasis.

AP19‐00664

Balanced and unbalanced augmentation of sympathetic nerve activity and its correlation to specific ECG features in cardiovascular diseases

Ayari Sugai, Osamu Saitoh, Ayaka Oikawa, Junya Watanabe, Hiroshi Furushima, Masaomi Chinushi

Niigata University, Japan

Introduction:

A sudden change in the autonomic nerve activity and its balance between the right‐­ and left‐­ sides are considered to be related to specific ECG characteristics in some cardiovascular diseases (LQTS, Takotsubo cardiomyopathy, early repolarization syndrome, etc.). However, this issue has not been well studied in systematic experimental protocol.

Methods:

Experiments were performed using a canine model (n = 18). To simulate right‐­ and left‐­ sides unbalanced augmentation of sympathetic nerve activity, electrical stimulation (5 ms, 20 Hz) was applied either from left‐­ or right‐­ side stellate ganglion (LSG and RSG). To simulate the right‐­ and left‐­ side balanced augmentation of sympathetic nerve activity, electrical stimulation (2 ms, 20 Hz) was delivered from the inside lumen of renal artery (RN). BP, HR, ECG were continuously monitored during and after cessation of the stimulation.

Result:

1. BP/HR were increased by all stimulation maneuvers (RSG: +35 ± 28/58 ± 21%, LSG: +60 ± 41/17 ± 24%, RN: +32 ± 15/45 ± 27%). 2. QTc interval was relatively stable (RSG: +8 ± 20%, LSG: +6 ± 16%, RN: +5 ± 6%), and giant negative T wave was not observed in any stimulations. 3. PVC/NSVT were induced 100% in LSG, 83% in RN and 22% in RSG. J‐­waves became visible 89% in LSG but none in other stimulation maneuvers. The appearance of J‐­waves was associated with ST‐­segment elevation in 44% of them.

Conclusion:

The balanced and unbalanced augmentation of sympathetic nerve activity failed to induce typical ECG configurations in cardiovascular diseases. Heterogeneous distribution of the myocardial repolarization can be a cause of the LSG stimulation induced J‐­waves.

AP19‐­00671

The role and mechanism of macrophage colony‐­stimulating factor (M‐­CSF) in ventricular electrical remodeling after myocardial infarction in mice by regulating cardiac macrophages

Shujuan Zhang

Renmin Hospital of Wuhan University, China

Introduction:

To investigate the effect and mechanism of macrophage colony‐­stimulating factor (M‐­ CSF) on ventricular arrhythmias (VAs) after MI in mice by regulating cardiac macrophages.

Methods:

Firstly, C57BL/6J wild type mice were randomly divided into 3 Groups to investigate the relationship between M‐­CSF, macrophages and VAs after MI. Then C57BL/6J wild mice were randomly divided into Sham group, MC group and MM group. The Sham group mice were given ligation line through LAD without ligation, the MC group and MM group were ligated with LAD to prepare MI model, the M‐­CSF (500 μg/kg/d) was administered intraperitoneally for 5 days in MM group. Three groups of mice were fed for 1 week. At the end of the experiment, the relevant indexes of ventricular structural remodeling, serum and tissue inflammatory factors, M1 and M2 macrophage levels and ERP, APD90, threshold of action potential electrical alternating and the VAs inducibility, the concentrations of Cx43 and TH in myocardial tissues were measured.

Result:

The levels of M‐­CSF significantly increased in MI/3d and MI/7d group than the Sham group, and the level of M‐­CSF in MI/3d group was higher than the MI/7d group. The M2 macrophage increased continuously after MI, while the M1 macrophage increased in the acute phase after MI, then decreased timely. The level of M2 macrophage in the MM group was significantly higher while the level of M1 macrophage was significantly lower than in the MC group. The ventricular function were significantly increased and also the ventricular ejection fraction, the infarction size significantly decreased. The levels of pro‐­inflammatory cytokines in serum and tissues all decreased while the levels of anti‐­inflammatory cytokines significantly increased (all P < .05). The cell apoptosis, hypertrophy and fibrosis were all significantly improved. And the ERP of the MM group was significantly longer than in the MC group while the APD90 significantly shortened, the threshold of action potential electrical alternating significantly increased, the VAs inducibility in the MM group was markedly lower than in the MC group. The TH level of the infarction area of the MM group was significantly lower than in the MC group while the Cx43 level significantly increased.

Conclusion:

M‐­CSF could significantly improve the ventricular electrical remodeling after MI in mice by regulating the levels of macrophages of different polarization types in heart tissue (increasing the level of M2 type macrophages and inhibiting the increase of M1 type macrophages), and then could inhibit the inducibility of VAs, which may have important relationship with M‐­CSF could significantly improve ventricular structural remodeling and function, inhibit sympathetic hyperdistribution in the peripheral area of infarction, and increase the expression level of Cx43.

Keywords: macrophage colony‐­stimulating factor, ventricular electrical remodeling, ventricular arrhythmias, myocardial infarction.

AP19‐­00694

Loss of MD1 increases vulnerability to ventricular arrhythmia in diet‐­induced obesity mice via enhanced activation of the TLR4/MyD88/CaMKII signaling pathway

Wei Shuai, Bin Kong, Hui Fu, Caijie Shen, He Huang

Renmin Hospital of Wuhan University, Afghanistan

Introduction:

Obesity is an important risk factor for ventricular arrhythmia (VA), and myeloid differentiation protein 1 (MD1) had been reported decrease in obese hearts. Nevertheless, underlying mechanisms linking MD1 and VA have not been fully studied. This study aims to investigate the regulatory role of MD1 in VA caused by diet‐­induced obesity.

Methods:

MD1 knock‐­out (KO) and wild type (WT) mice from experimental groups were fed with a high‐­fat diet (HFD) from 6‐­week‐­old for 20 weeks. The body weight gain, fast glucose and serum lipid levels were measured and recorded. In addition, pathological analysis, echocardiography, electrocardiography, langendorff‐­perfused heart and molecular analysis were performed to detect HFD‐­ induced vulnerability to VA and its underlying mechanisms.

Result:

After a 20‐­week HFD feeding, the mice showed an increase in body weight, glycemic, lipid levels, QTc interval, LVEDd, LVEDs and LVFS (Table 1). HFD feeding also increased vulnerability to VA, as shown by the prolonged action potential duration (APD), enhanced APD alternans threshold and greater incidence of VA (Figure 1). Moreover, HFD feeding caused LV hypertrophy and fibrosis (Figure 2), and decreased the protein expressions of Kv4.2, Kv4.3, Kv1.5, Kv2.1 and Cav1.2 channels (Figure 3). At last, above‐­mentioned HFD‐­induced adverse effects were further exacerbated in KO mice compared with WT mice. Mechanistically, the expressions of TLR4, MyD88, CaMKII and CaMKII phosphorylation (p‐­CaMKII) were obviously enhanced in KO‐­HFD mice, indicating MD1 deletion could strongly active the TLR4/MyD88/CaMKII signaling pathway (Figure 4).

Table 1 The characteristic of HFD‐­induced obese mice model

	WT‐­ND	KO‐­ND	WT‐­HFD	KO‐­HFD
BW, g	29.48±0.32	31.18±0.24	35±0.33*	45.36±0.47* #
Glucose, mmol/l	6.31±0.13	6.34±0.11	7.41±0.15*	9.73± 0.26* #
TC, mmol/L	1.96±0.16	1.93±0.06	3.8±0.19*	5.13± 0.21* #
TG, mmol/L	0.65±0.05	0.73±0.05	1.42±0.1*	1.93± 0.25* #
LDL‐­c, mmol/L	0.84±0.07	0.8±0.03	1.7±0.08*	1.92±0.07* #
RR interval, ms	132.39 ± 9.09	133.54 ± 6.49	141.02 ± 8.8	152.84 ± 7
PR interval, ms	39.78 ± 0.9	39.68 ± 0.71	39.07 ± 0.74	40.56 ± 1.86
QRS duration, ms	9.97 ± 0.25	10.5 ± 0.48	10.34 ± 0.36	10.68 ± 0.26
QTc interval, ms	43.36 ± 0.39	44 ± 1.3	51.12 ± 1.03*	56.91 ± 2.15* #
LVEDd, mm	3.69 ± 0.25	3.76 ± 0.21	4.11 ± 0.15*	4.6 ± 0.15* #
LVEDs, mm	1.84 ± 0.18	1.93 ± 0.13	2.34 ± 0.11*	2.99 ± 0.26* #
LVFS, %	50.24 ± 0.64	48.45 ± 1.97	43.05 ± 0.55*	35.12 ± 1.62* #
LVEF, %	78.25 ± 3.96	76.14 ± 2.61	79.29 ± 2.29	75.5 ± 4.69

N = 8 for each group. Data are presented as mean ± SEM. BW: body weight; TC, total cholesterol; TG, triglyceride; LDL‐­c: low density lipoprotein cholesterin; HFD, high‐­fat diet; HR, heart rate; LVEDd, left ventricular end‐­diastolic diameter; LVEDs, left ventricular end‐­systolic diameter; LVFS, left ventricular fraction shortening; LVEF, left ventricular ejection fraction.

* P < .05 vs. WT‐­ ND group

# P < .05 vs. WT‐­HFD group.

FIGURE 1 Loss of MD1 prolonged APD and increased susceptibility to arrhythmia in HFD‐­fed heart. (A, B) Representative action potential figures and statistical analysis of the APD20, APD50, APD90 in WT and KO mouse hearts after 20 weeks ND or HFD feeding (n = 8). (C, D) Representative electric alternans figures and statistical analysis of the ALT thresholds in WT and KO mouse hearts after 20 weeks ND or HFD feeding (n = 8). (E, F) Representative arrhythmia induced by burst‐­pacing stimulations and statistical analysis of WT and KO mouse hearts after 20 weeks ND or HFD feeding (n = 10‐­11). Data are expressed as mean ± SEM. *P < .05 vs. WT‐­ ND group, #P < .05 vs. WT‐­HFD group

FIGURE 2 Deletion of MD1 aggravated HFD‐­induced maladaptive LV hypertrophy and fibrosis. (A) Representative western blots of MD1 expression in LV tissues from WT and MD1‐­KO mice (n = 6). (B‐­D) HW, HW/BW, HW/TL values of the indicated groups (n = 8). (E) Gross hearts and H&E staining performed in WT and KO mouse hearts 20 weeks after the ND or HFD feeding (n = 6). (F) Statistical analysis of the cardiomyocyte cross‐­sectional area (CSA) from H&E‐­stained slices of LV from WT and KO mouse after 20 weeks ND or HFD feeding (n = 100+ cardiomyocytes in four samples). (G, H) Representative western blots and statistical analysis of the hypertrophy markers ANP, BNP and β‐­MHC in WT and KO mouse hearts after 20 weeks ND or HFD feeding (n = 4). (I) PSR staining of histological sections prepared from LV samples of WT and KO mouse hearts after 20 weeks ND or HFD feeding (n = 6). (J) Statistical analysis of the LV collagen volume (%) in PSR‐­stained slices of WT and KO mouse after 20 weeks ND or HFD feeding (n = 25+ fields in four samples). (K, L) Representative western blots and statistical analysis of the fibrosis markers collagen I, collagen III and TGFβ1 in WT and KO mouse hearts after 20 weeks ND or HFD feeding (n = 4). Data are expressed as mean ± SEM, *P < .05 vs. WT‐­ND group, #P < .05 vs. WT‐­HFD group

