Quantitative Assessment of Mitochondrial Morphology and Electrophysiological Function in the Diabetic Heart.

Mitochondria within a cardiomyocyte form a highly dynamic network that undergoes fusion and fission events in response to acute and chronic stressors, such as hyperglycemia and diabetes mellitus. Changes in mitochondrial architecture and morphology not only reflect their capacity for oxidative phosphorylation and ATP synthesis but also impact their subcellular localization and interaction with other organelles. The role of these ultrastructural abnormalities in modulating electrophysiological properties and excitation-contraction coupling remains largely unknown and warrants direct investigation considering the growing appreciation of the functional and structural coupling between the mitochondrial network, the calcium cycling machinery, and sarcolemmal ion channels in the cardiac myocyte. In this Methods in Molecular Biology chapter, we provide a protocol that allows for a quantitative assessment of mitochondrial shape and morphology in control and diabetic hearts that had undergone detailed electrophysiological measurements using high resolution optical action potential (AP) mapping.

Chronic diastolic stretch unmasks conduction defects in an in vitro model of arrhythmogenic cardiomyopathy.

We seek to elucidate the precise nature of mechanical loading that precipitates conduction deficits in a concealed-phase model of arrhythmogenic cardiomyopathy (ACM). ACM is a progressive disorder often resulting from mutations in desmosomal proteins. Exercise has been shown to worsen disease progression and unmask arrhythmia vulnerability, yet the underlying pathomechanisms may depend on the type and intensity of exercise. Because exercise causes myriad changes to multiple inter-dependent hemodynamic parameters, it is difficult to isolate its effects to specific changes in mechanical load. Here, we use engineered heart tissues (EHTs) with iPSC-derived cardiomyocytes expressing R451G desmoplakin, an ACM-linked mutation, which results in a functionally null model of desmoplakin (DSP). We also use a novel bioreactor to independently perturb tissue strain at different time points during the cardiac cycle. We culture EHTs under three strain regimes: normal physiological shortening; increased diastolic stretch, simulating high preload; and isometric culture, simulating high afterload. DSPR451G EHTs that have been cultured isometrically undergo adaptation, with no change in action potential parameters, conduction velocity, or contractile function, a phenotype confirmed by global proteomic analysis. However, when DSPR451G EHTs are subjected to increased diastolic stretch, they exhibit concomitant reductions in conduction velocity and the expression of connexin-43. These effects are rescued by inhibition of both lysosome activity and ERK signaling. Our results indicate that the response of DSPR451G EHTs to mechanical stimuli depends on the strain and the timing of the applied stimulus, with increased diastolic stretch unmasking conduction deficits in a concealed-phase model of ACM.

Humanized Dsp ACM Mouse Model Displays Stress-Induced Cardiac Electrical and Structural Phenotypes.

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder characterized by fibro-fatty infiltration with an increased propensity for ventricular arrhythmias and sudden death. Genetic variants in desmosomal genes are associated with ACM. Incomplete penetrance is a common feature in ACM families, complicating the understanding of how external stressors contribute towards disease development. To analyze the dual role of genetics and external stressors on ACM progression, we developed one of the first mouse models of ACM that recapitulates a human variant by introducing the murine equivalent of the human R451G variant into endogenous desmoplakin (DspR451G/+). Mice homozygous for this variant displayed embryonic lethality. While DspR451G/+ mice were viable with reduced expression of DSP, no presentable arrhythmogenic or structural phenotypes were identified at baseline. However, increased afterload resulted in reduced cardiac performance, increased chamber dilation, and accelerated progression to heart failure. In addition, following catecholaminergic challenge, DspR451G/+ mice displayed frequent and prolonged arrhythmic events. Finally, aberrant localization of connexin-43 was noted in the DspR451G/+ mice at baseline, becoming more apparent following cardiac stress via pressure overload. In summary, cardiovascular stress is a key trigger for unmasking both electrical and structural phenotypes in one of the first humanized ACM mouse models.

The inspection paradox: An important consideration in the evaluation of rotor lifetimes in cardiac fibrillation.

