Rationale:: Na _v 1.5 ( SCN5A ) is the primary cardiac voltage-gated Na _v channel. Na _v 1.5 is critical for cardiac excitability and conduction, and human SCN5A mutations cause sinus node dysfunction, atrial fibrillation, conductional abnormalities, and ventricular arrhythmias. Further, defects in Na _v 1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Na _v 1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Na _v 1.5 membrane targeting, assembly, and regulation are still largely undefined. Objective:: Define the in vivo mechanisms underlying Na _v 1.5 membrane regulation. Methods and Results:: Here, we define the molecular basis of an Na _v channel regulatory platform in heart. Using new cardiac-selective ankyrin-G ^-/- mice (conditional knock-out mouse), we report that ankyrin-G targets Na _v 1.5 and its regulatory protein calcium/calmodulin–dependent kinase II to the intercalated disc. Mechanistically, β _IV -spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin–dependent kinase II-δ; however, β _IV -spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Na _v 1.5 expression/membrane localization and reduced I _Na associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Na _v channel antagonists. Moreover, we report that ankyrin-G links Na _v channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to β-adrenergic stimulation. Conclusions:: Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Na _v 1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Na _v 1.5.

Background—: Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting >2 million patients in the United States alone. Despite decades of research, surprisingly little is known regarding the molecular pathways underlying the pathogenesis of AF. ANK2 encodes ankyrin-B, a multifunctional adapter molecule implicated in membrane targeting of ion channels, transporters, and signaling molecules in excitable cells. Methods and Results—: In the present study, we report early-onset AF in patients harboring loss-of-function mutations in ANK2 . In mice, we show that ankyrin-B deficiency results in atrial electrophysiological dysfunction and increased susceptibility to AF. Moreover, ankyrin-B ^+/− atrial myocytes display shortened action potentials, consistent with human AF. Ankyrin-B is expressed in atrial myocytes, and we demonstrate its requirement for the membrane targeting and function of a subgroup of voltage-gated Ca ²⁺ channels (Ca _v 1.3) responsible for low voltage-activated L-type Ca ²⁺ current. Ankyrin-B is associated directly with Ca _v 1.3, and this interaction is regulated by a short, highly conserved motif specific to Ca _v 1.3. Moreover, loss of ankyrin-B in atrial myocytes results in decreased Ca _v 1.3 expression, membrane localization, and function sufficient to produce shortened atrial action potentials and arrhythmias. Finally, we demonstrate reduced ankyrin-B expression in atrial samples of patients with documented AF, further supporting an association between ankyrin-B and AF. Conclusions—: These findings support that reduced ankyrin-B expression or mutations in ANK2 are associated with AF. Additionally, our data demonstrate a novel pathway for ankyrin-B–dependent regulation of Ca _v 1.3 channel membrane targeting and regulation in atrial myocytes.

Kinase/phosphatase balance governs cardiac excitability in health and disease. Although detailed mechanisms for cardiac kinase regulation are established, far less is known regarding cardiac protein phosphatase 2A (PP2A) regulation. This is largely due to the complexity of the PP2A holoenzyme structure (combinatorial assembly of three subunit enzyme from >17 subunit genes) and the inability to segregate “global” PP2A function from the activities of multiple “local” holoenzyme populations. Here we report that PP2A catalytic, regulatory, and scaffolding subunits are tightly regulated at transcriptional, translational, and post-translational levels to tune myocyte function at base line and in disease. We show that past global read-outs of cellular PP2A activity more appropriately represent the collective activity of numerous individual PP2A holoenzymes, each displaying a specific subcellular localization (dictated by select PP2A regulatory subunits) as well as local specific post-translational catalytic subunit methylation and phosphorylation events that regulate local and rapid holoenzyme assembly/disassembly (via leucine carboxymethyltransferase 1/phosphatase methylesterase 1 (LCMT-1/PME-1). We report that PP2A subunits are selectively regulated between human and animal models, across cardiac chambers, and even within specific cardiac cell types. Moreover, this regulation can be rapidly tuned in response to cellular activation. Finally, we report that global PP2A is altered in human and experimental models of heart disease, yet each pathology displays its own distinct molecular signature though specific PP2A subunit modulatory events. These new data provide an initial view into the signaling pathways that govern PP2A function in heart but also establish the first step in defining specific PP2A regulatory targets in health and disease.

The precise mechanisms by which oxidative stress (OS) causes atrial fibrillation (AF) are not known. Since AF frequently originates in the posterior left atrium (PLA), we hypothesized that OS, via calmodulin-dependent protein kinase II (CaMKII) signaling, creates a fertile substrate in the PLA for triggered activity and reentry. In a canine heart failure (HF) model, OS generation and oxidized-CaMKII–induced (Ox-CaMKII–induced) RyR2 and Na _v 1.5 signaling were increased preferentially in the PLA (compared with left atrial appendage). Triggered Ca ²⁺ waves (TCWs) in HF PLA myocytes were particularly sensitive to acute ROS inhibition. Computational modeling confirmed a direct relationship between OS/CaMKII signaling and TCW generation. CaMKII phosphorylated Na _v 1.5 (CaMKII-p-Na _v 1.5 [S571]) was located preferentially at the intercalated disc (ID), being nearly absent at the lateral membrane. Furthermore, a decrease in ankyrin-G (AnkG) in HF led to patchy dropout of CaMKII-p-Na _v 1.5 at the ID, causing its distribution to become spatially heterogeneous; this corresponded to preferential slowing and inhomogeneity of conduction noted in the HF PLA. Computational modeling illustrated how conduction slowing (e.g., due to increase in CaMKII-p-Na _v 1.5) interacts with fibrosis to cause reentry in the PLA. We conclude that OS via CaMKII leads to substrate for triggered activity and reentry in HF PLA by mechanisms independent of but complementary to fibrosis.

Background—: The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. Methods and Results—: Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin–deficient mice. Conclusions—: Our findings identify βII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.