Wen Dun

Ca2+/Calmodulin-Dependent Protein Kinase II–Based Regulation of Voltage-Gated Na+ Channel in Cardiac Disease

Background —Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked with potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite over a decade of investigation. Post-translational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel post-translational modifications and disease. We recently identified a novel pathway for post-translational regulation of the primary cardiac voltage-gated Na + channel (Na v 1.5) by CaMKII. However, a role for this pathway in cardiac disease has not been evaluated. Methods and Results —We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Na v 1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Na v 1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large animal model of acquired heart disease and in failing human myocardium. Conclusions —We identify the mechanism for two human arrhythmia variants that affect Na v 1.5 channel activity through direct effects on channel post-translational modification. We propose that the CaMKII phosphorylation motif in the Na v 1.5 DI-DII cytoplasmic loop is a critical nodal point for pro-arrhythmic changes to Na v 1.5 in congenital and acquired cardiac disease.Background— Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked to potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite more than a decade of investigation. Posttranslational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel posttranslational modifications and disease. We recently identified a novel pathway for posttranslational regulation of the primary cardiac voltage-gated Na+ channel (Nav1.5) by Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, a role for this pathway in cardiac disease has not been evaluated. Methods and Results— We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Nav1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Nav1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5, resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel, resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large-animal model of acquired heart disease and in failing human myocardium. Conclusions— We identify the mechanism for 2 human arrhythmia variants that affect Nav1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.

CaMKII-Based Regulation of Voltage-Gated Na+ Channel in Cardiac Disease

Background —Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked with potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite over a decade of investigation. Post-translational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel post-translational modifications and disease. We recently identified a novel pathway for post-translational regulation of the primary cardiac voltage-gated Na + channel (Na v 1.5) by CaMKII. However, a role for this pathway in cardiac disease has not been evaluated. Methods and Results —We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Na v 1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Na v 1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5 resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large animal model of acquired heart disease and in failing human myocardium. Conclusions —We identify the mechanism for two human arrhythmia variants that affect Na v 1.5 channel activity through direct effects on channel post-translational modification. We propose that the CaMKII phosphorylation motif in the Na v 1.5 DI-DII cytoplasmic loop is a critical nodal point for pro-arrhythmic changes to Na v 1.5 in congenital and acquired cardiac disease.Background— Human gene variants affecting ion channel biophysical activity and/or membrane localization are linked to potentially fatal cardiac arrhythmias. However, the mechanism for many human arrhythmia variants remains undefined despite more than a decade of investigation. Posttranslational modulation of membrane proteins is essential for normal cardiac function. Importantly, aberrant myocyte signaling has been linked to defects in cardiac ion channel posttranslational modifications and disease. We recently identified a novel pathway for posttranslational regulation of the primary cardiac voltage-gated Na+ channel (Nav1.5) by Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, a role for this pathway in cardiac disease has not been evaluated. Methods and Results— We evaluated the role of CaMKII-dependent phosphorylation in human genetic and acquired disease. We report an unexpected link between a short motif in the Nav1.5 DI-DII loop, recently shown to be critical for CaMKII-dependent phosphorylation, and Nav1.5 function in monogenic arrhythmia and common heart disease. Experiments in heterologous cells and primary ventricular cardiomyocytes demonstrate that the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Nav1.5, resulting in abnormal channel activity and cell excitability. In silico analysis reveals that these variants functionally mimic the phosphorylated channel, resulting in increased susceptibility to arrhythmia-triggering afterdepolarizations. Finally, we report that this same motif is aberrantly regulated in a large-animal model of acquired heart disease and in failing human myocardium. Conclusions— We identify the mechanism for 2 human arrhythmia variants that affect Nav1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.

Contactin-2 expression in the cardiac Purkinje fiber network.

Background—Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system, and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes. Methods and Results—Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express &bgr;-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca2+ transients in Cntn2-EGFP+ cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations. Conclusions—Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells.

The Purkinje cell; 2008 style

Cardiac Purkinje fibers, due to their unique anatomical location, cell structure and electrophysiologic characteristics, play an important role in cardiac conduction and arrhythmogenesis. Purkinje cell action potentials are longer than their ventricular counterpart, and display two levels of resting potential. Purkinje cells provide for rapid propagation of the cardiac impulse to ventricular cells and have pacemaker and triggered activity, which differs from ventricular cells. Additionally, a unique intracellular Ca2+ release coordination has been revealed recently for the normal Purkinje cell. However, since the isolation of single Purkinje cells is difficult, particularly in small animals, research using Purkinje cells has been restricted. This review concentrates on comparison of Purkinje and ventricular cells in the morphology of the action potential, ionic channel function and molecular determinants by summarizing our present day knowledge of Purkinje cells.

