Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltagegated Na + channels underlie cellular excitability and control neuronal communication, cardiac excitationcontraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na + channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β IV -spectrin as a multifunctional regulatory platform for Na + channels in mice. We found that β IV -spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β IV -spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na + channels, via direct phosphorylation by β IV -spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.

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 with cardiac pathologies including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction in cardiac electrical 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 post-translational 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 electrical 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 that include the Na/Ca exchanger, RyR2, 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 electrical activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.

The coordinated sorting of ion channels to specific plasma membrane domains is necessary for excitable cell physiology. KATP channels, assembled from pore-forming (Kir6.x) and regulatory sulfonylurea receptor subunits, are critical electrical transducers of the metabolic state of excitable tissues, including skeletal and smooth muscle, heart, brain, kidney, and pancreas. Here we show that the C-terminal domain of Kir6.2 contains a motif conferring membrane targeting in primary excitable cells. Kir6.2 lacking this motif displays aberrant channel targeting due to loss of association with the membrane adapter ankyrin-B (AnkB). Moreover, we demonstrate that this Kir6.2 C-terminal AnkB-binding motif (ABM) serves a dual role in KATP channel trafficking and membrane metabolic regulation and dysfunction in these pathways results in human excitable cell disease. Thus, the KATP channel ABM serves as a previously unrecognized bifunctional touch-point for grading KATP channel gating and membrane targeting and may play a fundamental role in controlling excitable cell metabolic regulation.K ATP channels are critical electrical transducers of the metabolic state of excitable tissues including skeletal and smooth muscle, heart, brain, kidney, and pancreas (1). Mechanistically, decreased metabolism opens K ATP channels, resulting in K ϩ efflux, membrane hyperpolarization, and suppression of action potential formation (1). Conversely, increased metabolism closes K ATP channels, resulting in membrane depolarization, stimulation of electrical activity (2), and subsequent triggering of diverse cellular responses, such as release of hormones and neurotransmitters, or muscle contraction.Given such critical roles in the regulation of electrical excitability in many cell types, it is not surprising that K ATP channel dysfunction results in disease. Human mutations in K ATP channel genes are associated with neonatal diabetes and hyperinsulinemia (3), epilepsy (4), and dilated cardiomyopathy (5). However, despite the clear importance of K ATP function for normal vertebrate physiology, little is resolved regarding the mechanisms responsible for K ATP channel membrane targeting and/or membrane organization.Here we identify a critical motif in the Kir6.2 C-terminal domain that is essential for normal Kir6.2 membrane targeting. We demonstrate that the Kir6.2 C-terminal motif interacts with the cytoskeletal adapter ankyrin-B (AnkB) and that Kir6.2 displays aberrant membrane trafficking when the motif is disrupted or in cells lacking ankyrin-B expression. We demonstrate that the Kir6.2 C-terminal motif displays an unexpected secondary role in Kir6.2 membrane function (K ATP channel activity) by altering K ATP channel ATP sensitivity. Finally, we demonstrate that a human neonatal diabetes disease mutation located in the Kir6.2 C-terminal motif results in a complex ␤ cell phenotype, likely due to the dual role of the C-terminal motif in both Kir6.2 targeting and K ATP channel membrane activity. Together, our findings define a pathway for K ATP ch...

Rationale: Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules.

Nav channels are essential for metazoan membrane depolarization, and Nav channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Nav channelopathies are primarily caused by variants that directly affect Nav channel permeability or gating. However, a new class of human Nav channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Nav channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Nav channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Nav channel Nav1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Nav1.5 for FHFs. Consistent with these data, Nav1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Nav1.5 p.H1849R as a gain-of-function for INa by altering steady-state inactivation and slowing the rate of Nav1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Nav1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Nav channelopathy based on altered Nav1.5 association with FHF proteins.