Background and Objective: Renewal theory is a statistical approach to model the formation and destruction of phase singularities (PS), which occur at the pivots of spiral waves. A common issue arising during observation of renewal processes is an inspection paradox, due to oversampling of longer events. The objective of this study was to characterise the effect of a potential inspection paradox on the perception of PS lifetimes in cardiac fibrillation. Methods: A multisystem, multi-modality study was performed, examining computational simulations (Aliev-Panfilov (APV) model, Courtmanche-Nattel model), experimentally acquired optical mapping Atrial and Ventricular Fibrillation (AF/VF) data, and clinically acquired human AF and VF. Distributions of all PS lifetimes across full epochs of AF, VF, or computational simulations, were compared with distributions formed from lifetimes of PS existing at 10,000 simulated commencement timepoints. Results: In all systems, an inspection paradox led towards oversampling of PS with longer lifetimes. In APV computational simulations there was a mean PS lifetime shift of +84.9% (95% CI, ± 0.3%) (p < 0.001 for observed vs overall), in Courtmanche-Nattel simulations of AF +692.9% (95% CI, ±57.7%) (p < 0.001), in optically mapped rat AF +374.6% (95% CI, ± 88.5%) (p = 0.052), in human AF mapped with basket catheters +129.2% (95% CI, ±4.1%) (p < 0.05), human AF-HD grid catheters 150.8% (95% CI, ± 9.0%) (p < 0.001), in optically mapped rat VF +171.3% (95% CI, ±15.6%) (p < 0.001), in human epicardial VF 153.5% (95% CI, ±15.7%) (p < 0.001). Conclusion: Visual inspection of phase movies has the potential to systematically oversample longer lasting PS, due to an inspection paradox. An inspection paradox is minimised by consideration of the overall distribution of PS lifetimes.

Atrial AMP-activated protein kinase is critical for prevention of dysregulation of electrical excitability and atrial fibrillation.

Metabolic stress is an important cause of pathological atrial remodeling and atrial fibrillation. AMPK is a ubiquitous master metabolic regulator, yet its biological function in the atria is poorly understood in both health and disease. We investigated the impact of atrium-selective cardiac AMPK deletion on electrophysiological and structural remodeling in mice. Loss of atrial AMPK expression caused atrial changes in electrophysiological properties and atrial ectopic activity prior to the onset of spontaneous atrial fibrillation. Concomitant transcriptional downregulation of connexins and atrial ion channel subunits manifested with delayed left atrial activation and repolarization. The early molecular and electrophysiological abnormalities preceded left atrial structural remodeling and interstitial fibrosis. AMPK inactivation induced downregulation of transcription factors (Mef2c and Pitx2c) linked to connexin and ion channel transcriptional reprogramming. Thus, AMPK plays an essential homeostatic role in atria, protecting against adverse remodeling potentially by regulating key transcription factors that control the expression of atrial ion channels and gap junction proteins.

Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact.

AIMS: A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus-9 (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. METHODS AND RESULTS: Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3 ± 1.2 Hz in hPLN-R14del vs. 25.7 ± 1.3 Hz in WT, P < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital polymerase chain reaction and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end-diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9 ± 2.3 Hz vs. 21.3 ± 1.5 Hz; P < 0.01). CONCLUSIONS: This study is the first to show that disruption of hPLN-R14del allele by AAV9-CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease.

Right predominant electrical remodeling in a pure model of pulmonary hypertension promotes reentrant arrhythmias.

BACKGROUND: Electrophysiological (EP) properties have been studied mainly in the monocrotaline model of pulmonary arterial hypertension (PAH). Findings are confounded by major extrapulmonary toxicities, which preclude the ability to draw definitive conclusions regarding the role of PAH per se in EP remodeling. OBJECTIVE: The purpose of this study was to investigate the EP substrate and arrhythmic vulnerability of a new model of PAH that avoids extracardiopulmonary toxicities. METHODS: Sprague-Dawley rats underwent left pneumonectomy (Pn) followed by injection of the vascular endothelial growth factor inhibitor Sugen-5416 (Su/Pn). Five weeks later, cardiac magnetic resonance imaging was performed in vivo, optical action potential (AP) mapping ex vivo, and molecular analyses in vitro. RESULTS: Su/Pn rats exhibited right ventricular (RV) hypertrophy and were highly prone to pacing-induced ventricular tachycardia/fibrillation (VT/VF). Underlying this susceptibility was disproportionate RV-sided prolongation of AP duration, which promoted formation of right-sided AP alternans at physiological rates. While propagation was impaired at all rates in Su/Pn rats, the extent of conduction slowing was most severe immediately before the emergence of interventricular lines of block and onset of VT/VF. Measurement of the cardiac wavelength revealed a decrease in Su/Pn relative to control. Nav1.5 and total connexin 43 expression was not altered, while connexin 43 phosphorylation was decreased in PAH. Col1a1 and Col3a1 transcripts were upregulated coinciding with myocardial fibrosis. Once generated, VT/VF was sustained by multiple reentrant circuits with a lower frequency of RV activation due to wavebreak formation. CONCLUSION: In this pure model of PAH, we document RV-predominant remodeling that promotes multiwavelet reentry underlying VT. The Su/Pn model represents a severe form of PAH that allows the study of EP properties without the confounding influence of extrapulmonary toxicity.

