B. Cicero Willis

Dominant Frequency Increase Rate Predicts Transition from Paroxysmal to Long-Term Persistent Atrial Fibrillation

Background— Little is known about the mechanisms underlying the transition from paroxysmal to persistent atrial fibrillation (AF). In an ovine model of long-standing persistent AF we tested the hypothesis that the rate of electric and structural remodeling, assessed by dominant frequency (DF) changes, determines the time at which AF becomes persistent. Methods and Results— Self-sustained AF was induced by atrial tachypacing. Seven sheep were euthanized 11.5±2.3 days after the transition to persistent AF and without reversal to sinus rhythm; 7 sheep were euthanized after 341.3±16.7 days of long-standing persistent AF. Seven sham-operated animals were in sinus rhythm for 1 year. DF was monitored continuously in each group. Real-time polymerase chain reaction, Western blotting, patch clamping, and histological analyses were used to determine the changes in functional ion channel expression and structural remodeling. Atrial dilatation, mitral valve regurgitation, myocyte hypertrophy, and atrial fibrosis occurred progressively and became statistically significant after the transition to persistent AF, with no evidence for left ventricular dysfunction. DF increased progressively during the paroxysmal-to-persistent AF transition and stabilized when AF became persistent. Importantly, the rate of DF increase correlated strongly with the time to persistent AF. Significant action potential duration abbreviation, secondary to functional ion channel protein expression changes (CaV1.2, NaV1.5, and KV4.2 decrease; Kir2.3 increase), was already present at the transition and persisted for 1 year of follow up. Conclusions— In the sheep model of long-standing persistent AF, the rate of DF increase predicts the time at which AF stabilizes and becomes persistent, reflecting changes in action potential duration and densities of sodium, L-type calcium, and inward rectifier currents.

Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes

Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers.

Extracellular Matrix–Mediated Maturation of Human Pluripotent Stem Cell–Derived Cardiac Monolayer Structure and Electrophysiological Function

Background—Human pluripotent stem cell–derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Methods and Results—Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of &bgr;1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Conclusions—Maturation of human stem cell–derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers.

Arrhythmogenesis in a catecholaminergic polymorphic ventricular tachycardia mutation that depresses ryanodine receptor function

Significance Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic syndrome characterized by life-threatening cardiac arrhythmias triggered by physical exercise or emotional stress. Although patients with CPVT have no abnormalities in cardiac structure, they present ventricular fibrillation or sudden death as the first symptom. Most CPVT cases are linked to mutations in cardiac ryanodine receptor (RyR2), an intracellular Ca2+ channel that provides the majority of Ca2+ that enables heart contraction. The current mechanism for CPVT arrhythmogenesis requires RyR2 affected by gain-of-function mutations. However, loss-of-function mutations of RyR2 have also been found in CPVT patients. We generated an animal model of CPVT that harbors a loss-of-function mutation that was originally described in humans and elucidated novel mechanisms by which hypoactive RyR2 channels trigger malignant arrhythmias. Current mechanisms of arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia (CPVT) require spontaneous Ca2+ release via cardiac ryanodine receptor (RyR2) channels affected by gain-of-function mutations. Hence, hyperactive RyR2 channels eager to release Ca2+ on their own appear as essential components of this arrhythmogenic scheme. This mechanism, therefore, appears inadequate to explain lethal arrhythmias in patients harboring RyR2 channels destabilized by loss-of-function mutations. We aimed to elucidate arrhythmia mechanisms in a RyR2-linked CPVT mutation (RyR2-A4860G) that depresses channel activity. Recombinant RyR2-A4860G protein was expressed equally as wild type (WT) RyR2, but channel activity was dramatically inhibited, as inferred by [3H]ryanodine binding and single channel recordings. Mice heterozygous for the RyR2-A4860G mutation (RyR2-A4860G+/−) exhibited basal bradycardia but no cardiac structural alterations; in contrast, no homozygotes were detected at birth, suggesting a lethal phenotype. Sympathetic stimulation elicited malignant arrhythmias in RyR2-A4860G+/− hearts, recapitulating the phenotype originally described in a human patient with the same mutation. In isoproterenol-stimulated ventricular myocytes, the RyR2-A4860G mutation decreased the peak of Ca2+ release during systole, gradually overloading the sarcoplasmic reticulum with Ca2+. The resultant Ca2+ overload then randomly caused bursts of prolonged Ca2+ release, activating electrogenic Na+-Ca2+ exchanger activity and triggering early afterdepolarizations. The RyR2-A4860G mutation reveals novel pathways by which RyR2 channels engage sarcolemmal currents to produce life-threatening arrhythmias.

Galectin-3 Regulates Atrial Fibrillation Remodeling and Predicts Catheter Ablation Outcomes

Summary Atrial fibrillation (AF) usually starts as paroxysmal but can evolve relentlessly to the persistent and permanent forms. However, the mechanisms governing such a transition are unknown. The authors show that intracardiac serum levels of galectin (Gal)-3 are greater in patients with persistent than paroxysmal AF and that Gal-3 independently predicts atrial tachyarrhythmia recurrences after a single ablation procedure. Using a sheep model of persistent AF the authors further demonstrate that upstream therapy targeting Gal-3 diminishes both electrical remodeling and fibrosis by impairing transforming growth factor beta–mediated signaling and reducing myofibroblast activation. Accordingly, Gal-3 inhibition therapy increases the probability of AF termination and reduces the overall burden of AF. Therefore the authors postulate that Gal-3 inhibition is a potential new upstream therapy to prevent AF progression.

