David S. Auerbach

Mechanisms of Wave Fractionation at Boundaries of High-Frequency Excitation in the Posterior Left Atrium of the Isolated Sheep Heart During Atrial Fibrillation

Background— High-frequency fractionated electrograms recorded during atrial fibrillation (AF) in the posterior left atrium (PLA) and elsewhere are being used as target sites for catheter ablation. We tested the hypothesis that highly periodic electric waves emerging from AF sources at or near the PLA give rise to the most fractionated activity in adjacent locations. Methods and Results— Sustained AF was induced in 8 isolated sheep hearts (0.5 &mgr;mol/L acetylcholine). Endocardial videoimaging (DI-4-ANEPPS) and electric mapping of the PLA enabled spatial characterization of dominant frequencies (DFs) and a regularity index (ratio of DF to total power). Regularity index showed that fractionation was lowest within the area with the maximal DF (DFmax domain; 0.19±0.02) and highest within a band of ≈3 mm (0.16±0.02; P=0.047) at boundaries with lower-frequency domains. The numbers of spatiotemporal periodic episodes (25.9±2.3) and rotors per experiment (1.9±0.7) were also highest within the DFmax domain. Most commonly, breakthrough waves at the PLA traveled toward the rest of the atria (76.8±8.1% outward versus 23.2±8.1% inward; P<0.01). In both experiments and simulations with an atrial ionic model, fractionation at DFmax boundaries was associated with increased beat-to-beat variability of conduction velocity and directionality with wavebreak formation. Conclusions— During stable AF, the PLA harbors regular, fast, and highly organized activity; the outer limit of the DFmax domain is the area where the most propagation pattern variability and fractionated activity occur. These new concepts introduce a new perspective in the clinical use of high-frequency fractionated electrograms to localize sources of AF precisely at the PLA and elsewhere.

Spatial Distribution of Fibrosis Governs Fibrillation Wave Dynamics in the Posterior Left Atrium During Heart Failure

Heart failure (HF) commonly results in atrial fibrillation (AF) and fibrosis, but how the distribution of fibrosis impacts AF dynamics has not been studied. HF was induced in sheep by ventricular tachypacing (220 bpm, 6 to 7 weeks). Optical mapping (Di-4-ANEPPS, 300 frames/sec) of the posterior left atrial (PLA) endocardium was performed during sustained AF (burst pacing) in Langendorff-perfused HF (n=7, 4 &mgr;mol/L acetylcholine; n=3, no acetylcholine) and control (n=6) hearts. PLA breakthroughs were the most frequent activation pattern in both groups (72.0±4.6 and 90.2±2.7%, HF and control, respectively). However, unlike control, HF breakthroughs preferentially occurred at the PLAs periphery near the pulmonary vein ostia, and their beat-to-beat variability was greater than control (1.93±0.14 versus 1.47±0.07 changes/[beats/sec], respectively, P<0.05). On histological analysis (picrosirius red), the area of diffuse fibrosis was larger in HF (23.4±0.4%) than control (14.1±0.6%; P<0.001, n=4). Also the number and size of fibrous patches were significantly larger and their location was more peripheral in HF than control. Computer simulations using 2-dimensional human atrial models with structural and ionic remodeling as in HF demonstrated that changes in AF activation frequency and dynamics were controlled by the interaction of electrical waves with clusters of fibrotic patches of various sizes and individual pulmonary vein ostia. During AF in failing hearts, heterogeneous spatial distribution of fibrosis at the PLA governs AF dynamics and fractionation.

Dynamic reciprocity of sodium and potassium channel expression in a macromolecular complex controls cardiac excitability and arrhythmia

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (IK1), which is important for maintenance of the cell resting membrane potential, and the sodium current (INa), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, IK1 modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, IK1–INa interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that IK1–INa interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and NaV1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Nav1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Nav1.5–Kir2.1 interactions. The reciprocal modulation between Nav1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.

