Electric Field-Based Spatial Analysis of Noncontact Unipolar Electrograms to Map Regional Activation-Repolarization Intervals.

BACKGROUND: Spatial heterogeneity in repolarization plays an important role in generating and sustaining cardiac arrhythmias. Reliable determination of repolarization times remains challenging. OBJECTIVES: The goal of this study was to improve processing of densely sampled noncontact unipolar electrograms to yield reliable high-resolution activation and repolarization maps. METHODS: Endocardial noncontact unipolar electrograms were both simulated and recorded in pig left ventricle. Electrical activity on the endocardial surface was processed in terms of a pseudo-electric field. Activation and repolarization times were calculated by using an amplitude-weighted average on QRS and T waves (ie, the E-field method). This was compared vs the conventional Wyatt method on unipolar electrograms. Timing maps were validated against timing on endocardial action potentials in a simulation study. In vivo, activation and repolarization times determined by using this alternative E-field method were validated against simultaneously recorded endocardial monophasic action potentials (MAPs). RESULTS: Simulation showed that the E-field method provides viable measurements of local endocardial action potential activation and repolarization times. In vivo, correlation of E-field activation times with MAP activation times (rE = 0.76; P < 0.001) was similar to those of Wyatt (rWyatt = 0.80, P < 0.001; P[h1:rE > rWyatt] = 0.82); for repolarization times, correlation improved significantly (rE = 0.96, P < 0.001; rWyatt = 0.82, P < 0.001; P[h1:rE > rWyatt] < 0.00001). This resulted in improved correlations of activation-repolarization intervals to endocardial action potential duration on MAP (rE = 0.96, P < 0.001; rWyatt = 0.86, P < 0.001; P[h1:rE > rWyatt] < 0.00001). Spatial beat-to-beat variation of repolarization could only be calculated by using the E-field methodology and correlated well with the MAP beat-to-beat variation of repolarization (rE = 0.76; P = 0.001). CONCLUSIONS: The E-field method substantially enhances information from endocardial noncontact electrogram data, allowing for dense maps of activation and repolarization times and derived parameters.

The ryanodine receptor microdomain in cardiomyocytes.

The ryanodine receptor type 2 (RyR) is a key player in Ca2+ handling during excitation-contraction coupling. During each heartbeat, RyR channels are responsible for linking the action potential with the contractile machinery of the cardiomyocyte by releasing Ca2+ from the sarcoplasmic reticulum. RyR function is fine-tuned by associated signalling molecules, arrangement in clusters and subcellular localization. These parameters together define RyR function within microdomains and are subject to disease remodelling. This review describes the latest findings on RyR microdomain organization, the alterations with disease which result in increased subcellular heterogeneity and emergence of microdomains with enhanced arrhythmogenic potential, and presents novel technologies that guide future research to study and target RyR channels within specific microdomains.

Heterogeneity of Repolarization and Cell-Cell Variability of Cardiomyocyte Remodeling Within the Myocardial Infarction Border Zone Contribute to Arrhythmia Susceptibility.

BACKGROUND: After myocardial infarction, the infarct border zone (BZ) is the dominant source of life-threatening arrhythmias, where fibrosis and abnormal repolarization create a substrate for reentry. We examined whether repolarization abnormalities are heterogeneous within the BZ in vivo and could be related to heterogeneous cardiomyocyte remodeling. METHODS: Myocardial infarction was induced in domestic pigs by 120-minute ischemia followed by reperfusion. After 1 month, remodeling was assessed by magnetic resonance imaging, and electroanatomical mapping was performed to determine the spatial distribution of activation-recovery intervals. Cardiomyocytes were isolated and tissue samples collected from the BZ and remote regions. Optical recording allowed assessment of action potential duration (di-8-ANEPPS, stimulation at 1 Hz, 37 °C) of large cardiomyocyte populations while gene expression in cardiomyocytes was determined by single nuclear RNA sequencing. RESULTS: In vivo, activation-recovery intervals in the BZ tended to be longer than in remote with increased spatial heterogeneity evidenced by a greater local SD (3.5±1.3 ms versus remote: 2.0±0.5 ms, P=0.036, npigs=5). Increased activation-recovery interval heterogeneity correlated with enhanced arrhythmia susceptibility. Cellular population studies (ncells=635-862 cells per region) demonstrated greater heterogeneity of action potential duration in the BZ (SD, 105.9±17.0 ms versus remote: 73.9±8.6 ms; P=0.001; npigs=6), which correlated with heterogeneity of activation-recovery interval in vivo. Cell-cell gene expression heterogeneity in the BZ was evidenced by increased Euclidean distances between nuclei of the BZ (12.1 [9.2-15.0] versus 10.6 [7.5-11.6] in remote; P<0.0001). Differentially expressed genes characterizing BZ cardiomyocyte remodeling included hypertrophy-related and ion channel-related genes with high cell-cell variability of expression. These gene expression changes were driven by stress-responsive TFs (transcription factors). In addition, heterogeneity of left ventricular wall thickness was greater in the BZ than in remote. CONCLUSIONS: Heterogeneous cardiomyocyte remodeling in the BZ is driven by uniquely altered gene expression, related to heterogeneity in the local microenvironment, and translates to heterogeneous repolarization and arrhythmia vulnerability in vivo.