Impaired Right Ventricular Calcium Cycling Is an Early Risk Factor in R14del-Phospholamban Arrhythmias.

The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.

Arrhythmia Mechanism and Dynamics in a Humanized Mouse Model of Inherited Cardiomyopathy Caused by Phospholamban R14del Mutation.

BACKGROUND: Arginine (Arg) 14 deletion (R14del) in the calcium regulatory protein phospholamban (hPLNR14del) has been identified as a disease-causing mutation in patients with an inherited cardiomyopathy. Mechanisms underlying the early arrhythmogenic phenotype that predisposes carriers of this mutation to sudden death with no apparent structural remodeling remain unclear. METHODS: To address this, we performed high spatiotemporal resolution optical mapping of intact hearts from adult knock-in mice harboring the human PLNWT (wildtype [WT], n=12) or the heterozygous human PLNR14del mutation (R14del, n=12) before and after ex vivo challenge with isoproterenol and rapid pacing. RESULTS: Adverse electrophysiological remodeling was evident in the absence of significant structural or hemodynamic changes. R14del hearts exhibited increased arrhythmia susceptibility compared with wildtype. Underlying this susceptibility was preferential right ventricular action potential prolongation that was unresponsive to ß-adrenergic stimulation. A steep repolarization gradient at the left ventricular/right ventricular interface provided the substrate for interventricular activation delays and ultimately local conduction block during rapid pacing. This was followed by the initiation of macroreentrant circuits supporting the onset of ventricular tachycardia. Once sustained, these circuits evolved into high-frequency rotors, which in their majority were pinned to the right ventricle. These rotors exhibited unique spatiotemporal dynamics that promoted their increased stability in R14del compared with wildtype hearts. CONCLUSIONS: Our findings highlight the crucial role of primary electric remodeling caused by the hPLNR14del mutation. These inherently arrhythmogenic features form the substrate for adrenergic-mediated VT at early stages of PLNR14del induced cardiomyopathy.

Renewal Theory as a Universal Quantitative Framework to Characterize Phase Singularity Regeneration in Mammalian Cardiac Fibrillation.

BACKGROUND: Despite a century of research, no clear quantitative framework exists to model the fundamental processes responsible for the continuous formation and destruction of phase singularities (PS) in cardiac fibrillation. We hypothesized PS formation/destruction in fibrillation could be modeled as self-regenerating Poisson renewal processes, producing exponential distributions of interevent times governed by constant rate parameters defined by the prevailing properties of each system. METHODS: PS formation/destruction were studied in 5 systems: (1) human persistent atrial fibrillation (n=20), (2) tachypaced sheep atrial fibrillation (n=5), (3) rat atrial fibrillation (n=4), (5) rat ventricular fibrillation (n=11), and (5) computer-simulated fibrillation. PS time-to-event data were fitted by exponential probability distribution functions computed using maximum entropy theory, and rates of PS formation and destruction (λf/λd) determined. A systematic review was conducted to cross-validate with source data from literature. RESULTS: In all systems, PS lifetime and interformation times were consistent with underlying Poisson renewal processes (human: λf, 4.2%/ms±1.1 [95% CI, 4.0-5.0], 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 atrial fibrillation: λf, 33%/ms±8.8 [95% CI, 11-55], λd, 38%/ms [95% CI, 22-55]; rat ventricular fibrillation: λf, 38%/ms±24 [95% CI, 22-55], λf, 46%/ms±21 [95% CI, 31-60]; simulated fibrillation λd, 6.6-8.97%/ms [95% CI, 4.1-6.7]; R2≥0.90 in all cases). All PS distributions identified through systematic review were also consistent with an underlying Poisson renewal process. CONCLUSIONS: Poisson renewal theory provides an evolutionarily preserved universal framework to quantify formation and destruction of rotational events in cardiac fibrillation.

