Dual Dysfunction of Kir2.1 Underlies Conduction and Excitation-Contraction Coupling Defects Promoting Arrhythmias in a Mouse Model of Andersen-Tawil Syndrome Type 1

Andersen-Tawil Syndrome (ATS) is associated with life threatening arrhythmias of unknown mechanism. We report on a mouse model carrying the trafficking-deficient mutant Kir2.1Δ314-315. The mouse recapitulates the electrophysiological phenotype of type 1 (ATS1), with slower conduction velocities in response to flecainide, QT prolongation exacerbated by isoproterenol, and increased vulnerability to calcium-mediated arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia (CPVT). Kir2.1Δ314-315 expression significantly reduced inward rectifier K+ and Na+ inward currents, depolarized resting membrane potential and prolonged action potential duration. Immunolocalization in wildtype cardiomyocytes and skeletal muscle cells revealed a novel sarcoplasmic reticulum (SR) microdomain of functional Kir2.1 channels contributing to intracellular Ca2+ homeostasis. Kir2.1Δ314-315 cardiomyocytes showed defects in SR Kir2.1 localization and function, which contributed to abnormal spontaneous Ca2+ release events. This is the first in-vivo demonstration of a dual arrhythmogenic mechanism of ATS1 defects in Kir2.1 channel function at the sarcolemma and the SR, with overlap between ATS1 and CPVT.

Atrial fibrillation impairs ventricular function by altering excitation-contraction coupling in the human heart

Atrial fibrillation (AF) often co-exists in patients with heart failure (HF). Recent clinical evidence suggests that the arrhythmic component of AF alone may contribute to ventricular dysfunction. However, the pathophysiological effects of a non-tachycardic AF on the human ventricle are unknown. To investigate the effects of normofrequent AF on the human ventricle we investigated ventricular myocardium from patients with preserved ejection fraction with sinus rhythm (SR) or AF in the absence of HF (compensated hypertrophy, EF>50%, matched clinical characteristics). In histological analysis we detected no difference between SR (n=9) vs. AF (n=6) regarding the amount and distribution of fibrosis. For functional investigation, Ca-handling was studied (Fura-2 AM). While systolic Ca-transient amplitude was in trend reduced in isolated human ventricular AF cardiomyocytes, we found a significantly prolonged Ca-elimination time (n=17–22 cells/4 pat.). Using caffeine application, a decreased SR Ca-load in AF was detected, which may be explained by a significant decrease in SERCA2a activity (ksys-kCaff, n=10–12/4 pat.). Patch-clamp experiments revealed a prolonged action potential duration in AF cardiomyocytes (n=5/15 cells). For the standardized evaluation of the mechanisms of persistent normofrequent arrhythmia, we simulated AF in vitro by using arrhythmic (1 Hz, 40% R-R-variability) or rhythmic (1 Hz) field stimulation. We performed contractility experiments using in-toto isolated human ventricular trabeculae from explanted human hearts. After 8h of pacing, arrhythmically stimulated human trabeculae showed a significantly reduced systolic force, an increase in diastolic tension and a prolonged relaxation (n=11–12 trabeculae/11 pat.). For studying the cellular effects of persistent normofrequent arrhythmia in a model suitable for chronic pacing (up to 7 days), we utilized human iPSC cardiomyocytes (iPSC-CM) from healthy donors (n=6). After 7 days, arrhythmic paced iPSC-CM showed a significantly reduced systolic Ca-transient amplitude, a prolonged Ca-elimination time (n=35/45 cells) as well as a reduced SR Ca-load and a trend towards a lower SERCA2a activity compared to control (n=11 cells). Confocal line-scans (Fluo-4 AM) showed an increased diastolic SR Ca-release, which might also explain the reduced SR Ca-content (n=45/35 cells). Moreover, in irregularly paced iPSC-CM we found significant increased levels of cytosolic Na (n=69 cells each) and in patch-clamp experiments a significantly prolonged action potential duration (n=14/11 cells/3 diff.). This study demonstrates that a normofrequent arrhythmic ventricular excitation as it occurs in AF impairs human ventricular myocardial function by altering cardiomyocyte excitation-contraction coupling. Thus, this study provides the first translational mechanistic characterization and the potential negative impact of isolated AF in the absence of tachycardia on the human ventricle. Funding Acknowledgement Type of funding source: None

Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates

Background: The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. Methods: hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. Results: Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% ( P <0.001, n=15), increased Ca2+ alternans by 300% ( P <0.001, n=18), and promoted spontaneous calcium release activity (n=14, P <0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1β (98%, P <0.0001) and IL-6 (460%, P <0.02) compared with nonfailing hMSCs. IL-1β or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti–IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. Conclusions: We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti–IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.