FIGURE 3 Loss of MD1 decreased the protein expression of ion channels in HFD‐­fed heart. (A, B) Representative western blots and statistical analysis of Kv4.2, Kv4.3, Kv1.5, Kv2.1 and Cav 2.1 in WT and KO mouse hearts after 20 weeks ND or HFD feeding. Data are expressed as mean ± SEM, n = 4 mice per group. *P < .05 vs. WT‐­ND group, #P < .05 vs. WT‐­HFD group

FIGURE 4 MD1 regulated the activation of TLR4/MyD88/CaMKII signaling pathway in HFD‐­fed heart. (A, B) Representative western blots and statistical analysis of TLR4, MyD88, CaMKII and p‐­CaMKII in WT and KO mouse LV tissues after 20 weeks ND or HFD feeding. Data are expressed as mean ± SEM, n = 4 mice per group. *P < .05 vs. WT‐­ND group, #P < .05 vs. WT‐­HFD group

Conclusion:

MD1 deficiency increased HFD‐­induced vulnerability to VA. This is mainly caused by the aggravated maladaptive LV hypertrophy, fibrosis and decreased protein expressions of ion channels, which are induced by the enhanced activation of the TLR4/MyD88/CaMKII signaling pathway.

AP19‐­00695

Myeloid differentiation protein 1 protected myocardial function against high‐­fat stimulation induced pathological remodeling

Caijie Shen, Bin Kong, Wei Shuai, He Huang

Renmin Hospital of Wuhan University, Afghanistan

Introduction:

Myeloid differentiation 1 (MD‐­1) is a secreted protein that regulates the immune response of B cell through interacting with radioprotective 105 (RP105). The disrupted immune response may contribute to the development of cardiac diseases, while the roles of MD‐­1 remain elusive. Our studies aimed to explore the functions and molecular mechanisms of MD‐­1 in obesity‐­induced cardiomyopathy.

Methods:

H9C2 myocardial cells were treated with free fatty acid (FFA) containing palmitic acid and oleic acid to challenge high‐­fat stimulation and adenoviruses harboring human MD‐­1 coding sequences or shRNA for MD‐­1 overexpression or knockdown in vitro. MD‐­1 over‐­expression or knockdown transgenic mice were generated to assess the effects of MD1 on a high‐­fat diet (HD) induced cardiomyopathy in vivo.

Result:

Our results showed that MD‐­1 was down‐­regulated in H9C2 cells exposed to FFA stimulation for 48 hours (Figure 1). Both in vivo and in vitro, silencing of MD1 accelerated myocardial function injury induced by HD stimulation through increased cardiac hypertrophy and fibrosis (Figures 2, 3, 4), while overexpression of MD1 alleviated the effects of HD by inhibiting the process of cardiac remodeling (Figured 5, 6). Moreover, the MAPK and NF‐­κB pathways were overactivated in MD1 deficient mice and H9C2 cells after high‐­fat treatment. Inhibition of MAPK and NF‐­κB pathways played a cardioprotective role against the adverse effects of MD1 silencing on high‐­fat stimulation‐­induced pathological remodeling (Figures 7, 8).

Conclusion:

MD1 protected myocardial function against high‐fat stimulation‐­induced cardiac pathological remodeling through negative regulation for MAPK/NF‐κB signaling pathways, providing feasible strategies for obesity cardiomyopathy

FIGURE 1 Free fatty acid (FFA) induced lipid accumulation and MD‐­1 differential expression in cardiomyocytes. (A) Proliferation curve of H9C2 measured with MTT assay. (B) Oil red O staining of H9C2. Cellular lipid accumulation was stained as bronzing droplets in the images. (C) qPCR and western blot analysis of MD‐­1 level. The mRNA and protein levels of MD‐­1 were determined following the treatment of FFA at the indicated doses. GAPDH was used as an internal control. (D) qPCR and western blot analysis of MD‐­1 level. The mRNA and protein levels of MD‐­1 were determined following the treatment of 1.0 mmol/L FFA for the indicated time. *P < .05, **P < .01, ***P < .001

FIGURE 2 MD‐­1 protected cardiomyocytes to resist pathological remodelling induced by free fatty acid (FFA) stimulation in vitro. (A) qPCR and western blot analysis of MD‐­1 level in H9C2. The mRNA and protein levels of MD‐­1 in H9C2 were determined after cardiomyocytes transfection with MD‐­1 overexpression (Ad‐MD‐­1) or knockdown (Ad‐shMD‐­1) recombinant adenovirus for 48 h. (B) TRITC Phalloidin staining of H9C2 to measure the cross‐section area of cardiomyocytes after adenovirus transfection and FFA treatment for 48 h. (C) qPCR and western blot analysis of ANP, BNP and β‐MHC levels in H9C2 cells as described in B. (D) qPCR and western blot analysis of COL1A1, COL3A1 and CTGF expressions in H9C2 cells as described in B. *P < .05, **P < .01, ***P < .001

FIGURE 3 Silencing of MD‐­1 promoted high‐fat diet induced myocardial damage. (A) Western blot validated the knockout efficiency in MD‐­1−/− mice compared to wild‐type mice (WT). (B) The content of serum creatine kinase‐MB (CK‐MB), a common serum marker for myocardial damage in MD‐­1−/− mice and WT mice after high‐fat diet feed for 20 weeks (n = 12). (C‐F) BW, HW, HW/BW and HW/TL for the indicated groups (n = 12). (G) Glucose tolerance testing on MD‐­1−/− mice and WT mice after high‐fat diet feed for 20 weeks (n = 12). (H) qPCR analysis of hypertrophy markers ANP, BNP and β‐MHC in MD‐­1−/− hearts and WT mice hearts after high‐fat diet feed for 20 weeks (n = 4). *P < .05, **P < .01, ***P < .001

FIGURE 4 Silencing of MD‐­1 accelerated high‐fat diet induced myocardial dysfunction. (A‐D) Echocardiographic results for MD‐­1−/− mice and wild‐type (WT) mice after high‐fat diet feed for 20 weeks (n = 12). (E) Masson's trichrome staining of heart tissues from MD‐­1−/− mice and WT mice after high‐fat diet feed for 20 weeks (n = 4). (F) qPCR analysis of fibrosis markers COL1A1, COL3A1 and CTGF in MD‐­1−/− hearts and WT mice hearts after high‐fat diet feed for 20 weeks (n = 4). *P < .05, **P < .01, ***P < .001

FIGURE 5 Overexpression of MD‐­1 alleviated high‐fat diet induced myocardial damage. (A) Western blot validated the overexpression efficiency in MD‐­1 transgenic mice (TG) compared to wild‐type mice (WT). (B) The level of serum CK‐MB in TG and WT mice after high‐fat diet feed for 20 weeks (n = 12). (C‐F) BW, HW, HW/BW and HW/TL for the indicated groups (n = 12). (G) Glucose tolerance testing on TG and WT mice after high‐fat diet feed for 20 weeks (n = 12). (H) qPCR analysis of hypertrophy markers ANP, BNP and β‐MHC in TG and WT mice hearts after high‐fat diet feed for 20 weeks (n = 4). *P < .05, **P < .01, ***P < .001

FIGURE 6 Overexpression of MD‐­1 protected myocardial function against high‐fat stimulation. (A‐D) Echocardiographic results for MD‐­1 transgenic mice (TG) and wild‐type mice (WT) after high‐fat diet feed for 20 weeks (n = 12). (E) Masson's trichrome staining of heart tissues from TG mice and WT mice after high‐fat diet feed for 20 weeks (n = 4). (F) qPCR analysis of fibrosis markers COL1A1, COL3A1 and CTGF in TG hearts and WT mice hearts after high‐fat diet feed for 20 weeks (n = 4). *P < .05, **P < .01, ***P < .001

FIGURE 7 MD‐­1 mediated high‐fat diet‐induced cardiac pathological remodelling via negatively regulating MAPK and NF‐κB signalling. (A, C) Western blot analysis for the phosphorylation levels of MEK, ERK, JNK, p38, IκBα and p65, relative to levels of the total one, in MD‐­1−/− hearts and wild‐type (WT) mice hearts after high‐fat diet feed for 20 weeks. (B, D) Western blot analysis for the phosphorylation levels of MEK, ERK, JNK, p38, IκBα and p65, relative to levels of the total one, in TG hearts and WT mice hearts after high‐fat diet feed for 20 weeks. (E, F) Representative images of NF‐κB p65 immunohistochemistry analysis for heart tissues from TG or MD‐­1−/− mice models after high‐fat diet feed for 20 weeks. *P < .05, **P < .01, ***P < .001

FIGURE 8 Inactivation of MAPK and NF‐κB signalling pathways by inhibitors rescues the adverse effects of MD‐­1 deficiency on high‐fat stimulation induced cardiac remodelling. (A) Western blot analysis for phosphorylation levels of MEK1/2 and IκBα, relative to levels of the total one, in H9C2 cells pre‐treated with 10 μmol/L U0126 and 10 μmol/L Bay 11‐7082 for 30 min before FFA stimulation. (B) Western blot analysis for phosphorylation levels of MEK1/2, ERK1/2 and IκBα, relative to levels of the total one, in MD‐­1 deficient H9C2 cells (Ad‐shMD‐­1) treated with U0126, Bay 11‐7082 and FFA stimulation. (C) qPCR analysis for the mRNA levels of ANP, BNP, β‐MHC, COL1A1, COL3A1 and CTGF in MD‐­1 deficient H9C2 cells (Ad‐shMD‐­1) treated with U0126, Bay 11‐7082 and FFA stimulation. *P < .05, **P < .01, ***P < .001

AP19‐­00716

Relationship between Angptl 2 level and severity of coronary heart disease

He Huang, Rui Wang, Yu Liu, Bin Kong

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China, China

Introduction:

To analyze the relationship between Angptl 2 level and coronary lesions heart disease.