Regional beat-to-beat variability of repolarization increases during ischemia and predicts imminent arrhythmias in a pig model of myocardial infarction.

Ventricular arrhythmia (VT/VF) can complicate acute myocardial ischemia (AMI). Regional instability of repolarization during AMI contributes to the substrate for VT/VF. Beat-to-beat variability of repolarization (BVR), a measure of repolarization lability increases during AMI. We hypothesized that its surge precedes VT/VF. We studied the spatial and temporal changes in BVR in relation to VT/VF during AMI. In 24 pigs, BVR was quantified on 12-lead electrocardiogram recorded at a sampling rate of 1 kHz. AMI was induced in 16 pigs by percutaneous coronary artery occlusion (MI), whereas 8 underwent sham operation (sham). Changes in BVR were assessed at 5 min after occlusion, 5 and 1 min pre-VF in animals that developed VF, and matched time points in pigs without VF. Serum troponin and ST deviation were measured. After 1 mo, magnetic resonance imaging and VT induction by programmed electrical stimulation were performed. During AMI, BVR increased significantly in inferior-lateral leads correlating with ST deviation and troponin increase. BVR was maximal 1 min pre-VF (3.78 ± 1.36 vs. 5 min pre-VF, 1.67 ± 1.56, P < 0.0001). After 1 mo, BVR was higher in MI than in sham and correlated with the infarct size (1.43 ± 0.50 vs. 0.57 ± 0.30, P = 0.009). VT was inducible in all MI animals and the ease of induction correlated with BVR. BVR increased during AMI and temporal BVR changes predicted imminent VT/VF, supporting a possible role in monitoring and early warning systems. BVR correlated to arrhythmia vulnerability suggesting utility in risk stratification post-AMI.NEW & NOTEWORTHY The key finding of this study is that BVR increases during AMI and surges before ventricular arrhythmia onset. This suggests that monitoring BVR may be useful for monitoring the risk of VF during and after AMI in the coronary care unit settings. Beyond this, monitoring BVR may have value in cardiac implantable devices or wearables.

Reversal of Right Ventricular Remodeling After Correction of Pulmonary Regurgitation in Tetralogy of Fallot.

In the sheep model with pathophysiologic changes similar to patients with repaired TOF, severe PR leads to fibrotic changes in the RV. Pulmonary valve replacement reverses these fibrotic changes. Early valve replacement led to a quick RV recovery, and in time there was no difference in outcome between early and late valve replacement. These data support the benefit of valve replacement for RV function and suggest that there is a margin in the timing of the surgery. The fibrotic changes correlated well with the circulating biomarker PICP, which can have an added value in the clinical follow-up of patients with repaired TOF.

InsP3R-RyR channel crosstalk augments sarcoplasmic reticulum Ca2+ release and arrhythmogenic activity in post-MI pig cardiomyocytes.

Ca2+ transients (CaT) underlying cardiomyocyte (CM) contraction require efficient Ca2+ coupling between sarcolemmal Ca2+ channels and sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channels (RyR) for their generation; reduced coupling in disease contributes to diminished CaT and arrhythmogenic Ca2+ events. SR Ca2+ release also occurs via inositol 1,4,5-trisphosphate receptors (InsP3R) in CM. While this pathway contributes negligeably to Ca2+ handling in healthy CM, rodent studies support a role in altered Ca2+ dynamics and arrhythmogenic Ca2+ release involving InsP3R crosstalk with RyRs in disease. Whether this mechanism persists in larger mammals with lower T-tubular density and coupling of RyRs is not fully resolved. We have recently shown an arrhythmogenic action of InsP3-induced Ca2+ release (IICR) in end stage human heart failure (HF), often associated with underlying ischemic heart disease (IHD). How IICR contributes to early stages of disease is however not determined but highly relevant. To access this stage, we chose a porcine model of IHD, which shows substantial remodelling of the area adjacent to the infarct. In cells from this region, IICR preferentially augmented Ca2+ release from non-coupled RyR clusters that otherwise showed delayed activation during the CaT. IICR in turn synchronised Ca2+ release during the CaT but also induced arrhythmogenic delayed afterdepolarizations and action potentials. Nanoscale imaging identified co-clustering of InsP3Rs and RyRs, thereby allowing Ca2+-mediated channel crosstalk. Mathematical modelling supported and further delineated this mechanism of enhanced InsP3R-RyRs coupling in MI. Our findings highlight the role of InsP3R-RyR channel crosstalk in Ca2+ release and arrhythmia during post-MI remodelling.