Comparative Analysis of Genetic Variations in the Nav1.5 Sodium Channel Subunits that Underlie Brugada Syndrome Using Patient-Specific iPSC-CMs

Background: Brugada syndrome (BrS) is an autosomal dominant disorder that causes a high predisposition to sudden cardiac death. Several genes have been reported to be associated with BrS. Considering that the heterogeneity in clinical manifestations may result from genetic variations, the application of patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (CMs) may help to reveal cell phenotype characteristics resulting from different genetic backgrounds. The present study was to compare the structural and electrophysiological characteristics of sodium channel subunits with different genetic variations and evaluate the safety of quinidine for use with BrS patient-specific iPSC-derived cardiomyocytes.Methods: Two BrS patient-specific iPS cell lines were constructed that carried missense mutations in SCN5A and SCN1B. One iPS cell line from a healthy volunteer was used as a control. The differentiated cardiomyocytes from the three groups were evaluated by flow cytometry, immunofluorescence staining, electron microscopy, as well as calcium transient and patch clamp analyses to assess different pathological phenotypes. Finally, we evaluated the drug responses to varying concentrations of quinidine by measuring the action potential.Results: Compared to the control group, BrS-CMs showed a significant reduction in sodium current, prolonged action potential duration and varying degrees of decreased Vmax, but no structural difference was observed. After applying different concentrations of quinidine, the disease-specific groups and the control group had a downward trend in maximal upstroke velocity, resting membrane potential and action potential amplitude, and exhibited prolonged action potential duration without increasing incidence of arrhythmic events.Conclusion: Both patient-specific iPSC-CMs recapitulated the BrS phenotype at the cellular level. Although the SCN5A variation led to a markedly lower sodium current than what was observed with the SCN1B variation, their responses to quinidine were quite similar. The present study provides an advantageous platform for exploring disease mechanisms and evaluating drug safety in vitro.

NOS1AP polymorphisms reduce NOS1 activity and interact with prolonged repolarization in arrhythmogenesis

Aims NOS1AP single-nucleotide polymorphisms (SNPs) correlate with QT prolongation and cardiac sudden death in patients affected by long QT syndrome type 1 (LQT1). NOS1AP targets NOS1 to intracellular effectors. We hypothesize that NOS1AP SNPs cause NOS1 dysfunction and this may converge with prolonged action-potential duration (APD) to facilitate arrhythmias. Here we test (i) the effects of NOS1 inhibition and their interaction with prolonged APD in a guinea pig cardiomyocyte (GP-CMs) LQT1 model; (ii) whether pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from LQT1 patients differing for NOS1AP variants and mutation penetrance display a phenotype compatible with NOS1 deficiency. Methods and results In GP-CMs, NOS1 was inhibited by S-Methyl-L-thiocitrulline acetate (SMTC) or Vinyl-L-NIO hydrochloride (L-VNIO); LQT1 was mimicked by IKs blockade (JNJ303) and β-adrenergic stimulation (isoproterenol). hiPSC-CMs were obtained from symptomatic (S) and asymptomatic (AS) KCNQ1-A341V carriers, harbouring the minor and major alleles of NOS1AP SNPs (rs16847548 and rs4657139), respectively. In GP-CMs, NOS1 inhibition prolonged APD, enhanced ICaL and INaL, slowed Ca2+ decay, and induced delayed afterdepolarizations. Under action-potential clamp, switching to shorter APD suppressed ‘transient inward current’ events induced by NOS1 inhibition and reduced cytosolic Ca2+. In S (vs. AS) hiPSC-CMs, APD was longer and ICaL larger; NOS1AP and NOS1 expression and co-localization were decreased. Conclusion The minor NOS1AP alleles are associated with NOS1 loss of function. The latter likely contributes to APD prolongation in LQT1 and converges with it to perturb Ca2+ handling. This establishes a mechanistic link between NOS1AP SNPs and aggravation of the arrhythmia phenotype in prolonged repolarization syndromes.

Abstract 117: Chronic Efficacy of Liposome-Encapsulated Hemoglobin (HbV) on Rat’s Lethal 85% Hemorrhage: Intraosseus HbV Transfusion Acutely Rescues Through Anti-Arrhythmogenic Effects on Myocardium That Continues in Chronic Phase