Methods:

A total of 262 Han Hohhot residents who underwent coronary angiography in our hospital from January to November, 2016, were collected. According to the results of coronary angiography, the patients were divided into four groups according to the coronary angiography results: the normal group (N), the single lesion group (S), the double‐­lesion group (D), and the three‐­lesion group (T). According to the Gensini score of each coronary lesion, the patients with coronary heart disease of group S, D and T, were divided into three groups: the mild‐­stenosis group (MI, <30 points), the moderate‐­ stenosis group (MO, 30‐­90 points), and the severe‐­stenosis group (SE, >90 points). The fasting elbow venous blood was collected in the hospital after 12‐­hour fasting for detecting the level of Angptl 2 was tested within 12 hours in fasting state by ELISA. The clinical data Angptl 2, as well as some inflammation factors, were compared among different groups to find the relationship with and coronary angiography results.

Result:

Compared with group N, the serum level of Angptl 2 in the patients group T was highest, followed by group D and Swith coronary heart disease was significantly higher (P < .05). As the number of coronary lesions increased, the level of Angptl 2 also increased, which was positively correlated with the degree of coronary stenosis. As and with the increase of Gensini score increased, the Angptl 2 level also increased, indicating that the Gensini score increased as the number and extent of coronary lesions.

Conclusion:

The level of Angptl 2 can reflect the extent of coronary lesions and can be used as a clinical indicator for the detection of coronary lesions.

AP19‐­00723

Empagliflozin, a sodium glucose co‐­transporter‐­2 inhibitor, alleviates atrial remodeling and improves mitochondrial function in high‐­fat diet diabetic rats

Tong Liu, Qingmiao Shao, Lei Meng, Sharen Lee, Gary Tse, Mengqi Gong, Zhiwei Zhang, Jichao Zhao, Yungang Zhao, Guangping Li

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China, China

Introduction:

Sodium‐­glucose co‐­transporter‐­2 (SGLT‐­2) inhibitors were recently reported to have cardioprotective effects in diabetes mellitus (DM) patients. However, the role of SGLT‐­2 inhibition in atrial remodeling, especially of the arrhythmogenic substrate, remains unclear. To gain insights on the effects of SGLT‐­2 inhibition on atrial fibrillation (AF), we investigated the effects of empagliflozin (EMPA), a commercially available and highly selective SGLT‐­2 inhibitor, on atrial remodeling in high‐­ fat diet diabetic rats.

Methods:

A total of 96 rats were randomized into 4 groups as follows: control group; DM group; low dose of empagliflozin (10 mg/kg/d); and high dose of empagliflozin (30 mg/kg/d). Biochemical examination, echocardiography, hemodynamic examination, histology, electrophysiology, western blot, mitochondrial respiratory function and membrane potential were assessed amongst the groups

Result:

Diabetic rats exhibited left ventricular hypertrophy and left atrial dilation with obvious hemodynamic abnormalities. All changes in the diabetic rates were attenuated by empagliflozin. Compared with the control group, higher atrial fibrillation inducibility was observed in the DM group, and was markedly reduced under empagliflozin treatment. Moreover, empagliflozin improved mitochondrial biogenesis by peroxisome proliferator–activated receptor‐­c coactivator 1α (PGC‐­1α)/nuclear respiratory factor‐­1(NRF‐­1) / mitochondrial transcription factor A (Tfam) signaling.

Conclusion:

Empagliflozin can alleviate atrial remodeling by reversing electrophysiological abnormalities, improving mitochondrial function and mitochondrial biogenesis under DM, hence may be potentially used in the prevention of DM‐­induced atrial fibrillation.

AP19‐­00794

Role of glucose fluctuations in regulation of vascular BK channel via PKCα/NF‐­ κB/MuRF1 signaling

Zhen ye Zhang, Ling Ling Qian, Ning Wang, Ling Feng Miao, Xin Ma, Shi Peng Dang, Ying Wu, Xiao Yu Liu, Xiao Yan Li, Qiang Chai, Min Pan, Fu Yi, Tian You Ling, Ru Xing Wang

Wuxi People Hospital, China

Introduction:

Recent studies have demonstrated that glucose fluctuations have a more harmful effect on the cardiovascular system than persistent high blood glucose, and may be related with coronary diseases. The aim of this study was to investigate the molecular mechanisms of glucose fluctuations on BK channel dysfunction.

Methods:

The rat model with diabetes mellitus (DM) was established through injection of streptozotocin. Diabetic rats with glucose fluctuations were induced by fasting and additional insulin injections. Rat coronary arteries were isolated and coronary vascular tension was tested after 3 weeks. Rat coronary artery smooth muscle cells were isolated and whole‐­cell BK channel currents were recorded using patch clamp technique. Human coronary artery smooth muscle cells in vitro were used to explore the underlying mechanisms.

Result:

Compared with the controlled DM (C‐­DM) group, coronary constriction and BK channel dysfunction were aggravated in the uncontrolled DM (U‐­DM) group and more pronounced in the glucose fluctuation (GF‐­DM) group. The levels of muscle ring finger protein 1 (MuRF1), nuclear factor (NF)‐­κB and protein kinase C (PKC) α were significantly increased in the GF‐­DM group. Activation of PKCα and NF‐­κB induced by glucose fluctuations promoted BK channel dysfunction, while inhibition of reactive oxygen species, PKCα, NF‐­κB and MuRF1 reversed this effect.

Conclusion:

Glucose fluctuations aggravate BK channel dysfunction via the PKCα/NF‐­κB/MuRF1 signaling pathway.

AP19‐­00799

Association between PRKAG3 polymorphisms and sporadic Wolff‐­Parkinson‐­White syndrome

Ken‐Pen Weng

Kaohsiung Veterans General Hospital, Taiwan

Introduction:

The aim of this study was to investigate whether mutation in AMPK subunit genes (PRKAG3‐­230) is associated with sporadic, isolated WPW syndrome.

Methods:

This study consisted of 87 patients with symptomatic WPW syndrome, and 93 normal controls. PRKAG3‐­230 genotypes were determined by real‐­time PCR assay. Genotype and allele frequencies of PRKAG3‐­230 between patients with WPW syndrome and normal controls were analyzed.

Result:

PRKAG3‐­230 were genotyped in 87 patients with WPW syndrome (M/F 53/34, age 24.4 (18.0 years) and 93 normal controls (M/F 57/36, age 16.8 (4.2 years). There were no significant differences in two groups in terms of age and sex. The patients with CG and CG+CC genotypes were with a significantly increased risk of WPW syndrome as compared to those with GG genotype (OR = 1.99, 95% CI: 1.01‐­3.89, P = .045; OR = 1.99, 95% CI: 1.04‐­3.78, P = .037, respectively). The allelic types were not associated with the risk of WPW syndrome. The patients with manifest type with CG and CG+CC genotypes were with a significantly increased risk of WPW syndrome as compared to those with GG genotype (OR = 2.86, 95% CI: 1.16‐­7.05, P = .022; OR = 2.84, 95% CI: 1.19‐­6.80, P = .019, respectively). The patients with right‐­side accessory pathways with CG and CG+CC genotypes were with a significantly increased risk of WPW syndrome as compared to those with GG genotype (OR = 3.07, 95% CI: 1.25‐­7.51, P = .014; OR = 2.84, 95% CI: 1.19‐­6.80, P = .019, respectively). The allelic types were not associated with the risk of WPW types and locations.

Conclusion:

This study shows that PRKAG3‐­230 may be associated with sporadic WPW syndrome among the Taiwanese population. Further studies are warranted to elucidate the role of mutations in AMPK subunit genes other than PRKAG3‐­230 in sporadic WPW syndrome.

AP19‐­00805

Impairment of TRPC1‐­BK complex in diabetic rat coronary artery

Man‐Qing Sun, Ling‐Ling Qian, Ling‐Feng Miao, Ying Wu, Ru‐Xing Wang

Wuxi People's Hospital, China

Introduction:

This study aimed to investigate the dysregulation of the transient receptor potential canonical channel 1 (TRPC1) and the large conductance Ca2+‐­activated K+ (BK) channels complex (TRPC1‐­BK complex) in diabetic coronary vasculopathy.

Methods:

DM was induced with streptozotocin (STZ) in male Sprague‐­Dawley rats. Diabetic and control coronary arteries were respectively achieved 2 months after STZ or vehicle treatment. Using quantitative real‐­time PCR, immunoblotting, fluorescent assay, patch clamp techniques and vascular tension measurements, we investigated TRPC1 channel and BK channel activities and coronary vasoreactivity in both control and diabetic rats.

Result:

We confirmed TRPC1 channel and BK channel were physically associated in the coronary artery SMCs of both control and diabetic rats. The expression of TRPC1 channel was significantly increased while BK‐­β1 subunit was decreased without changing of BK‐­α subunits, in diabetic coronary artery SMCs. Cytosolic calcium concentrations were higher in diabetic coronary artery SMCs than control group, and pre‐­incubation with TRPC1 channel blocker SKF96365 decreased cytosolic calcium concentrations in both groups. BK channel current densities were reduced (P < .05) and the constriction of coronary artery induced by BK inhibitor iberiotoxin was weakened in diabetic groups than control group. The dilatation of coronary artery was stronger in diabetic group than control group in presence of SKF96365.

Conclusion:

TRPC1 channels and BK channels are co‐­localized on diabetic coronary artery SMCs. The imbalance of TRPC1‐­BK complex was due to increased TRPC1 channel expression and decreased BK channel expression in diabetic coronary artery SMCs, in turn contributing to diabetic rat coronary artery dysfunction.

AP19‐­00807

Cytochrome P450 epoxygenase activity contributes to n‐­3 polyunsaturated fatty acids‐­ induced BK channels activation in diabetic rats

Ling‐Ling Qian, Heng‐Jian Chen, Da‐Yun Xia, Xu Tang, Ru‐Xing Wang

Wuxi People's Hospital, China

Introduction:

n‐­3 polyunsaturated fatty acids (n‐­3 PUFAs), which mainly include docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are known to protect the coronary large conductance Ca2+‐­activated K+ (BK) channel function in diabetic rats. However, the mechanisms remain unclear. The objective of this study was to examine the regulation role of cytochrome P450 epoxygenase in n‐­3 PUFAs ‐­induced BK channels activation in diabetic rats

Methods:

The effects DHA and EPA on coronary artery tension were measured using a myograph system. BK channel currents were determined by the patch clamp technique. The mRNA and protein expressions of BK channel subunits were measured using qRT‐­PCR and western blots. The Ca2+ concentrations of the coronary smooth muscle cells were measured using and fluorescence Ca2+ indicator.