Connexin hemichannels as candidate targets for cardioprotective and anti-arrhythmic treatments.

Connexins are crucial cardiac proteins that form hemichannels and gap junctions. Gap junctions are responsible for the propagation of electrical and chemical signals between myocardial cells and cells of the specialized conduction system in order to synchronize the cardiac cycle and steer cardiac pump function. Gap junctions are normally open, while hemichannels are closed, but pathological circumstances may close gap junctions and open hemichannels, thereby perturbing cardiac function and homeostasis. Current evidence demonstrates an emerging role of hemichannels in myocardial ischemia and arrhythmia, and tools are now available to selectively inhibit hemichannels without inhibiting gap junctions as well as to stimulate hemichannel incorporation into gap junctions. We review available experimental evidence for hemichannel contributions to cellular pro-arrhythmic events in ventricular and atrial cardiomyocytes, and link these to insights at the level of molecular control of connexin-43-based hemichannel opening. We conclude that a double-edged approach of both preventing hemichannel opening and preserving gap junctional function will be key for further research and development of new connexin-based experimental approaches for treating heart disease.

InsP3R-RyR Ca2+ channel crosstalk facilitates arrhythmias in the failing human ventricle.

Dysregulated intracellular Ca2+ handling involving altered Ca2+ release from intracellular stores via RyR channels underlies both arrhythmias and reduced function in heart failure (HF). Mechanisms linking RyR dysregulation and disease are not fully established. Studies in animals support a role for InsP3 receptor Ca2+ channels (InsP3R) in pathological alterations in cardiomyocyte Ca2+ handling but whether these findings translate to the divergent physiology of human cardiomyocytes during heart failure is not determined. Using electrophysiological and Ca2+ recordings in human ventricular cardiomyocytes, we uncovered that Ca2+ release via InsP3Rs facilitated Ca2+ release from RyR and induced arrhythmogenic delayed after depolarisations and action potentials. InsP3R-RyR crosstalk was particularly increased in HF at RyR clusters isolated from the T-tubular network. Reduced SERCA activity in HF further facilitated the action of InsP3. Nanoscale imaging revealed co-localisation of InsP3Rs with RyRs in the dyad, which was increased in HF, providing a mechanism for augmented Ca2+ channel crosstalk. Notably, arrhythmogenic activity dependent on InsP3Rs was increased in tissue wedges from failing hearts perfused with AngII to promote InsP3 generation. These data indicate a central role for InsP3R-RyR Ca2+ signalling crosstalk in the pro-arrhythmic action of GPCR agonists elevated in HF and the potential for their therapeutic targeting.

Temporal Changes in Beat-to-Beat Variability of Repolarization Predict Imminent Nonsustained Ventricular Tachycardia in Patients With Ischemic and Nonischemic Dilated Cardiomyopathy.

Background An increase in beat-to-beat variability of repolarization (BVR) predicts arrhythmia onset in experimental models, but its clinical translation is not well established. We investigated the temporal changes in BVR before nonsustained ventricular tachycardia (nsVT) in patients with implantable cardioverter defibrillator (ICD). Methods and Results Patients with nsVT on 24-hour Holter before ICD implantation for ischemic cardiomyopathy (ischemic cardiomyopathy+nsVT, n=43) or dilated cardiomyopathy (dilated cardiomyopathy+nsVT, n=37), matched ICD candidates without nsVT (ischemic cardiomyopathy-nsVT, n=29 and dilated cardiomyopathy-nsVT, n=26), and patients without ICD without structural heart disease (n=50) were studied. Digital Holter recordings from these patients were analyzed using a modified fiducial segment averaging technique to detect the QT interval. The nsVT episodes were semi-automatically identified and QT-BVR was assessed 1-, 5-, and 30-minutes before nsVT, and at rest (at 3:00 am). Resting BVR was higher in ICD patients compared with controls without structural heart disease. In ICD patients with nsVT, BVR increased significantly 1-minute pre-nsVT in ischemic cardiomyopathy (2.21±0.59 ms, versus 5 minutes pre-nsVT: 1.78±0.50 ms, P<0.001) and dilated cardiomyopathy (2.09±0.57 ms, versus 5-minutes pre-nsVT: 1.58±0.51 ms, P<0.001), but not in patients without nsVT. In multivariable Cox regression analysis, pre-nsVT BVR was a significant predictor for appropriate therapy during follow-up. Conclusions Baseline BVR is elevated and temporal changes in BVR predict imminent nsVT events in patients with ICD independent of underlying cause. Real-time BVR monitoring could be used to predict impending ventricular arrhythmia and allow preventive therapy to be incorporated into ICDs.