Altered Cardiac Electrophysiology and SUDEP in a Model of Dravet Syndrome

Objective Dravet syndrome is a severe form of intractable pediatric epilepsy with a high incidence of SUDEP: Sudden Unexpected Death in epilepsy. Cardiac arrhythmias are a proposed cause for some cases of SUDEP, yet the susceptibility and potential mechanism of arrhythmogenesis in Dravet syndrome remain unknown. The majority of Dravet syndrome patients have de novo mutations in SCN1A, resulting in haploinsufficiency. We propose that, in addition to neuronal hyperexcitability, SCN1A haploinsufficiency alters cardiac electrical function and produces arrhythmias, providing a potential mechanism for SUDEP. Methods Postnatal day 15-21 heterozygous SCN1A-R1407X knock-in mice, expressing a human Dravet syndrome mutation, were used to investigate a possible cardiac phenotype. A combination of single cell electrophysiology and in vivo electrocardiogram (ECG) recordings were performed. Results We observed a 2-fold increase in both transient and persistent Na+ current density in isolated Dravet syndrome ventricular myocytes that resulted from increased activity of a tetrodotoxin-resistant Na+ current, likely Nav1.5. Dravet syndrome myocytes exhibited increased excitability, action potential duration prolongation, and triggered activity. Continuous radiotelemetric ECG recordings showed QT prolongation, ventricular ectopic foci, idioventricular rhythms, beat-to-beat variability, ventricular fibrillation, and focal bradycardia. Spontaneous deaths were recorded in 2 DS mice, and a third became moribund and required euthanasia. Interpretation These data from single cell and whole animal experiments suggest that altered cardiac electrical function in Dravet syndrome may contribute to the susceptibility for arrhythmogenesis and SUDEP. These mechanistic insights may lead to critical risk assessment and intervention in human patients.

A Major Role for hERG in Determining Frequency of Reentry in Neonatal Rat Ventricular Myocyte Monolayer

Rationale: The rapid delayed rectifier potassium current, IKr, which flows through the human ether-a-go-go-related (hERG) channel, is a major determinant of the shape and duration of the human cardiac action potential (APD). However, it is unknown whether the time dependency of IKr enables it to control APD, conduction velocity (CV), and wavelength (WL) at the exceedingly high activation frequencies that are relevant to cardiac reentry and fibrillation. Objective: To test the hypothesis that upregulation of hERG increases functional reentry frequency and contributes to its stability. Methods and Results: Using optical mapping, we investigated the effects of IKr upregulation on reentry frequency, APD, CV, and WL in neonatal rat ventricular myocyte (NRVM) monolayers infected with GFP (control), hERG (IKr), or dominant negative mutant hERG G628S. Reentry frequency was higher in the IKr-infected monolayers (21.12±0.8 Hz; n=43 versus 9.21±0.58 Hz; n=16; P<0.001) but slightly reduced in G628S-infected monolayers. APD80 in the IKr-infected monolayers was shorter (>50%) than control during pacing at 1 to 5 Hz. CV was similar in both groups at low frequency pacing. In contrast, during high-frequency reentry, the CV measured at varying distances from the center of rotation was significantly faster in IKr-infected monolayers than controls. Simulations using a modified NRVM model predicted that rotor acceleration was attributable, in part, to a transient hyperpolarization immediately following the AP. The transient hyperpolarization was confirmed experimentally. Conclusions: hERG overexpression dramatically accelerates reentry frequency in NRVM monolayers. Both APD and WL shortening, together with transient hyperpolarization, underlies the increased rotor frequency and stability.

Scn1b deletion leads to increased tetrodotoxin-sensitive sodium current, altered intracellular calcium homeostasis and arrhythmias in murine hearts

Na+ current (INa) results from the integrated function of a molecular aggregate (the voltage‐gated Na+ channel complex) that includes the β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including Brugada syndrome and sudden unexpected death in patients with epilepsy. We used Scn1b null mice to understand better the relation between Scn1b expression, and cardiac electrical function. Loss of Scn1b caused, among other effects, increased amplitude of tetrodotoxin‐sensitive INa, delayed after‐depolarizations, triggered beats, delayed Ca2+ transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca2+ homeostasis were prevented by 100 nm tetrodotoxin. We propose that life‐threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na+ channel α subunit, can be partly consequent to disrupted intracellular Ca2+ homeostasis.

A null mutation of the neuronal sodium channel NaV1.6 disrupts action potential propagation and excitation-contraction coupling in the mouse heart