Result:

Both DHA and EPA relaxed coronary arterial rings pre‐­constricted by endothelin‐­1 in control and diabetic rats, which was significantly inhibited by preincubation with a specific BK channel blocker iberiotoxin. Consistent with DHA, EPA activated BK channels currents in a concentration dependent manner in fresh isolated coronary smooth muscle cells. The activation effects were inhibited by pre‐­ incubation with a cytochrome P450 epoxygenase inhibitor, SKF525A. Oral administration of n‐­3 PUFAs enhanced BK‐­β1 expression without altering BK‐­α levels, activated BK channels currents, decreased cytosolic Ca2+ concentrations in coronary smooth muscle cells and protect coronary vasoreactivity in both control and diabetic rats; however, all these effects were significant weaken in diabetic rats in addition to SKF525A intraperitoneal injection.

Conclusion:

These results suggest that n‐­3 PUFAs ‐­mediated coronary vasodilatation is mediated by activation of BK channels through cytochrome P450 epoxygenase in diabetic rats.

AP19‐­00808

The reversal of atrial fibrillation after depression via the chronic stimulation of sigma‐­1 receptor and the associated roles of myocardial inflammation and gap junctions

Xin Liu, Bo Yang

Renmin hospital of Wuhan University, China

Introduction:

Sigma‐­1 receptor, known as a putative chaperone protein, regulates ion channels as well as ER stress response and plays a protective role in cardiogenic disease. The present study assessed whether sigma‐­1 receptor (S1R) stimulation could alleviate atrial fibrillation (AF) after depression by regulating myocardial inflammation and gap junctions.

Methods:

One hundred male rats were randomly divided into four treatment groups for 4 weeks: saline [control (CTL)], saline + intragastric administration of SA4503 agonist of the S1R, [control + SA4503 (CTS)], chronic unpredictable mild stress (CUMS) to produce major depression disorder (MDD), and CUMS + intragastric administration of SA4503 [MDD + SA4503 (MDS)]. After 4 weeks, depression‐­like behaviors including sucrose preference, body weight, and immobility time were measured to evaluate the success of model preparation. Microelectrode array (MEA) technology was used for extracellular electrophysiological recordings.

Result:

The results showed that the total activation time (TAT) significantly increased and the excitation propagation was markedly disordered in the MDD group compared to the CTL group (P < .01 for both groups). The rats in the MDD group also displayed a higher frequency of AF incidence, heavier fibrosis, greater expression of inflammatory factors (TGF‐­α and IL‐­6), and lower expression of gap junction proteins (CX40 and CX43) compared to those in the CTL group (P < .01 for all groups). Furthermore, chronic S1R stimulation partially alleviated the above indices in the MDS group (P < .01 for all groups).

Conclusion:

The results indicates that myocardial inflammation and gap junctions may be key contributors to AF after depression. By increasing atrial conduction velocity, recovering excitation propagation disorder, and reducing myocardial inflammation, S1R stimulation has the potential for treatment of AF.

AP19‐­00817

Electrophysiological parameters and anatomic findings of left bundle branch pacing in an in vivo canine model

Xueying Chen, Yangang Su, Junbo Ge

Zhongshan Hospital of Fudan University, Shanghai Institute of Cardiovascular Diseases, China

Introduction:

Left bundle branch pacing (LBBP), an alternative to His bundle pacing (HBP), could maintain the electrical synchrony of the left ventricle in a low and stable threshold. However, the definitions of LBBP have not been well established.

Methods:

A canine model (n = 3; male; weight 30‐­40 kg) was subjected to receive HBP and LBBP procedure. The characteristics of the electrocardiogram (ECG) and intracardiac electrogram (EGM) and the pacing parameters between HBP and LBBP were collected and compared. The hearts were isolated and stained by Lugol's iodine (5%) to show the relative locations between the leads and the conduction system.

Result:

The means of potential to ventricle (PV) interval in HBP was significantly different to that in LBBP (26.67 ± 3.06 vs 12.67 ± 1.15, P = .002). The pacing parameters including pacing threshold, R wave amplitude and impedance were significantly different in HBP as compared to those in LBBP, respectively (2.30 ± 0.66 vs 0.67 ± 0.15, P = .014; 2.67 ± 0.42 vs 11.33 ± 3.06, P = .008; 423.3 ± 40.4 vs 660.0 ± 45.8, P = .003). The paced morphology of ECG was the same as the intrinsic in HBP while it was a RBBB pattern in LBBP. The anatomic findings showed that the HBP lead was located at the right septum across the tricuspid and the LBBP lead was placed deeply and vertically with the ring of the lead inside the septum. The means of the lead depth were significantly different between HBP lead and LBBP lead (1.83 ± 0.29 vs 12.33 ± 1.53 mm, P < .0001). While from the left septum, the LBB and Purkinje network were stained dark brown and the tip of the LBBP lead's helix could be seen around the left bundle branch (LBB).

Conclusion:

In this small size in vivo canine model study, HBP and LBBP could be performed feasibly and their characteristics could be summarized as: (a) with His and LBB potentials; (b) a RBBB paced morphology in LBBP; and 3) pacing parameters significantly different between HBP and LBBP. Moreover, the precise anatomic findings were direct evidences of the depth and the location of the leads around the conduction system.

Table 1 The characteristics between HBP and LBBP

Characteristics	HBP (n = 3)	LBBP (n = 3)	P value
Potential to ventricle interval (ms)	26.67±3.06	12.67±1.15	.002
Pacing threshold (V/0.5ms)	2.30±0.66	0.67±0.15	.014
R wave amplitude (mV)	2.67±0.42	11.33±3.06	.008
Impedance (Ω)	423.3±40.4	660.0±45.8	.003
QRS duration (ms)	72.67±3.51	80.33±5.69	.118
Sti‐­LVAT (ms)	52.33±3.51	39.67±1.53	.005
Lead depth in the septum (mm)	1.83±0.29	12.33±1.53	<.0001
Septum thickness around the lead (mm)	7.57±0.40	11.17±2.47	.067

HBP, His bundle pacing; LBBP, left bundle branch pacing; Sti‐­LVAT, stimulus to left

ventricular activation time

FIGURE 1 The intrinsic 12‐­lead ECG and the EGM recorded by the HBP and the LBBP lead: The intrinsic QRS duration was 69 ms, Po His and Po LBB during the intrinsic rhythm with the potential to ventricle interval were 24 and 12 ms, respectively

FIGURE 2 The 12‐­lead ECG and the EGM recorded by the HBP lead: The Po LBB was recorded through the anterograde conduction by HBP with a pacing stimulus to potential interval of 12 ms. The Sti‐­LVAT was 52 ms and the paced QRS duration was 69 ms

FIGURE 3 The 12‐­lead ECG and the EGM recorded by the LBBP lead: The Po His was recorded through the retrograde conduction by LBBP with a pacing stimulus to potential interval of 12 ms. The Sti‐­LVAT was 40 ms and the paced QRS duration was 74 ms

FIGURE 4 The 12‐­lead ECG and the EGM recorded by unipolar pacing from the ring electrode of the LBBP lead: The Po His was recorded through the retrograde conduction with a pacing stimulus to potential interval of 40 ms. The Sti‐­LVAT was 78 ms and the paced QRS duration was 104 ms

FIGURE 5 The fluoroscopic images of the HBP lead and the LBBP lead: A. Ventriculography through the sheath to show the right ventricular septum's edge (Arrow) and the LBBP lead's depth and direction inside the interventricular septum at right anterior oblique (RAO) 30o; (B) The positions of the leads at left anterior oblique (LAO) 30o

FIGURE 6 The intraventricular septum stain by Lugol's iodine (5%): A. The locations of the HBP lead and the LBBP lead observed from the right septum; B. The LBB and Purkinje network were stained dark brown and the tip of the LBBP lead's helix (White arrow) could be seen around the LBB in sub‐­endocardium of the left septum

AP19‐­00822

Doxorubicin and dysregulation of cytosolic calcium dynamics in heart

Praloy Chakraborty, Mohammed Ali Azam, Keith Dadson, Stéphane Massé, Patrick F. H. Lai, Phyllis Billia, Kumaraswamy Nanthakumar

Toronto General Hospital, Canada

Introduction:

Anthracyclines are the most commonly used antineoplastic drugs to treat a host of hematological and solid tumors. The most feared complication of this group of antineoplastic agents is cardiotoxicity, characterized by systolic dysfunction and heart failure. Myocyte loss due to apoptosis is the hall mark of anthracycline induced cardiomyopathy. Calcium (Ca2+) is essential for excitation–transcription (ET) coupling pathways. Disturbances in cellular Ca2+ handling has been shown to be critically involved in structural and functional remodeling processes associated with heart failure. Doxorubicin has been shown to alter the expression and function of proteins associated with cytosolic calcium handling. However, there is limited published data on the effect of doxorubicin in cardiac cytosolic calcium dynamics physiology. The study was planned to determine the effects of doxorubicin in cardiac cytosolic calcium dynamics in an in vivo mouse model

Methods:

A single dose of doxorubicin (10 mg/kg) (n = 12) or saline (n = 10) was administered intraperitoneally in mice. Fourteen days later, the mice were sacrificed, and hearts were perfused in a Langendorff setup. After stabilization, Rhod‐­2AM and blebbistatin were infused and ventricular epicardial calcium fluorescence was optically measured following a pace‐­and‐­pause protocol. Calcium transient duration 50 (CaTD50) (Figure 1A), calcium transient duration 80 (CaTD80) (Figure 1B), calcium alternans ratio (1C), and spontaneous calcium elevation (Figure 1D) were measured using custom MATLAB codes

Result:

Diastolic spontaneous calcium elevation (P = .034) (1D) and calcium alternans (P = .002) (1C) were higher in doxorubicin‐­treated mice hearts compared to controls. In vivo treatment with doxorubicin prolonged both CaTD50 (P = .016) (1A) and CaTD80 (P = .005) (1B) and decreased CaTD50‐­to‐­CaTD80 ratio (P = .016) compared to control hearts.

Conclusion:

Doxorubicin treatment is associated with abnormal cytosolic calcium handling characterized by spontaneous diastolic calcium release, decreased diastolic clearance and calcium alternans. The pattern of abnormality suggests dysfunction of both Ryanodine Receptor and SERCA2a. Apart from abnormal excitation contraction and extraction excitation–transcription coupling, the abnormal calcium handling may also play crucial role in genesis of arrhythmia, a complication of heart failure

AP19‐­00823

Impaired response of KCNQ1 channel to beta‐­adrenergic stimulation in human iPSC‐­ derived cardiomyocytes carrying a CALM2‐­N98S mutation associated with long QT syndrome

Yuta Yamamoto, Takeru Makiyama, Yimin Wuriyanghai, Hirohiko Kohjitani, Sayako Hirose, Jingshan Gao, Asami Kashiwa, Huang Hai, Takanori Aizawa, Tomohiko Imamura, Taisuke Ishikawa, Hideki Motomura, Yoshinori Yoshida, Seiko Ohno, Minoru Horie, Naomasa Makita, Takeshi Kimura

Kyoto University, Japan

Introduction:

Recently, mutations in CALM genes (CALM1‐­3) encoding calmodulin (CaM) are reported to be associated with severe early‐­onset arrhythmias known as the calmodulinopathy. Calmodulin modulates various proteins including several ion channels in cardiomyocytes. KCNQ1 channel is known to have the interaction with CaM, which regulates the channel gating, assembly and surface localization. However, the interaction between mutant CaM causing calmodulinopathy and KCNQ1 channel in cardiomyocytes remains unknown. The present study aimed to evaluate the KCNQ1 channel function in human iPS cell‐­derived cardiomyocytes (hiPSC‐­CMs) generated from calmodulinopathy patient.