MSK-Mediated Phosphorylation of Histone H3 Ser28 Couples MAPK Signalling with Early Gene Induction and Cardiac Hypertrophy.

Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response including activator protein 1 (AP-1) complex factors is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the mitogen and stress-activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights into the role of MAPK pathways in signalling to the epigenome and regulation of gene expression during cardiac hypertrophy.

Human iPSC model reveals a central role for NOX4 and oxidative stress in Duchenne cardiomyopathy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disorder caused by mutations in the Dystrophin gene. Cardiomyopathy is a major cause of early death. We used DMD-patient-specific human induced pluripotent stem cells (hiPSCs) to model cardiomyopathic features and unravel novel pathologic insights. Cardiomyocytes (CMs) differentiated from DMD hiPSCs showed enhanced premature cell death due to significantly elevated intracellular reactive oxygen species (ROS) resulting from depolarized mitochondria and increased NADPH oxidase 4 (NOX4). CRISPR-Cas9 correction of Dystrophin restored normal ROS levels. ROS reduction by N-acetyl-L-cysteine (NAC), ataluren (PTC124), and idebenone improved hiPSC-CM survival. We show that oxidative stress in DMD hiPSC-CMs was counteracted by stimulating adenosine triphosphate (ATP) production. ATP can bind to NOX4 and partially inhibit the ROS production. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose targeting ROS production and preventing detrimental effects of NOX4 on DMD CMs as promising therapeutic strategy.

Incomplete Assembly of the Dystrophin-Associated Protein Complex in 2D and 3D-Cultured Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are increasingly used to study genetic diseases on a human background. However, the lack of a fully mature adult cardiomyocyte phenotype of hiPSC-CM may be limiting the scope of these studies. Muscular dystrophies and concomitant cardiomyopathies result from mutations in genes encoding proteins of the dystrophin-associated protein complex (DAPC), which is a multi-protein membrane-spanning complex. We examined the expression of DAPC components in hiPSC-CM, which underwent maturation in 2D and 3D culture protocols. The results were compared with human adult cardiac tissue and isolated cardiomyocytes. We found that similarly to adult cardiomyocytes, hiPSC-CM express dystrophin, in line with previous studies on Duchenne's disease. ß-dystroglycan was also expressed, but, contrary to findings in adult cardiomyocytes, none of the sarcoglycans nor α-dystroglycan were, despite the presence of their mRNA. In conclusion, despite the robust expression of dystrophin, the absence of several other DAPC protein components cautions for reliance on commonly used protocols for hiPSC-CM maturation for functional assessment of the complete DAPC.

Ventricular Arrhythmias in Ischemic Cardiomyopathy-New Avenues for Mechanism-Guided Treatment.

Ischemic heart disease is the most common cause of lethal ventricular arrhythmias and sudden cardiac death (SCD). In patients who are at high risk after myocardial infarction, implantable cardioverter defibrillators are the most effective treatment to reduce incidence of SCD and ablation therapy can be effective for ventricular arrhythmias with identifiable culprit lesions. Yet, these approaches are not always successful and come with a considerable cost, while pharmacological management is often poor and ineffective, and occasionally proarrhythmic. Advances in mechanistic insights of arrhythmias and technological innovation have led to improved interventional approaches that are being evaluated clinically, yet pharmacological advancement has remained behind. We review the mechanistic basis for current management and provide a perspective for gaining new insights that centre on the complex tissue architecture of the arrhythmogenic infarct and border zone with surviving cardiac myocytes as the source of triggers and central players in re-entry circuits. Identification of the arrhythmia critical sites and characterisation of the molecular signature unique to these sites can open avenues for targeted therapy and reduce off-target effects that have hampered systemic pharmacotherapy. Such advances are in line with precision medicine and a patient-tailored therapy.