Michael A. Makara

Ankyrin-G Coordinates Intercalated Disc Signaling Platform to Regulate Cardiac Excitability In Vivo

Rationale: Nav1.5 (SCN5A) is the primary cardiac voltage-gated Nav channel. Nav1.5 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 Nav1.5 regulation are linked with malignant arrhythmias associated with human heart failure. Consequently, therapies to target select Nav1.5 properties have remained at the forefront of cardiovascular medicine. However, despite years of investigation, the fundamental pathways governing Nav1.5 membrane targeting, assembly, and regulation are still largely undefined. Objective: Define the in vivo mechanisms underlying Nav1.5 membrane regulation. Methods and Results: Here, we define the molecular basis of an Nav channel regulatory platform in heart. Using new cardiac-selective ankyrin-G-/- mice (conditional knock-out mouse), we report that ankyrin-G targets Nav1.5 and its regulatory protein calcium/calmodulin–dependent kinase II to the intercalated disc. Mechanistically, &bgr;IV-spectrin is requisite for ankyrin-dependent targeting of calcium/calmodulin–dependent kinase II-&dgr;; however, &bgr;IV-spectrin is not essential for ankyrin-G expression. Ankyrin-G conditional knock-out mouse myocytes display decreased Nav1.5 expression/membrane localization and reduced INa associated with pronounced bradycardia, conduction abnormalities, and ventricular arrhythmia in response to Nav channel antagonists. Moreover, we report that ankyrin-G links Nav channels with broader intercalated disc signaling/structural nodes, as ankyrin-G loss results in reorganization of plakophilin-2 and lethal arrhythmias in response to &bgr;-adrenergic stimulation. Conclusions: Our findings provide the first in vivo data for the molecular pathway required for intercalated disc Nav1.5 targeting/regulation in heart. Further, these new data identify the basis of an in vivo cellular platform critical for membrane recruitment and regulation of Nav1.5.

Glial ankyrins facilitate paranodal axoglial junction assembly

Neuron-glia interactions establish functional membrane domains along myelinated axons. These include nodes of Ranvier, paranodal axoglial junctions and juxtaparanodes. Paranodal junctions are the largest vertebrate junctional adhesion complex, and they are essential for rapid saltatory conduction and contribute to assembly and maintenance of nodes. However, the molecular mechanisms underlying paranodal junction assembly are poorly understood. Ankyrins are cytoskeletal scaffolds traditionally associated with Na+ channel clustering in neurons and are important for membrane domain establishment and maintenance in many cell types. Here we show that ankyrin-B, expressed by Schwann cells, and ankyrin-G, expressed by oligodendrocytes, are highly enriched at the glial side of paranodal junctions where they interact with the essential glial junctional component neurofascin 155. Conditional knockout of ankyrins in oligodendrocytes disrupts paranodal junction assembly and delays nerve conduction during early development in mice. Thus, glial ankyrins function as major scaffolds that facilitate early and efficient paranodal junction assembly in the developing CNS.

Dysfunction in the βII Spectrin–Dependent Cytoskeleton Underlies Human Arrhythmia

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 &bgr;II spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. &bgr;II spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/&bgr;II spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac &bgr;II spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, &bgr;II 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 &agr;II spectrin. Finally, we observe accelerated heart failure phenotypes in &bgr;II spectrin–deficient mice. Conclusions— Our findings identify &bgr;II 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.

EHD3-Dependent Endosome Pathway Regulates Cardiac Membrane Excitability and Physiology

Rationale: Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology. Objective: To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology. Methods and Results: We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger–mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes. Conclusions: These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability.

Neuronal Na+ channel blockade suppresses arrhythmogenic diastolic Ca2+ release

AIMS Sudden death resulting from cardiac arrhythmias is the most common consequence of cardiac disease. Certain arrhythmias caused by abnormal impulse formation including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with delayed afterdepolarizations resulting from diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum (SR). Despite high response of CPVT to agents directly affecting Ca2+ cycling, the incidence of refractory cases is still significant. Surprisingly, these patients often respond to treatment with Na+ channel blockers. However, the relationship between Na+ influx and disturbances in Ca2+ handling immediately preceding arrhythmias in CPVT remains poorly understood and is the object of this study. METHODS AND RESULTS We performed optical Ca2+ and membrane potential imaging in ventricular myocytes and intact cardiac muscles as well as surface ECGs on a CPVT mouse model with a mutation in cardiac calsequestrin. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; nNav) colocalize with ryanodine receptor Ca2+ release channels (RyR2). Disruption of the crosstalk between nNav and RyR2 by nNav blockade with riluzole reduced and also desynchronized DCR in isolated cardiomyocytes and in intact cardiac tissue. Such desynchronization of DCR on cellular and tissue level translated into decreased arrhythmias in CPVT mice. CONCLUSIONS Thus, our study offers the first evidence that nNav contribute to arrhythmogenic DCR, thereby providing a conceptual basis for mechanism-based antiarrhythmic therapy.