FIGURE 1 Slowly activating delayed‐­rectifier K+ currents (IKS) analysis

FIGURE 2 Action potential (AP) recordings

Methods:

The hiPSC clones were generated from a 12‐­year‐­old boy with long‐­QT syndrome (LQTS) carrying a missense CALM2 mutation (c.293A>G, p.N98S), whose ECG showed marked QT prolongation in epinephrine stress test. After cardiac differentiation, KCNQ1 channel current (IKS) was analyzed using a patch‐­clamp technique. Action potentials were recorded using optical mapping (Fluovolt, transmembrane voltage dye). These electrophysiological characteristics of N98S‐­hiPSC‐­CMs were compared with those of control derived from healthy individual.

Result:

In IKS analysis, 500 nM isoproterenol (ISO) significantly increased peak current of control at 0, 20, 40 mV test potentials. In contrast, the response of peak current to ISO was impaired in N98S‐­ hiPSC‐­CMs (Figure 1). The action potential durations at 90% repolarization (APD90) of N98S‐­hiPSC‐­CMs (n = 13) were significantly prolonged compared to those of control (n = 18) regardless of the presence or absence of ISO at 1.33 Hz pacing (Baseline and ISO: Control, 249.4 ± 29.2 and 169.8 ± 11.7 ms vs N98S, 378.7 ± 19.8 and 321.2 ± 22.0 ms; Figure 2). There was significant difference in the percentage of APD90 shortening between control and N98S.

Conclusion:

This study elucidated the KCNQ1 channel dysfunction using hiPSC model that may explain the clinical phenotype of the LQTS patient carrying the CALM2‐­N98S mutation.

AP19‐­00827

A novel mutation in SCN1B associated with Brugada Syndrome

Wang Linlin

Nanjing Brain Hospital, the Affiliated Hospital of Nanjing Medical University, China

Introduction:

Brugada syndrome (BrS) is a highly arrhythmogenic cardiac disorder characterized by ventricular fibrillation. Here, we identified and characterized a novel SCN1B mutation, A197V, associated with BrS. We aimed to identify and characterize a novel SCN1B mutation, A197V, associated with BrS.

Methods:

Whole‐­exome sequencing was employed to explore the potential causative genes in 8 unrelated clinically diagnosed BrS patients. A novel A197V variant was detected in exon 4 of SCN1B in a 46‐­year‐­old patient, who was admitted due to syncope. Wild type (WT) and mutant (A197V) genes were co‐­expressed with SCN5A in human embryonic kidney cells (HEK293 cells) and studied using whole‐­cell patch clamp and immunodetection techniques.

FIGURE 1 Electrocardiogram (ECG) recording and genetic analysis of the proband with Brugada syndrome. A: Twelve‐­lead ECG recording of the proband at baseline, showing prominent coved ST‐­segment elevation, following a negative T wave in V1‐­V2 leads (Type I BrS ECG). B: Family pedigree. Plus signs indicate the carriers of the mutation A197V. The arrow identifies the proband. C: Polymerase chain reaction– based sequence of SCN1B exon 4 showing wild‐­type (WT) and heterozygous C to T transversion at nucleotide 723 (arrow). It predicts a substitution of valine for alanine at position 197 (A197). D: Predicted topology of Navβ1. Red circle indicates the location of the mutant

FIGURE 2 Effect of SCN1B/A197V on sodium channel current (INa) expressed in HEK293 cells. A: Representative sodium channel current (INa) traces from cells expressing NaV1.5 in the absence and presence of SCN1B wild type (WT) and variant. INa was evoked from a holding potential of ‐­Coexpression of SCN1B/A197V resulted in a marked decrease in INa current density. Statistical description is provided in Table 1. C: Bar graph of peak current density indicated significant reduction for SCN5A/WT and SCN5A/WT+SCN1B/A197V when compared with SCN5A/WT+SCN1B/WT

FIGURE 3 A, B Voltage dependence of inactivation and activation of SCN5A/WT and co‐­transfection of either SCN1B/WT or SCN1B/A197V. The activation voltage dependence curve was not shifted by A197V mutation, but the inactivation voltage dependence curve was significantly shifted to the left in 5A/WT+1B/A197V compared to 5A/WT+1B/WT. Biophysical properties are provided in Table 2. C: Window current was estimated by the overlap of activation and inactivation curves. The Na+ window current was decreased in SCN1B/A197V compared to SCN1B/WT

FIGURE 4 Recovery from inactivation of 3 groups determined using the 2‐­inactivation. Biophysical properties are provided in Table 2

FIGURE 5 Fluorescence analysis and immunostaining of cell surface Nav1.5 expression. Nav1.5 protein was immunostained with anti‐­SCN5A primary antibody (red).A: Nav1.5 channels were enriched in the plasma membrane of the cell in SCN5A/WT+SCN1B/WT. Scale bar: 10 um. B: A197V mutant channels were distributed in the cytoplasm to a large extent, suggesting a trafficking impairment. C: Plasma membrane fraction of Nav1.5 showed aberrant membrane translocation in SCN5A/WT+SCN1B/A197V

Result:

Coexpression of 5A/WT+1B/A197V resulted in a marked decrease in current density compared to 5A/WT+1B/WT. The activation velocity was decelerated by A197V mutation. No significant changes were observed in recovery from inactivation parameters. Cell surface protein analyses confirmed that Nav1.5 channel membrane distribution was affected by A197V mutation.

Conclusion:

The current study is the first to report mutation in SCN1B/ A197V, serving as a substrate responsible for BrS.

AP19‐­00829

Glucose fluctuations promoted aortic fibrosis through ROS/p38 MAPK /Runx2 signaling pathway

Zhen ye Zhang, Ning Wang, Ling Ling Qian, Ling Feng Miao, Shi Peng Dang, Ying Wu, Ru Xing Wang

Wuxi People Hospital, China

Introduction:

Glucose fluctuations may be responsible for the onset of arterial hypertension. However, the mechanisms still remain unclear. The purpose of this study was to investigate the mechanisms of aortic fibrosis and aortic stiffening induced by glucose fluctuations.

FIGURE 1 Effects of glucose fluctuations on aortic function (A) Levels of blood glucose in C‐­STZ, U‐­STZ and STZ‐­ GF groups were 5 mmol/L, >25 mmol/L and 5‐­25 mmol/L respectively (n = 8 per group). (B) Body weight in three groups (n = 8 per group). (C‐­E) The systolic blood pressure (BP), mean BP and diastolic BP of C‐­STZ, U‐­STZ and STZ‐­GF groups respectively (n = 5 per group). *P < .05

FIGURE 2 Glucose fluctuations promoted aortic fibrosis in diabetic rats (A) Representative images of Masson trichrome staining of rat aortas and the quantitative ratio of the area of fibrosis (n = 3 per group). (B) Representative images of immunofluorescence staining of collagen I (n = 3 per group). (C) The mRNA levels of collagen I (n = 3 per group). (D‐­E) The protein expression of collagen I in rat aortas of three groups (n = 5 per group). *P < .05

Methods:

Sprague Dawley (SD) rats with injection of streptozotocin (STZ) were randomly divided into three groups: controlled STZ‐­induced diabetes (C‐­STZ); uncontrolled STZ‐­induced diabetes (U‐­ STZ); STZ‐­induced diabetes with glucose fluctuations (STZ‐­GF). Fluctuated blood glucose was induced by repeated fasting along with additional insulin injections. After 3 weeks, rat blood pressure was tested and aortic fibrosis was detected by Masson trichrome staining. The levels of p38 mitogen activated protein kinase (p38 MAPK), runt‐­related transcription factor 2 (Runx2), collagen type 1 (collagen I) and NADPH oxidases (NOXs) were determined by western blot. The rat vascular smooth muscle cells (VSMCs) in vitro were used to explore the underlying mechanisms.

Result:

Compared with C‐­STZ group, aortic fibrosis and aortic stiffening were aggravated in U‐­STZ group, which were more pronounced in STZ‐­GF group. The levels of p38 MAPK, Runx2, collagen I were significantly increased in STZ‐­GF group. In vitro, applications of inhibitors of reactive oxygen species (ROS) and p38 MAPK reversed glucose fluctuations‐­induced aortic fibrosis.

Conclusion:

Blood glucose fluctuations aggravate aortic fibrosis via ROS/p38 MAPK /Runx2 signaling pathway.

AP19‐­00865

Concordant increase of post‐­pacing action potential duration and contractility predicts occurrence of ventricular arrhythmia in Brugada syndrome model

Chih‐Min Liu, Fong‐Jhih Lin, Yao‐Chang Chen, Yung‐Kuo Lin, Yen‐Yu Lu, Cheng‐I Wu, Satoshi Higa, Shih‐Ann Chen, Yi‐Jen Chen

Taipei Veterans General Hospital, Taiwan

Introduction:

Mechano‐­electrical coupling from integration of action potential duration (APD) and contractility plays an important role in arrhythmogenesis but not clear in Brugada syndrome. We investigate whether distinctive mechano‐­electrical coupling may contribute to the genesis of ventricular arrhythmias (VAs) in Brugada syndrome.

Methods:

Conventional microelectrodes were used to record the electrical and mechanical activity simultaneously in right ventricular outflow tract rabbit tissue preparations before and after receiving transient outward current enhancer (NS 5806, 10 μmol/L), a ATP sensitive potassium channel opener (Pinacidil, 2 μmol/L), and a sodium channel blocker (Pilsocanide, 5 μmol/L) for Brugada model construction. Events of VAs were recorded under rapid electrical pacing (cycle lengths from 1000 to 100 ms) with or without NCX inhibitor (KB‐­R7943, 10 μmol/L).