Evidence supports the expression of brain‐type sodium channels in the heart. Their functional role, however, remains controversial. We used global NaV1.6‐null mice to test the hypothesis that NaV1.6 contributes to the maintenance of propagation in the myocardium and to excitation‐contraction (EC) coupling. We demonstrated expression of transcripts encoding full‐length NaV1.6 in isolated ventricular myocytes and confirmed the striated pattern of NaV1.6 fluorescence in myocytes. On the ECG, the PR and QRS intervals were prolonged in the null mice, and the Ca2+ transients were longer in the null cells. Under patch clamping, at holding potential (HP) = –120 mV, the peak INa was similar in both phenotypes. However, at HP = –70 mV, the peak INa was smaller in the nulls. In optical mapping, at 4 mM [K+]o, 17 null hearts showed slight (7%) reduction of ventricular conduction velocity (CV) compared to 16 wild‐type hearts. At 12 mM [K+]o, CV was 25% slower in a subset of 9 null vs. 9 wild‐type hearts. These results highlight the importance of neuronal sodium channels in the heart, whereby NaV1.6 participates in EC coupling, and represents an intrinsic depolarizing reserve that contributes to excitation.—Noujaim, S. F., Kaur, K., Milstein, M., Jones, J. M., Furspan, P., Jiang, D., Auerbach, D. S., Herron, T., Meisler, M. H., Jalife, J. A null mutation of the neuronal sodium channel NaV1.6 disrupts action potential propagation and excitation‐contraction coupling in the mouse heart. FASEB J. 26, 63–72 (2012). www.fasebj.org

Structural heterogeneity promotes triggered activity, reflection and arrhythmogenesis in cardiomyocyte monolayers.

Non‐technical summary  The heartbeat depends on the spread of electrical waves through the cardiac muscle. If that spread becomes disturbed, arrhythmias and death may ensue. Patients with heart disease are predisposed to cardiac arrhythmias by unidentified mechanisms. Using both experiments and computer models we demonstrate that structural defects in the heart leading to contiguous areas of physical narrowing and expansion of the musculature can alter the spread of the waves, in such a way that some waves may return abnormally along the same narrow pathway as the original electrical wave (reflection), leading to extra beats and arrhythmia initiation. The possibility of reflection is enhanced when structural defects combine with alterations in the sodium channels responsible for the electrical waves, such as seen in inherited and acquired cardiac electrical diseases. Our results provide a novel mechanism whereby a substrate (structural heterogeneity) and a trigger (abnormal sodium channel activity) combine to promote life‐threatening arrhythmia initiation.

Dmpk gene deletion or antisense knockdown does not compromise cardiac or skeletal muscle function in mice

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which dominant-active DM protein kinase (DMPK) transcripts accumulate in nuclear foci, leading to abnormal regulation of RNA processing. A leading approach to treat DM1 uses DMPK-targeting antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. However, basal levels of DMPK protein are reduced by half in DM1 patients. This raises concern that intolerance for further DMPK loss may limit ASO therapy, especially since mice with Dmpk gene deletion reportedly show cardiac defects and skeletal myopathy. We re-examined cardiac and muscle function in mice with Dmpk gene deletion, and studied post-maturity knockdown using Dmpk-targeting ASOs in mice with heterozygous deletion. Contrary to previous reports, we found no effect of Dmpk gene deletion on cardiac or muscle function, when studied on two genetic backgrounds. In heterozygous knockouts, the administration of ASOs reduced Dmpk expression in cardiac and skeletal muscle by > 90%, yet survival, electrocardiogram intervals, cardiac ejection fraction and muscle strength remained normal. The imposition of cardiac stress by pressure overload, or muscle stress by myotonia, did not unmask a requirement for DMPK. Our results support the feasibility and safety of using ASOs for post-transcriptional silencing of DMPK in muscle and heart.

Genetic biomarkers for the risk of seizures in long QT syndrome

Objectives: The coprevalence, severity, and biomarkers for seizures and arrhythmias in long QT syndrome (LQTS) remain incompletely understood. Methods: Using the Rochester-based LQTS Registry, this study included large cohorts of LQTS1–3 participants (LQTS+, n = 965) and those without a LQTS mutation (LQTS−, n = 936). Results: Compared to LQTS− participants, there was a higher prevalence of LQTS1, LQTS2, and LQTS+ participants classified as having seizures (p < 0.001, i.e., history of seizures/epilepsy or antiseizure medication). LQTS+ participants with longer corrected QT interval (QTc) durations were more likely to have seizures. LQTS2 mutations in the KCNH2 pore domain were positive predictors for both arrhythmias and seizures. In contrast, mutations in the cyclic nucleotide binding domain (cNBD) of KCNH2 conferred a negative risk of seizures, but not arrhythmias. LQTS2, KCNH2-pore, KCNH2-cNBD, QTc duration, and sex were independent predictors of seizures. LQTS+ participants with seizures had significantly longer QTc durations, and a history of seizures was the strongest independent predictor of arrhythmias (hazard ratio 4.09, 95% confidence interval 2.63–6.36, p < 0.001). Conclusions: This study highlights potential biomarkers for neurocardiac electrical abnormalities in LQTS.