Protein assemblies of sodium and inward rectifier potassium channels control cardiac excitability and arrhythmogenesis

The understanding of how cardiac ion channels function in the normal and the diseased heart has greatly increased over the last four decades thanks to the advent of patch-clamp technology and, more recently, the emergence of genetics, as well as cellular and molecular cardiology. However, our knowledge of how these membrane-embedded proteins physically interact with each other within macromolecular complexes remains incomplete. This review focuses on how the main cardiac inward sodium channel (NaV1.5) and the strong inward rectifier potassium channel (Kir2.1) function within macromolecular complexes to control cardiac excitability. It has become increasingly clear that these two important ion channel proteins physically interact with multiple other protein partners and with each other from early stages of protein trafficking and targeting through membrane anchoring, recycling, and degradation. Recent findings include compartmentalized regulation of NaV1.5 channel expression and function through a PDZ (postsynaptic density protein, Drosophila disc large tumor suppressor, and zonula occludens-1 protein) domain-binding motif, and interaction of caveolin-3 with Kir2.1 and ankyrin-G as a molecular platform for NaV1.5 signaling. At the cardiomyocyte membrane, NaV1.5 and Kir2.1 interact through at least two distinct PDZ domain-scaffolding proteins (synapse-associated protein-97 and α1-syntrophin), thus modulating reciprocally their cell-surface expression at two different microdomains. Emerging evidence also shows that inheritable mutations in plakophilin-2, ankyrin-G, dystrophin, syntrophin, synapse-associated protein-97, and caveolin-3, among others, modify functional expression and/or localization in the cardiac cell of NaV1.5, Kir2.1 or both to give rise to arrhythmogenic diseases. Unveiling the mechanistic underpinnings of macromolecular interactions should increase our understanding of inherited and acquired arrhythmogenic cardiac diseases and may lead to advances in therapy.

Constitutive Intracellular Na+ Excess in Purkinje Cells Promotes Arrhythmogenesis at Lower Levels of Stress Than Ventricular Myocytes From Mice With Catecholaminergic Polymorphic Ventricular Tachycardia.

Background— In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca2+ dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2R4496C+/Cx40eGFP), we tested whether PC intracellular Ca2+ ([Ca2+]i) dysregulation results from a constitutive [Na+]i surplus relative to VMs. Methods and Results— Simultaneous optical mapping of voltage and [Ca2+]i in CPVT hearts showed that spontaneous Ca2+ release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca2+ imaging, early and delayed afterdepolarizations trailed spontaneous Ca2+ release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca2+ load, measured by caffeine-induced Ca2+ transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na+]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na+/Ca2+ exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na+]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca2+ release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na+]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca2+ spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na+]i played a central role. Conclusions— In CPVT mice, the constitutive [Na+]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.

Ventricular remodelling in rabbits with sustained high-fat diet

Excess weight gain and obesity are one of the most serious health problems in the western societies. These conditions enhance risk of cardiac disease and have been linked with increased prevalence for cardiac arrhythmias and sudden death. Our goal was to study the ventricular remodelling occurring in rabbits fed with high‐fat diet (HFD) and its potential arrhythmogenic mechanisms.

Scn2b Deletion in Mice Results in Ventricular and Atrial Arrhythmias

Background—Mutations in SCN2B, encoding voltage-gated sodium channel &bgr;2-subunits, are associated with human cardiac arrhythmias, including atrial fibrillation and Brugada syndrome. Because of this, we propose that &bgr;2-subunits play critical roles in the establishment or maintenance of normal cardiac electric activity in vivo. Methods and Results—To understand the pathophysiological roles of &bgr;2 in the heart, we investigated the cardiac phenotype of Scn2b null mice. We observed reduced sodium and potassium current densities in ventricular myocytes, as well as conduction slowing in the right ventricular outflow tract region. Functional reentry, resulting from the interplay between slowed conduction, prolonged repolarization, and increased incidence of premature ventricular complexes, was found to underlie the mechanism of spontaneous polymorphic ventricular tachycardia. Scn5a transcript levels were similar in Scn2b null and wild-type ventricles, as were levels of Nav1.5 protein, suggesting that similar to the previous work in neurons, the major function of &bgr;2-subunits in the ventricle is to chaperone voltage-gated sodium channel &agr;-subunits to the plasma membrane. Interestingly, Scn2b deletion resulted in region-specific effects in the heart. Scn2b null atria had normal levels of sodium current density compared with wild type. Scn2b null hearts were more susceptible to atrial fibrillation, had increased levels of fibrosis, and higher repolarization dispersion than wild-type littermates. Conclusions—Genetic deletion of Scn2b in mice results in ventricular and atrial arrhythmias, consistent with reported SCN2B mutations in human patients.

Cardiac Kir2.1 and NaV1.5 Channels Traffic Together to the Sarcolemma to Control Excitability

Rationale: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective: The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1&Dgr;314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1&Dgr;314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1&Dgr;314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1&Dgr;314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 υ-adaptin subunit in human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions: The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.