Result:

There were 13 (12.4%) episodes of triggered ventricular tachycardias (VTs) (Group 1) and 92 (87.6%) of non‐­inducible VTs (Group 2) in Brugada model rabbits. The first occurrence of post‐­pacing APD in group 1 had increasing APD90 (APD90, 48.4 ± 8.3% vs 11.6 ± 2.0%, P = .001) and contractility (348.2 ± 56.7% vs 178.2 ± 14.8%, P < .001) than group 2 (Table 1). Triggered VT was common (75%) in those with ∆APD90 > 15% and ∆Contractility > 270%, but not detectable in ∆APD90 < 15% and ∆Contractility < 270% (Figure). A cut‐­off value of product of “∆Contractility and ∆APD90” above 1.29 × 104 (%%) would predict 100% genesis of VTs. In those with pacing‐­induced VTs, KB‐­ R7943 significantly reduced the occurrence of VT from 100.0% to 23.1% (P < .001). After multivariate analysis, the “∆Contractility × ∆APD90” (OR: 35.77, P = .039) and “∆Contractility/∆APD90” between 2 and 7 (OR: 186.93, P = .033) were independent predictors of triggered VAs (Table 2).

Conclusion:

Concordant increase of post‐­pacing APD and contractility results in the occurrence of VAs in the triggered Brugada model. NCX inhibition may be a potential therapeutic strategy for Brugada syndrome.

Table 1 Basic electrophysiological characteristics of inducible and non‐­inducible ventricular arrhythmias without KB‐­R7943 infusion

	Inducible VT (N = 13, Group 1)	Non‐­inducible VT (N = 92, Group 2)	P value
Last pacing APA, mV	67.2 ± 3.6	68.0 ± 2.7	.859
Last pacing APD20, ms	23.3 ± 2.5	23.7 ± 1.3	.918
Last pacing APD50, ms	38.2 ± 3.4	37.9 ± 1.9	.949
Last pacing APD90, ms	58.9 ± 3.3	62.2 ± 2.2	.581
First post‐­pacing APA, mV	68.3 ± 3.4	68.8 ± 2.7	.912
First post‐­pacing APD20, ms	20.0 ± 2.2	17.8 ± 1.1	.483
First post‐­pacing APD50, ms	40.5 ± 4.8	31.5 ± 1.7	.072
First post‐­pacing APD90, ms	86.8 ± 6.6	68.4 ± 2.4	.009
First post‐­pacing APD20 changed interval, ms	‐­3.3 ± 1.6	‐­5.9 ± 0.7	.173
First post‐­pacing APD50 changed interval, ms	2.3 ± 4.2	‐­6.4 ± 0.8	.064
First post‐­pacing APD90 changed interval, ms	27.9 ± 5.4	6.2 ± 1.2	.002
*First post‐­pacing APD90 changed percentage, %	48.4 ± 8.3	11.6 ± 2.0	.001
Last pacing contractility, mg	15.8 ± 2.4	18.5 ± 1.0	.352
First post‐­pacing contractility, mg	70.2 ± 13.6	52.5 ± 4.0	.135
First post‐­pacing increasing contractility, mg	54.4 ± 12.1	34.0 ± 3.3	.126
**First post‐­pacing contractility enhancement, %	348.2 ± 56.7	178.2 ±14.8	<.001
***ΔContractility x ΔAPD90, %%	17286.0 ± 4006.6	2046.8 ± 351.1	.003
VT cycle, ms	125.9 ± 8.8	‐­	‐­

Values are number of the variables ± standard error of the mean. APA, amplitude; APD, action potential duration; VT, ventricular tachycardia.

* First post‐­pacing APD90 changed percentage (%) was calculated as (First post‐­pacing APD90 ‐­ Last pacing APD90) x 100 / Last pacing APD90.

**First post‐­pacing contractility enhancement (%) was calculated as (First post‐­pacing contractility ‐­ Last pacing contractility) x 100 / First post‐­pacing contractility.

*** ΔContractility x ΔAPD90 equals to First post‐­pacing APD90 changed percentage (%) x First post‐­pacing contractility enhancement (%).

Table 2 Risk of ventricular tachycardias during the overall episodes

	Univariate analysis			Multivariate analysis*
	Odd ratio	95% CI	P value	Odd ratio	95% CI	P value
Last pacing APA, mV	1.00	0.98‐­1.02	.909
Last pacing APD20, ms	0.99	0.95‐­1.04	.778
Last pacing APD50, ms	1.00	0.97‐­1.03	.806
Last pacing APD90, ms	0.99	0.97‐­1.02	.653
First post‐­pacing APA, mV	1.00	0.98‐­1.02	.724
First post‐­pacing APD20, ms	1.02	0.97‐­1.06	.475
First post‐­pacing APD50, ms	1.02	1.00‐­1.05	.102
First post‐­pacing APD90, ms	1.03	1.01‐­1.05	.005	0.99	0.91‐­1.08	.768
First post‐­pacing APD20 changed interval, ms	1.08	0.99‐­1.18	.096	1.06	0.80‐­1.40	.683
First post‐­pacing APD50 changed interval, ms	1.09	1.03‐­1.16	.005	1.25	0.94‐­1.66	.125
First post‐­pacing APD90 changed interval, ms	1.10	1.05‐­1.15	<.001	1.34	0.85‐­2.12	.210
First post‐­pacing APD90 changed percentage, per sd 24.0%	3.69	2.06‐­6.61	<.001	0.01	0.01‐­6.56	.151
First post‐­pacing contractility enhancement, per sd 160.8%	2.62	1.57‐­4.38	<.001	7.03	1.00‐­49.37	.050
ΔContractility x ΔAPD90, per sd 6827.8%%	10.58	3.55‐­31.57	<.001	35.77	1.19‐1073.25	.039
ΔContractility / ΔAPD90 (2‐­7)	7.28	2.36‐­22.50	.001	186.93	1.54‐22763.99	.033

APA, amplitude; APD, action potential duration.

*The multivariate regression model was calculated using multiple cox regression stepwise analysis (included all variables with P < .1).

AP19‐­00940

Thalidomide as a treatment for non‐­Langerhans cell histiocytosis with cardiac infiltration: a case report

Justin R. Tan, Michelle Espiritu, Timothy Dy

Internal Medicine, Philippines

Introduction:

Non‐­Langerhans Cell histiocytoses (non‐­LCH) are a group of disorders characterized by proliferation of histiocytes with absence of Langerhans cells. Majority of non‐­LCH manifest cutaneously, but may also have systemic manifestations such as the heart. In this case, we described our experience with thalidomide in treating an extracutaneous manifestation of non‐­LCH.

Methods:

This is a case of a 35 years old male, diagnosed with non‐­Langerhans cell histiocytosis who presented with heart failure symptoms. 2D‐­Echo revealed a homogenous echogenic density measuring 5.57 × 3.12 cm with an area of 13.7 cm2 from the base of the right atrium superiorly to the mid right ventricular free wall, inferiorly abutting the tricuspid annular area. A cardiac MRI was done revealing infiltrative intrapericardial enhancing soft tissue mass below the level of the right pulmonary artery, located between the SVC and ascending aorta, surrounding the right atrium and extending into the right AV groove. Patient was initially treated with thalidomide 200 mg/d which was subsequently decreased to 50 mg/d due to elevation of creatinine. A repeat 2D‐­Echo after 2 months showed a decrease in size of the echogenic density measuring 5.55 × 2.63 cm with an area of 12.3 cm². Medications were continued while a repeat 2D‐­Echo is scheduled upon follow‐­up. Erdheim‐­Chester disease, a specific form of non‐­LCH was also a consideration hence BRAF mutation testing will be done.

Result:

Thalidomide is an immunomodulatory drug that is used for treatment of several inflammatory skin diseases including Langerhans and Non‐­LCH. Thalidomide acts by inhibiting the production of TNF‐­ alpha and IL‐­6 hence its anti‐­inflammatory and antineoplastic properties.

Conclusion:

In conclusion, our case suggests that thalidomide can be used for extracutaneous manifestations of non‐­LCH base on its partial regression. Further testing of thalidomide for treatment of extracutaneous non‐­LCH lesions is required to support the use of this treatment option.

AP19‐­00966

Dose‐­response irradiation effect on rat heart

Myung‐Jin Cha, Ji Hyun Chang, Hak Jae Kim, Jeong‐Wook Seo, Seil Oh

Seoul National University Hospital, South Korea

Introduction:

Non‐­invasive radioablation has recently been used for treatment of refractory ventricular arrhythmia in humans. However, the pathological changes of myocardium brought on by irradiation has not been well known. Dose‐­responsive histologic changes were examined after irradiation of rat hearts.

Methods:

Whole hearts of 33 wild‐­type Lewis rats were irradiated with 20, 25, 30, 40, and 50 Gy of radiation (6 mice in each group and 3 mice for control). Hearts were explanted at 2‐­week (3 mice) or 3‐­week (3 mice) after irradiation

Result:

From irradiation to 3 weeks, there were no changes in body weights of rats irradiated with 20‐­30 Gy, while a 3 and 20% serial decrease in body weight was observed in rats irradiated with 40 and 50 Gy group, respectively. There was left ventricular functional changes, but LV chamber sizes were decreased. Pathology showed Pathology showed subepithelial lymphocytic infiltration and mucosal epithelial atrophy in the trachea and esophagus, especially in 50 Gy irradiated rats. There were prominent lymphocyte and endothelial damages in all‐­dose group, esp in 3‐­week harvest rats. Focal myocyte necrosis was showed in high‐­dose 3‐­week group.

Conclusion:

High dose irradiation effect on heart is mainly caused by endothelial cells and lymphocytes damage.

AP19‐­00991

In‐­vivo safety and efficacy of the hood: a novel cardio‐­embolic protection device for catheter‐­based ablation

Omar Yasin, Thomas Ladas, Martin van Zyl, Adetola Ladejobi, Alan Sugrue, Vaibhav Vaidya, Roshini Asirvatham, Noah Schneider, Lisa Yngsdal, Renee Taubel, Joanne Pedersen, Suraj Kapa, Vance Lehman, Samuel Asirvatham

Mayo Clinic, United States

Introduction:

Radiofrequency (RF) ablation of cardiac arrhythmias is associated with a risk of stroke or silent brain lesions detected on MRI despite peri and intraprocedural anticoagulation. These result from thermal coagulum and microbubble formation at the ablation site. We previously demonstrated the potential benefit of a novel “hood” device that can contain thermal coagulum and microbubbles at the catheter‐­tissue interface which can thereby decrease the rate of embolization (Figure 1A). In this study, we aim to demonstrate the safety of this device in chronic in‐­vivo animal studies. We hypothesize that ablation using the hood device is not associated with increased risk of stroke, brain infarcts and silent brain lesions detected on MRI.