Cardiomyocyte-Specific STIM1 (Stromal Interaction Molecule 1) Depletion in the Adult Heart Promotes the Development of Arrhythmogenic Discordant Alternans.

BACKGROUND: STIM1 (stromal interaction molecule 1) is a calcium (Ca2+) sensor that regulates cardiac hypertrophy by triggering store-operated Ca2+ entry. Because STIM1 binding to phospholamban increases sarcoplasmic reticulum Ca2+ load independent of store-operated Ca2+ entry, we hypothesized that it controls electrophysiological function and arrhythmias in the adult heart. METHODS: Inducible myocyte-restricted STIM1-KD (STIM1 knockdown) was achieved in adult mice using an αMHC (α-myosin heavy chain)-MerCreMer system. Mechanical and electrophysiological properties were examined using echocardiography in vivo and optical action potential (AP) mapping ex vivo in tamoxifen-induced STIM1flox/flox-Cretg/- (STIM1-KD) and littermate controls for STIM1flox/flox (referred to as STIM1-Ctl) and for Cretg/- without STIM deletion (referred to as Cre-Ctl). RESULTS: STIM1-KD mice (N=23) exhibited poor survival compared with STIM1-Ctl (N=22) and Cre-Ctl (N=11) with >50% mortality after only 8-days of cardiomyocyte-restricted STIM1-KD. STIM1-KD but not STIM1-Ctl or Cre-Ctl hearts exhibited a proclivity for arrhythmic behavior, ranging from frequent ectopy to pacing-induced ventricular tachycardia/ventricular fibrillation (VT/VF). Examination of the electrophysiological substrate revealed decreased conduction velocity and increased AP duration (APD) heterogeneity in STIM1-KD. These features, however, were comparable in VT/VF(+) and VT/VF(-) hearts. We also uncovered a marked increase in the magnitude of APD alternans during rapid pacing, and the emergence of a spatially discordant alternans profile in STIM1-KD hearts. Unlike conduction velocity slowing and APD heterogeneity, the magnitude of APD alternans was greater (by 80%, P<0.05) in VT/VF(+) versus VT/VF(-) STIM1-KD hearts. Detailed phase mapping during the initial beats of VT/VF identified one or more rotors that were localized along the nodal line separating out-of-phase alternans regions. CONCLUSIONS: In an adult murine model with inducible and myocyte-specific STIM1 depletion, we demonstrate for the first time the regulation of spatially discordant alternans by STIM1. Early mortality in STIM1-KD mice is likely related to enhanced susceptibility to VT/VF secondary to discordant APD alternans.

Recurrence quantification analysis of complex-fractionated electrograms differentiates active and passive sites during atrial fibrillation.

OBJECTIVES: To differentiate electrograms representing sites of active atrial fibrillation (AF) drivers from passive ones. BACKGROUND: Ablation of complex-fractionated atrial electrograms (CFAEs) is controversial due to difficulty in distinguishing CFAEs representing sites of active AF drivers from passive mechanisms. We hypothesized that active CFAE sites exhibit repetitive wavefront directionality, thereby inscribing an electrogram conformation (Egm-C) that is more recurrent compared with passive CFAE sites; and that can be differentiated from passive CFAEs using nonlinear recurrence quantification analysis (RQA). METHODS: We developed multiple computer models of active CFAE mechanisms (ie, rotors) and passive CFAE mechanisms (ie, wavebreak, slow conduction, and double potentials). CFAE signals were converted into discrete time-series representing Egm-C. The RQA algorithm was used to compare signals derived from active CFAE sites to those from passive CFAEs sites. The RQA algorithm was then applied to human CFAE signals collected during AF ablation (n = 17 patients). RESULTS: RQA was performed in silico on simulated bipolar CFAEs within active (n = 45) and passive (n = 60) areas. Recurrence of Egm-C was significantly higher in active compared with passive CFAE sites (31.8% ± 19.6% vs 0.3% ± 0.5%, respectively, P < .0001) despite no difference in mean cycle length (CL). Similarly, for human AF (n = 39 signals), Egm-C recurrence was higher in active vs passive CFAE areas despite similar CLs (%recurrence 13.6% ± 15.5% vs 0.1% ± 0.3%, P < .002; mean CL 102.5 ± 14.3 vs 106.6 ± 14.4, P = NS). CONCLUSION: Active CFAEs critical to AF maintenance exhibit higher Egm-C recurrence and can be differentiated from passive bystander CFAE sites using RQA.