Protein phosphatase 2A (PP2A) is a serine/threonine-selective holoenzyme composed of a catalytic, scaffolding, and regulatory subunit. In the heart, PP2A activity is requisite for cardiac excitation-contraction coupling and central in adrenergic signaling. We found that mice deficient in the PP2A regulatory subunit B56α (1 of 13 regulatory subunits) had altered PP2A signaling in the heart that was associated with changes in cardiac physiology, suggesting that the B56α regulatory subunit had an autoinhibitory role that suppressed excess PP2A activity. The increase in PP2A activity in the mice with reduced B56α expression resulted in slower heart rates and increased heart rate variability, conduction defects, and increased sensitivity of heart rate to parasympathetic agonists. Increased PP2A activity in B56α+/− myocytes resulted in reduced Ca2+ waves and sparks, which was associated with decreased phosphorylation (and thus decreased activation) of the ryanodine receptor RyR2, an ion channel on intracellular membranes that is involved in Ca2+ regulation in cardiomyocytes. In line with an autoinhibitory role for B56α, in vivo expression of B56α in the absence of altered abundance of other PP2A subunits decreased basal phosphatase activity. Consequently, in vivo expression of B56α suppressed parasympathetic regulation of heart rate and increased RyR2 phosphorylation in cardiomyocytes. These data show that an integral component of the PP2A holoenzyme has an important inhibitory role in controlling PP2A enzyme activity in the heart.

Background— Long QT syndrome confers susceptibility to ventricular arrhythmia, predisposing to syncope, seizures, and sudden death. While rare globally, long QT syndrome is ≈15× more common in First Nations of Northern British Columbia largely because of a known mutation in KCNQ1 . However, 2 large multigenerational families were affected, but negative for the known mutation. Methods and Results— Long QT syndrome panel testing was carried out in the index case of each family, and clinical information was collected. Cascade genotyping was performed. Biochemical and myocyte-based assays were performed to evaluate the identified gene variant for loss-of-function activity. Index cases in these 2 families harbored a novel ANK2 c.1937C>T variant (p.S646F). An additional 16 carriers were identified, including 2 with structural heart disease: one with cardiomyopathy resulting in sudden death and the other with congenital heart disease. For all carriers of this variant, the average QTc was 475 ms (±40). Although ankyrin-B p.S646F is appropriately folded and expressed in bacteria, the mutant polypeptide displays reduced expression in cultured H9c2 cells and aberrant localization in primary cardiomyocytes. Furthermore, myocytes expressing ankyrin-B p.S646F lack normal membrane targeting of the ankyrin-binding partner, the Na/Ca exchanger. Thus, ankyrin-B p.S646F is a loss-of-function variant. Conclusions— We identify the first disease-causing ANK2 variant localized to the membrane-binding domain resulting in reduced ankyrin-B expression and abnormal localization. Further study is warranted on the potential association of this variant with structural heart disease given the role of ANK2 in targeting and stabilization of key structural and signaling molecules in cardiac cells.

Arrhythmogenic cardiomyopathy (ACM) is an inherited arrhythmia syndrome characterized by severe structural and electrical cardiac phenotypes, including myocardial fibrofatty replacement and sudden cardiac death. Clinical management of ACM is largely palliative, owing to an absence of therapies that target its underlying pathophysiology, which stems partially from our limited insight into the condition. Following identification of deceased ACM probands possessing ANK2 rare variants and evidence of ankyrin-B loss of function on cardiac tissue analysis, an ANK2 mouse model was found to develop dramatic structural abnormalities reflective of human ACM, including biventricular dilation, reduced ejection fraction, cardiac fibrosis, and premature death. Desmosomal structure and function appeared preserved in diseased human and murine specimens in the presence of markedly abnormal β-catenin expression and patterning, leading to identification of a previously unknown interaction between ankyrin-B and β-catenin. A pharmacological activator of the WNT/β-catenin pathway, SB-216763, successfully prevented and partially reversed the murine ACM phenotypes. Our findings introduce what we believe to be a new pathway for ACM, a role of ankyrin-B in cardiac structure and signaling, a molecular link between ankyrin-B and β-catenin, and evidence for targeted activation of the WNT/β-catenin pathway as a potential treatment for this disease.