Methods:

RF ablation was performed in the left atria of three canine animals through trans‐­septal access after heparin was administered for goal activated clotting time of 300 seconds. Each animal had 5 total ablations using non‐­irrigated standard ablation catheters. Two of the three animals had ablations with our hood device at 30 W, 35 seconds, and temperature control at 50°C in animal 1 and 30 W, 45 seconds and 50°C in animal 2. Adequate positioning of our device to encompass the ablation site was confirmed on Fluoroscopy and intra‐­cardiac echocardiogram (Figure 1B). One animal served as control and RF ablation was performed without the hood at 30 W, 45 seconds and 50°C. Each animal underwent MRI of the brain at baseline and 3 days after the procedures. Diffusion‐­weighted imaging, T2 FLAIR, gradient echo and gadolinium enhanced T1 sequences were included. The frequency of brain lesions on MRI was compared before and after the procedure for each animal and with control.

FIGURE 1 Prototype device, positioning and MRI before and after left atrial ablation in second animal. A‐­ A prototype novel cardio‐­embolic protection device through which an ablation catheter can be advanced. B‐­ Intra‐­cardiac echocardiograph image demonstrating adequate position of the device in the left atrium. C‐­ A sample baseline axial diffusion‐­weighted imaging (DWI) MRI image of brain before ablation procedure. D‐­ Follow‐­up DWI of the same animal 3 days after ablation using the cardio‐­embolic protection device. E‐­ A sample axial T2 FLAIR image MRI of the same animal before ablation procedure. F‐­ Follow up T2 FLAIR image MRI of the same animal three days after ablation procedure. These images and all other images throughout the brain (not shown) were normal without evidence of infarct or brain lesion

Result:

Baseline brain MRI did not show infarcts in the animals (Figure 1C, E). All five ablation procedures were performed successfully in each animal model and all animals survived with no signs of stroke or any obvious signs of embolic phenomena. Follow up MRI did not show evidence of infarct in either of the two experimental animals or the control animal (Figure 1D, F). Left atrial ablation lesions were confirmed on gross examination of the explanted heart after day 3.

Conclusion:

The use of our hood cardio‐­embolic protection device is feasible and did not show increased risk of acute peri‐­procedural stroke or silent brain lesions detected on MRI following non‐­ irrigated left atrial ablation.

AP19‐­01029

Renewal theory provides a universal quantitative framework to characterise the continuous regeneration of phase singularities in cardiac fibrillation and accurately predicts spontaneous AF termination

Dhani Dharmaprani, Madeline Schopp, Pawel Kuklik, Darius Champan, Anandaroop Lahiri, Lukah Dykes, Feng Xiong, Martin Aguilar, Benjamin Strauss, Lewis Mitchell, Kenneth Pope, Christian Meyer, Stephan Willems, Fadi Akar, Stanley Nattel, Andrew McGavigan, Anand Ganesan

Flinders University, Australia

Introduction:

Atrial and ventricular fibrillation (AF/VF) are postulated to be maintained by rotors, with pivoting regions called phase singularities (PS). Despite a century of research, no universal quantitative framework exists to describe the generation of PS in cardiac fibrillation, and the role of this in maintaining AF/VF. Here, we develop a Poisson renewal theory framework to quantify the continuous formation and destruction of PS in cardiac fibrillation, and demonstrate for the first time that this regeneration process is responsible for the perpetuation of AF/VF. Further, we demonstrate that this framework can accurately predict spontaneous AF termination.

Methods:

PS formation/destruction was studied in 5 systems: (a) human persistent AF (n = 20), (b) tachypaced sheep AF (n = 5), (c) rat AF (n = 4), (d) rat VF (n = 11) and (e) computer simulated AF (SIM). PS survival data was fitted using maximum likelihood, and rates of PS formation and destruction (λf/λd) determined. A systematic review was conducted to cross‐­validate with source data from literature. The spatiotemporal stability of λf/λd was assessed through bivariate correlation between: (a) 5‐­minute long vs 30‐­second short duration recordings and (b) local vs global recordings of AF. The association between λf/λd and AF termination was investigated in n = 15 epochs of terminating human AF, and compared with n = 43 control epochs of sustained human AF.

Result:

PS lifetime and inter‐­formation times were consistent with underlying Poisson renewal processes (human: λf‐­4.5%/ms ± 1.1 (95% CI, 4.3, 5.0), λd‐­4.6%/ms ± 1.5 (95% CI, 4.3, 4.9); sheep: λf‐­ 4.4%/ms (95% CI, 4.1, 4.7), λd‐­ 4.6%/ms ± 1.4 (95% CI, 4.3, 4.8; rat AF: λf‐­ 33%/ms ± 8.8 (95% CI, 11, 55), λd‐­ 38%/ms (95% CI, 22, 55); rat VF: λf‐­ 38%/ms ± 24 (95% CI, 22, 55), λd‐­ 46%/ms ± 21 (95% CI, 31, 60); SIM λd 6.6‐­8.97%/ms (95% CI, 4.1, 6.7); R ² ≥ 0.90 in all cases). All PS distributions identified through systematic review were also consistent with an underlying Poisson renewal process. λf/λd was spatiotemporally stable (long vs short duration λd: R:0.99; local vs global λd: R: 0.74) and also accurately predicts spontaneous AF termination (λf_non‐­term: 4.6%/ms (95% CI, 4.3, 4.9); λf_term: 12.7%/ms (95% CI, 11.0, 14.3); λd_non‐­term: 4.7%/ms (95% CI, 4.5, 4.9); λd_non‐­term: 11.8%/ms (95% CI, 9.8, 13.8); P < .001).

Conclusion:

These data redefine AF/VF as continuous Poisson renewal processes that occur by repetitive formation and destruction of phase singularities. The rate constants of λf/λd are simple, robust, spatiotemporally stable metrics that accurately predict the likelihood of AF termination. The universality of this motif demonstrates that renewal processes are fundamental to understanding and quantifying fibrillatory dynamics with profound implications for mechanistic and clinical understanding of AF/VF.

Key words: Ventricular Fibrillation, Atrial Fibrillation, rotors, phase singularities, mapping

AP19‐­01085

Fibroblast growth factor‐­21 ameliorates cardiac dysfunction in diabetic cardiomyopathy via AMPK‐­mediated NLRP3 inflammasome pathway

Liu Yang, Peng Yu, Xiaoyang Lai

The Second Affiliated Hospital of Nanchang University, China

Introduction:

Diabetic cardiomyopathy (DCM) is one of the most severe macrovascular complications and a major cause of mortality in diabetes mellitus. To explore a new therapeutic strategy target the pathogenesis of DCM urgently needs to be resolved.

Methods:

DCM model was induced to mice in the DCM groups by streptozotocin injection. Neonatal rat cardiomyocytes were exposed to normal and high‐­concentration glucose and palmitic acid, while FGF21/AMPK expression was inhibited by siRNA interference. Real‐­time PCR, immunoblot and immunohistochemistry were performed for the expression of targeted genes/proteins.

Result:

The animal experiment showed that fenofibrate (FF) improved DCM by upregulation of FGF21. The above results suggest that FGF21 could improve DCM, but how FGF21 improved DCM and its mechanism are still unclear. In addition, exogenous administration of FGF21 DCM improvement by activating AMPK signaling pathway and inhibit the activation of NLRP3 inflammatory body in vivo. In high glucose treated primary myocardial cells, decreased FGF21 induced AMPK activation disorder and ultimately triggered NLRP3 inflammatory activation leading to pyroptosis. And, inhibition of AMPK activation by Compound C/SiRNA partially abolished FGF21‐­induced protection in cardiomyocytes.

Conclusion:

Thus, the results indicate that FGF21 mediates NLRP3 inflammasome via AMPK inhibition, and further consolidate the evidence for the FGF21/analog being a pharmacotherapeutic target for T2DM and its related DCM.

AP19‐­01096

Cardiac emerinopathy, novel non‐­syndromic X‐­linked left ventricular non‐­compaction associated with progressive atrial conduction disturbance

Taisuke Ishikawa, Hiroyuki Mishima, Julien Barc, Keiichi Hirono, Shigenori Terada, Shinya Kowase, Teruki Sato, Yasushi Mukai, Yoshiaki Yui, Kimie Ohkubo, Hiroki Kimoto, Hiroyuki Watanabe, Yukiko Hata, Takeshi Aiba, Seiko Ohno, Akiko Chishaki, Wataru Shimizu, Minoru Horie, Fukiko Ichida, Akihiko Nogami, Koh‐ichiro Yoshiura, Jean‐Jacques Schott, Naomasa Makita

National Cerebral and Cardiovascular Center, Japan

Introduction:

Left ventricular noncompaction (LVNC) is a rare cardiomyopathy often associated with other heart diseases, including other cardiomyopathies and arrhythmias, as well as neuromuscular diseases. More than 40 genes are involved in LVNC, but their causal relations and clinical presentations are highly variable. Recently, a strong genotype–phenotype correlation was reported in LVNC associated with some forms of cardiac conduction disturbance, including familial sick sinus syndrome (SSS) and atrioventricular block. We aimed to identify a genetic basis for the novel electromechanical disorder characterized by X‐­linked LVNC associated with cardiac conduction disturbance.

Methods:

Targeted exon sequencings were performed in three cohorts, including 87 probands diagnosed with familial SSS (n = 36) or a progressive cardiac conduction defect (n = 51), and pediatric LVNC probands (n = 102). Sanger sequencing, family cascade screening and clinical records were performed to ensure the involvement of EMD mutations as the X‐­linked inheritance of cardiac conduction disturbance and LVNC in these families.

Result:

Among 36 familial SSS and 51 PCCD probands, we found three hemizygous mutations (stop‐­ loss, splicing, missense) in EMD on chromosome Xq28, which encodes for the inner nuclear membrane protein emerin and is responsible for Emery–Dreifuss muscular dystrophy. To determine if EMD is a novel gene responsible for LVNC, we further genetically screened 102 pediatric LVNC patients, and identified a frameshift mutation in a boy with LVNC complicated by atrial standstill. The probands of four families were male, sharing a common clinical phenotype of LVNC associated with progressive atrial standstill but lacking skeletal muscle abnormalities and the elevation of serum creatine kinase level. They underwent pacemaker or defibrillator implantation. Two of them had episodes of cerebral infarction due to the synergistic thromboembolic risks attributable to LVNC and atrial standstill.

Conclusion:

Cardiac emerinopathy is a novel non‐­syndromic X‐­linked LVNC associated with progressive atrial conduction disturbance and increased risk of thromboembolism.

AP19‐­01099

Effect of hyperglicemia on the T wave inversion

Jennifer Michelle Widysanto, Antonia Anna Lukito

Siloam Hospital Lippo Village, Indonesia

Introduction:

Hyperglicemia is a poor predictor for CAD patients post PCI which is associated with an increased risk of death and major adverse cardiovascular events.