Introduction: Liposome-encapsulated hemoglobin vesicles (HbVs) can serve as a blood substitute with human blood. Method: To investigate the resuscitation effect of HbVs on lethal hemorrhage and their efficacy for decreasing myocardial arrhythmogenesis, optical mapping analysis (OMP) and an electrophysiological study (EPS) were performed in graded 85% hemorrhage in rats in both acute and chronic phase. Six rats were resuscitated by intraosseous infusion of 5% albumin solution (ALB group), while 6 rats by washed rat erythrocytes (wRBC group) and 6 rats by HbVs (HbV group). Survival effects over 24 hours were examined in > 10 rats in each group. After excising the heart, OMP and an EPS were performed. Results: All rats died in ALB group, whereas all survived subsequent 24 hours in wRBC group and HbV group. In acute phase, OMP showed impaired (prolonged) action potential duration dispersion (APDd) in left ventricle in ALB group. In contrast, myocardial APDds in left ventricle were substantially attenuated in HbV group and wRBC group. Lethal arrhythmias (ventricular tachycardia [VT] or ventricular fibrillation [VF], VT/VF) were provoked by EPS in ALB group. No VT/VF was induced in both wRBC group and HbV group. In chronic phase of two weeks after the resuscitation, OMP and an EPS were performed again in excising the heart. Anti-arrhythmic effects were confirmed by normal OMP findings (Normal APDd) and No VT/VF induced as shown in Figure. Conclusions: Lethal hemorrhage causes VT/VF in the presence of impaired APDd. Intraosseous HbVs infusion acutely rescues lethal hemorrhage through preventing lethal arrhythmias with preserved APDd. These effects were preserved in chronic phase.

Abstract 193: Intraosseous Transfusion With Liposome-Encapsulated Hemoglobin (HbV) Comparably Improves Rat Survival and its Arrythmogenesis Undergoing Progressive Lethal 85% Hemorrhage With Central Venous Infusion: Possible Implication for Pre-Hospital Settings

Liposome-encapsulated hemoglobin (HbV) can serve as blood substitute in hemorrhagic shock (HS). To investigate the resuscitation effect of HbV through intraosseous transfusion (IO) on lethal hemorrhage and its efficacy on myocardial arrhythmogenesis, optical mapping analysis (OMP) and electrophysiological study (EPS) were performed in graded blood exchange up to 85% blood loss in rats. And it was compared with central venous infusion (CVI). Total 90 rats were randomly allocated into 6 subgroups as followings; through IO or CVI, gradually exchanged blood with 5% albumin (Alb-IO/CVI-groups), exchanged with washed rat erythrocyte (wRBC-IO/CVI-groups) and with HbV (HbV-IO/CVI-groups). Survival effects were examined in each six rat groups. After excising the heart, OMP and EPS were performed. All rats died in Alb-IO/CVI-groups whereas excellent and comparably survived for following >48-hours in wRBC-IO/CVI-groups and HbV-IO/CVI-groups (Figure 1). OMP revealed impaired (prolonged) action potential duration (APD) dispersion in LV in Alb-IO/CVI-groups. In contrast, myocardial APD dispersions in LV were substantially attenuated in HbV-IO/CVI-groups and wRBC-IO/CVI-groups (Figure 2). Lethal arrhythmias (VT/VF) were provoked by EPS in Alb-IO/CVI-groups. No VT/VF was induced in both HbV-IO/CVI-groups and wRBC-IO/CVI-groups. Conclusions: IO vascular access could a reliable bridging method to resuscitate lethal HS by HbV. This suggests that IO with HbV resuscitation might be useful in pre-hospital settings in HS through preventing lethal arrhythmias.

Role of KATP channel in electrical depression and asystole during long-duration ventricular fibrillation in ex vivo canine heart

Long-duration ventricular fibrillation (LDVF) in the globally ischemic heart is characterized by transmurally heterogeneous decline in ventricular fibrillation rate (VFR), emergence of inexcitable regions, and eventual global asystole. Rapid loss of both local and global excitability is detrimental to successful defibrillation and resuscitation during cardiac arrest. We sought to assess the role of the ATP-sensitive potassium current ( IKATP) in the timing and spatial pattern of electrical depression during LDVF in a structurally normal canine heart. We analyzed endo-, mid-, and epicardial unipolar electrograms and epicardial optical recordings in the left ventricle of isolated canine hearts during 10 min of LDVF in the absence (control) and presence of an IKATP blocker glybenclamide (60 μM). In all myocardial layers, average VFR was the same or higher in glybenclamide-treated than in control hearts. The difference increased with time of LDVF and was overall significant in all layers ( P < 0.05). However, glybenclamide did not significantly affect the transmural VFR gradient. In epicardial optical recordings, glybenclamide shortened diastolic intervals, prolonged action potential duration, and decreased the percentage of inexcitable area (all differences P < 0.001). During 10 min of LDVF, asystole occurred in 55.6% of control and none of glybenclamide-treated hearts ( P < 0.05). In three hearts paced after the onset of asystole, there was no response to LV epicardial or atrial pacing. In structurally normal canine hearts, IKATP opening during LDVF is a major factor in the onset of local and global inexcitability, whereas it has a limited role in overall deceleration of VFR and the transmural VFR gradient.