Neuronal Na+ Channels Are Integral Components of Pro-Arrhythmic Na+/Ca2+ Signaling Nanodomain That Promotes Cardiac Arrhythmias During β-Adrenergic Stimulation

Summary Although triggered arrhythmias including catecholaminergic polymorphic ventricular tachycardia (CPVT) are often caused by increased levels of circulating catecholamines, the mechanistic link between β-adrenergic receptor (AR) stimulation and the subcellular/molecular arrhythmogenic trigger(s) is unclear. Here, we systematically investigated the subcellular and molecular consequences of β-AR stimulation in the promotion of catecholamine-induced cardiac arrhythmias. Using mouse models of cardiac calsequestrin-associated CPVT, we demonstrate that a subpopulation of Na+ channels, mainly the neuronal Na+ channels (nNav), colocalize with ryanodine receptor 2 (RyR2) and Na+/Ca2+ exchanger (NCX) and are a part of the β-AR-mediated arrhythmogenic process. Specifically, augmented Na+ entry via nNav in the settings of genetic defects within the RyR2 complex and enhanced sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA)-mediated SR Ca2+ refill is both an essential and a necessary factor for arrhythmogenesis. Furthermore, we show that augmentation of Na+ entry involves β-AR–mediated activation of CAMKII, subsequently leading to nNav augmentation. Importantly, selective pharmacological inhibition as well as silencing of Nav1.6 inhibit myocyte arrhythmic potential and prevent arrhythmias in vivo. Taken together, these data suggest that the arrhythmogenic alteration in Na+/Ca2+ handling evidenced ruing β-AR stimulation results, at least in part, from enhanced Na+ influx through nNav. Therefore, selective inhibition of these channels and of Nav1.6 in particular can serve as a potential antiarrhythmic therapy.

Eps15 Homology Domain-containing Protein 3 Regulates Cardiac T-type Ca2+ Channel Targeting and Function in the Atria

Background: Endosome-based protein trafficking in the heart is poorly understood. Results: Functional targeting and expression of the T-type Ca2+ channel (TTCC) in the atria requires the endosomal protein, EHD3. Conclusion: Impaired endosomal transport leads to cardiac rhythm and conduction disorders. Significance: Understanding endosome-based protein trafficking in the heart may provide new therapeutic targets for cardiovascular disease. Proper trafficking of membrane-bound ion channels and transporters is requisite for normal cardiac function. Endosome-based protein trafficking of membrane-bound ion channels and transporters in the heart is poorly understood, particularly in vivo. In fact, for select cardiac cell types such as atrial myocytes, virtually nothing is known regarding endosomal transport. We previously linked the C-terminal Eps15 homology domain-containing protein 3 (EHD3) with endosome-based protein trafficking in ventricular cardiomyocytes. Here we sought to define the roles and membrane protein targets for EHD3 in atria. We identify the voltage-gated T-type Ca2+ channels (CaV3.1, CaV3.2) as substrates for EHD3-dependent trafficking in atria. Mice selectively lacking EHD3 in heart display reduced expression and targeting of both Cav3.1 and CaV3.2 in the atria. Furthermore, functional experiments identify a significant loss of T-type-mediated Ca2+ current in EHD3-deficient atrial myocytes. Moreover, EHD3 associates with both CaV3.1 and CaV3.2 in co-immunoprecipitation experiments. T-type Ca2+ channel function is critical for proper electrical conduction through the atria. Consistent with these roles, EHD3-deficient mice demonstrate heart rate variability, sinus pause, and atrioventricular conduction block. In summary, our findings identify CaV3.1 and CaV3.2 as substrates for EHD3-dependent protein trafficking in heart, provide in vivo data on endosome-based trafficking pathways in atria, and implicate EHD3 as a key player in the regulation of atrial myocyte excitability and cardiac conduction.

Dysfunction of the β2-spectrin-based pathway in human heart failure.

β2-Spectrin is critical for integrating membrane and cytoskeletal domains in excitable and nonexcitable cells. The role of β2-spectrin for vertebrate function is illustrated by dysfunction of β2-spectrin-based pathways in disease. Recently, defects in β2-spectrin association with protein partner ankyrin-B were identified in congenital forms of human arrhythmia. However, the role of β2-spectrin in common forms of acquired heart failure and arrhythmia is unknown. We report that β2-spectrin protein levels are significantly altered in human cardiovascular disease as well as in large and small animal cardiovascular disease models. Specifically, β2-spectrin levels were decreased in atrial samples of patients with atrial fibrillation compared with tissue from patients in sinus rhythm. Furthermore, compared with left ventricular samples from nonfailing hearts, β2-spectrin levels were significantly decreased in left ventricle of ischemic- and nonischemic heart failure patients. Left ventricle samples of canine and murine heart failure models confirm reduced β2-spectrin protein levels. Mechanistically, we identify that β2-spectrin levels are tightly regulated by posttranslational mechanisms, namely Ca(2+)- and calpain-dependent proteases. Furthermore, consistent with this data, we observed Ca(2+)- and calpain-dependent loss of β2-spectrin downstream effector proteins, including ankyrin-B in heart. In summary, our findings illustrate that β2-spectrin and downstream molecules are regulated in multiple forms of cardiovascular disease via Ca(2+)- and calpain-dependent proteolysis.

Cellular and Molecular Pathobiology of the Cardiac Conduction System

Abstract The human heart contracts approximately two billion times over the average human lifetime with little margin for error. Proper cardiac function further requires the synchronized electrical activation of the myocardium controlled by networks of specialized cardiac cells governing automaticity and cardiac impulse conduction. Over the past decade, pathologists, molecular cardiologists, geneticists, and electro-physiologists have elucidated a wealth of information regarding the structural and molecular components responsible for regulating the properties of the myocardium, as well as the systems governing the cardiac conduction system. Furthermore, we are now beginning to understand the coordinated dysregulation of these pathways in human pathophysiology. This chapter will focus on the structural and functional pathways governing cardiac conduction as well as discuss the mechanisms underlying dysfunction of the conduction system in human pathophysiology.