Methods:

A hypertensive and diabetic 55 years old female came with mild dyspnea on exertion. Her blood glucose is not well controlled with HbA1C at 9.7% due to not adherence to therapy. ECG showed Q‐­ wave on V1‐­V3. Coronary CTA was performed and showed significant lesion at LAD and first diagonal (D1). Elective PCI with two DES strategy was done at bifurcation lesion of LAD‐­D1. On the follow‐­up consultation, her blood glucose was not improving with HbA1C at 10.6% and the follow‐­up ECG also keep showing deep T‐­wave inversion. Her HbA1C eventually improved to 9.6%, with random blood glucose at 158 mg/dL at the third follow‐­up visit (4 months after PCI) and the T‐­wave became normal at the same time.`

Result:

This patient has a problem of uncontrolled hyperglycemia after PCI with metformin, linagliptin, dapagliflozin and insulin glargine. The ECG changes after bifurcation stenting was not resolved during her blood glucose kept on at high level. When the blood glucose getting better, at the same time the ECG back to normal. The hyperglicemia effect on T‐­wave inversion might be due to several mechanisms, including shortening of the fibrinogen half‐­life, increased of fibrinopeptide A, fragments of pro‐­ thrombin in factor VII, and platelet aggregation, all of which could lead to increased activation of thrombosis. Hyperglycemia also associated with increased levels of inflammatory markers, enhanced expression of cytotoxic T‐­cells, and reduced expression of cytotoxic T‐­lymphocyte‐­associated protein, indicating the role of inflammatory immune process. Furthermore, it also could be associated with endothelial dysfunction, oxidative stress, and possibly, abolition of protective ischemic preconditioning.

Conclusion:

Hyperglicemia might have contribution in the persistent T‐­wave inversion after coronary stenting via several mechanisms. Therefore, blood glucose lowering to desired target is an important factor included in the management strategies in improving both short‐­term and long‐­term outcomes after coronary stenting in diabetic patients.

AP19‐­01101

An optimal approach for optogenetic to control function of cardiac and neural cell in vitro and in vivo

Yen Ling Sung, Shien‐Fong Lin

National Chiao Tung University, Taiwan

Introduction:

Optogenetic, which is a novel and useful tool due to its unique ability to act on specific neuronal groups, has gained wide application in basic and clinical medical researches. It can manipulate different groups of specific neurons at the same time at the right location. Therefore, our novel objective of the study is to use optogenetics to specifically dissect the complex network of sympathetic and parasympathetic nerve functions in cardiac diseases. However, the primary goal of this study was to produce neural progenitor cells from human induced pluripotent stem cells (iPSC) with a differentiation protocol established for the co‐­culture system. This research project is a bold first attempt to further develop these important experimental techniques toward optogenetic applications in neurocardiology.

Methods:

AAV vectors have successfully manipulated CNS function using a wide variety of approaches including expression of foreign genes, expression of endogenous genes. With the discovery and characterization of different AAV serotypes, the potential patterns of in vivo vector transduction have been expanded substantially, offering alternatives to the more studied AAV 2 serotype.

Result:

Schematic depicts neural differentiation process and the conversion of I into ChR2‐­expressed motor neurons. Optogenetically controlled with ChR2‐­expressing iPSC–derived motor neurons when they were under 430 nm blue light stimulation. Following optogenetic activation of neurons, the motor neuron derived from iPSC were quickly demonstrated action potentials, while immediately down after stimulation. The ChR2‐­expressed neurons exposed to 430 nm blue light stimulation enabled regulation of membrane voltage and depolarized the cells. The results confirm that we generated iPSC into functional neurons. The efficiency of transfection is a key point to dominated successful or fail treatment. Cardiomyocytes is non‐­monolayer cell type and it is connected to each layer and cells. However, it is interesting that when one of cardiomyocyte is stimulated, the neighborhood is also changed action potential at the same time. After AAV virus injection for 4‐­6 weeks, the heart is excised, perfused with Tyrode's buffer ex vivo. However, when the heart is illuminated by blue light, the heart is coordinated with stimulation.

Conclusion:

We derived motor neuron form iPSC and they can from physical and functional connections with smooth muscles cells. We aimed to further probe the effect of optogenetic in iPSC–derived motor neurons, specifically determining the sufficiency of this optogene function for the development of the in vitro phenotypes. We adopted a co‐­culture strategy for this purpose, providing a valuable tool in the future. The potential of iPSC could be further valorized by generating other cell types that may be relevant to the pathology and more clinically relevant.

AP19‐­01125

Algorithmic auto‐­recreation system of hiPSC‐­CMs simulation and prediction of drug testing

Hirohiko Kohjitani, Shigeya Kouda, Takeru Makiyama, Tomohiko Imamura, Takanori Aizawa, Asami Kashiwa, JingShan Gao, Hai Huang, Yimin Wuriyanghai, Yuta Yamamoto, Satoshi Sizuta, Takeshi Kimura, Akinori Noma

Kyoto University Hospital, Japan

Introduction:

Mathematical optimization is core technology which makes fundamental of “AI”. We use this for analysis of action potentials (APs) from human induced pluripotent stem cell derived cardiomyocytes (hiPSC‐­CMs) , in order to understand its heterogeneity and its complicated system of many ion‐­channels. HiPSC‐­CMs show different action potential (AP) morphology among cells and cell lines, and electrophysiological reaction for drug is also different among cells. In order to understand those phenomena, mathematical models for AP analyzation is needed. So, we aimed to construct precise model that is compatible for drug testing and molecular disease prediction using mathematical optimization method.

Methods:

For analysis of APs from hiPSC‐­CMs, we developed comprehensive hiPSC‐­CMs mathematical models, and applicated mathematical optimization for this model, and explored a method for simulational re‐­creation of APs. We recorded APs from 50 hiPSC‐­CMs and emulated all AP morphologies simulationally by changing conductance of each ion current, using autonomic mathematical fitting method. After that, we compared AP morphological change between in‐­silico and in‐­ vitro IKr‐­ blocking test.

Result:

All 50 AP morphologies were successfully recapitulated in ±1 mV error range of each points. Morphological change of AP after E4031 application was well recapitulated. APD90 was 269 ms (in‐­vitro AP) vs 274 ms (in‐­silico AP). After application of IKr‐­blocking, APD90 was prolonged to 463 vs 440 ms, respectively.

Conclusion:

In‐­silico approach that includes algorithmic emulating simulation system enables prediction of in‐­vitro drug testing using hiPSC‐­CMs.

AP19‐­01230

The physiological understanding of Coenzyme Q10 treatment in patients undergoing cardiac surgery: could an antioxidant reduce the incidence of reperfusion arrhythmia?

Louisa Fadjri Kusuma Wardhani, Ivana P. Dewi, Djoko Soemantri

Universitas Airlangga, Indonesia

Introduction:

Reperfusion arrhythmia, as part of myocardial injury, during cardiac operation has long been investigated. Oxygen free radicals, identified in re‐­oxygenated myocardium, are belief to play a significant role in reperfusion injury. Reactive oxygen intermediates arrhythmogenic oscillation in membrane potential. Coenzyme Q10, a lipid‐­soluble antioxidant, inhibiting lipid peroxidation in biological membranes and supplying ATP cell production, that is necessary as the basic energy source for the organism. This mechanism explains the role of CoQ10 in membrane stabilization and metabolite essential depletion prevention, which further induces the incidence of reperfusion arrhythmia.

Methods:

The aim of this study is to determine the effect of coenzyme Q10 treatment in reducing reperfusion arrhythmia among patients undergoing cardiac surgery. We searched Pubmed database using the following keywords: Coenzyme Q10, CoQ10, Ubiquinone, Heart Surgery, Cardiac Surgery, Arrhythmia, and Reperfusion Injury. Articles were then systematically collected, assessed, and analyzed for this review. Two good quality clinical studies met our inclusion criteria with total of 70 patients for analysis.

Result:

Both studies showed pre‐­cardiac surgery adjunctive treatment using Coenzyme Q10 were significantly associated with good outcome. Patients undertake Coenzyme Q10 were less likely to have reperfusion arrhythmia [OR (95% CI) 0.03 (0.00‐­0.16); P < .0001].

Conclusion:

These conclude that coenzyme Q10 could reduce the incidence of reperfusion arrhythmia in patients undergoing cardiac surgery.

Keywords: Coenzyme Q10, ubiquinone, cardiac surgery, arrhythmia, reperfusion injury

Table 1 Description of the study

Author (year)	Setting	Method	Patients	Outcome
Chello (1994)	Italy	Retrospective	40 patients	Pretreatment with CoQ10 significantly reduced reperfusion arrhythmia
Makhija (2008)	India	Retrospective	30 patients	A significantly greater amount of arrhythmia among control group compared with long term orally supplemented CoQ10 group

AP19‐­01233

Weight fluctuation induces atrial substrate by fibro‐­fatty depositions, myolysis, and residual electro‐­structural remodelling in an ovine model

Thomas Agbaedeng, Darragh Twomey, Shivshankar Thanigaimani, Adrian Elliott, Dominik Linz, Dennis Lau, Rajiv Mahajan, Prashanthan Sanders

The University of Adelaide, Australia

Introduction:

Obesity‐­mediated epicardial adipose tissue (EAT) expansion drives the deposition of fibro‐­fatty infiltrates, which form the unique substrate for atrial fibrillation (AF). The LEGACY study showed the benefits of weight loss but an attenuated response with weight fluctuation. How fluxes in weight impacts the atrial substrate remains unknown. Here, we investigated cardiac adiposity and the atrial substrate due to weight fluctuation.

Methods:

We studied 24 sheep in 3 equal groups over 80 weeks: 1. Obesity was induced by high calorie diet fed ad libitum; 2. Weight fluctuation by 20‐­week cycle of weight gain/loss; and 3. Lean controls maintained at baseline weight. All sheep underwent: daily weight measurement; haemodynamic and imaging assessments (CMRI & DEXA); electrophysiological studies; and histological, and structural analysis.

Result:

The Table shows the group differences. Compared to reference controls, obesity demonstrated: Increased atrial volume and pressure, abnormal atrial electrical properties, expanded EAT and ensuing fibro‐­fatty infiltrations, and myolysis of myocytes. Despite comparable weight and EAT with controls, weight fluctuation resulted in extensive and severe fibro‐­fatty infiltrations, and twofold greater myolysis that persisted. More importantly, fibro‐­fatty infiltrates strongly correlated with increased atrial volume and pressure; fractionated electrograms (r = .71, P < .001) and conduction slowing (r = −.59, P = .006). Similarly, atrial myolysis exhibited significant correlations with atrial enlargement and haemodynamics, and electrical substrates (P < .05 for all).

Conclusion:

Despite final weight loss and non‐­expanded epicardial fat depot, weight fluctuation demonstrates residual electro‐­structural, fibro‐­fatty deposition, and abnormal contractile substrates, similar to stable chronic obesity, but to a less severe extent. This may indicate an important mechanism for atrial fibrillation in subjects with weight fluctuation.