Ca2+ Sparks and Waves in Canine Purkinje Cells. A Triple Layered System of Ca2+ Activation

We have investigated the subcellular spontaneous Ca2+ events in canine Purkinje cells using laser scanning confocal microscopy. Three types of Ca2+ transient were found: (1) nonpropagating Ca2+ transients that originate directly under the sarcolemma and lead to (2) small Ca2+ wavelets in a region limited to ≈6-&mgr;m depth under the sarcolemma causing (3) large Ca2+ waves that travel throughout the cell (CWWs). Immunocytochemical studies revealed 3 layers of Ca2+ channels: (1) channels associated with type 1 IP3 receptors (IP3R1) and type 3 ryanodine receptors (RyR3) are prominent directly under the sarcolemma; (2) type 2 ryanodine receptors (RyR2s) are present throughout the cell but virtually absent in a layer between 2 and 4 &mgr;m below the sarcolemma (Sub-SL); (3) type 3 ryanodine receptors (RyR3) is the dominant Ca2+ release channel in the Sub-SL. Simulations of both nonpropagating and propagating transients show that the generators of Ca2+ wavelets differ from those of the CWWs with the threshold of the former being less than that of the latter. Thus, Purkinje cells contain a functional and structural Ca2+ system responsible for the mechanism that translates Ca2+ release occurring directly under the sarcolemma into rapid Ca2+ release in the Sub-SL, which then initiates large-amplitude long lasting Ca2+ releases underlying CWWs. The sequence of spontaneous diastolic Ca2+ transients that starts directly under the sarcolemma and leads to Ca2+ wavelets and CWWs is important because CWWs have been shown to cause nondriven electrical activity.

EH Domain Proteins Regulate Cardiac Membrane Protein Targeting

Rationale: Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. Objective: We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. Methods and Results: We report the initial characterization of a large family of membrane trafficking proteins in human heart. We used a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified 4 members of a unique Eps15 homology (EH) domain–containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are coexpressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Conclusions: Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin-based targeting network, and identify potential new targets to modulate membrane excitability in disease. Notably, these data provide the first link between EHD proteins and a human disease model.

Cardiac Purkinje cells.

Purkinje cells are specialized for rapid propagation in the heart. Furthermore, Purkinje fibers as the source as well as the perpetuator of arrhythmias is a familiar finding. This is not surprising considering their location in the heart and their unique cell ultrastructure, cell electrophysiology, and mode of excitation-contraction coupling. This review touches on each of these points as we outline what is known today about Purkinje fibers/cells.

Oxidized calmodulin kinase II regulates conduction following myocardial infarction: A computational analysis

Calmodulin kinase II (CaMKII) mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ). These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart.

Regulation of the ankyrin-B-based targeting pathway following myocardial infarction

AIMS Ion channel reorganization is a critical step in the pro-arrhythmogenic remodelling process that occurs in heart disease. Ankyrin-B (AnkB) is required for targeting and stabilizing ion channels, exchangers, and pumps. Despite a wealth of knowledge implicating the importance of AnkB in human cardiovascular physiology, nothing is known regarding the role of AnkB in common forms of acquired human disease. METHODS AND RESULTS We present the first report of AnkB regulation following myocardial infarction (MI). AnkB protein levels were reduced in the infarct border zone 5 days following coronary artery occlusion in the canine. We also observed a dramatic increase in AnkB mRNA levels 5 days post-occlusion. Surprisingly, the expression of the upstream AnkB cytoskeletal component beta2-spectrin was unchanged in post-infarct tissues. However, protein levels and/or membrane expression of downstream AnkB-associated ion channels and transporters Na+/K+ ATPase, Na+/Ca2+ exchanger, and IP3 receptor were altered 5 days post-occlusion. Interestingly, protein levels of the protein phosphatase 2A, an AnkB-associated signalling protein, were significantly affected 5 days post-occlusion. AnkB and PP2A protein levels recovered by 14 days post-occlusion, whereas Na+/K+ ATPase levels recovered by 2 months post-occlusion. CONCLUSION These findings reveal the first evidence of ankyrin remodelling following MI and suggest an unexpected divergence point for regulation between ankyrin and the underlying cytoskeletal network. These findings suggest a logical, but unexpected, molecular mechanism underlying ion channel and transporter remodelling following MI.

Calcium and potassium currents in cells from adult and aged canine right atria

BACKGROUND Action potential (AP) contours vary considerably between normal adult and aged right atrial fibers. The ionic bases for these differences remain unknown. Therefore we studied Ca(2+) and K(+) currents in cells from adult and aged canine right atria (RA). METHODS AND RESULTS We used whole cell patch clamp recording techniques to measure L-type Ca(2+) currents (I(CaL)) with either Ca(2+) or Ba(2+) (3 mM) as the charge carrier, and both the transient outward (I(to)) and sustained potassium currents (I(sus)) in cells dispersed from normal adult (Adult, 2-5 years) and older dogs (Aged, >8 years). There is a significant reduction in peak I(CaL) (47%) and I(BaL) (43%) in Aged cells, yet differences in I(BaL) disappear with maximal beta adrenergic stimulation (isoproterenol, 1 microM). Composite I(to) and I(sus) densities were significantly increased in the Aged versus Adult cell group (by 31 and 27% at +50 mV, respectively). I(to) decay during a maintained depolarization was slowed in Aged cells. Furthermore, I(to) steady-state inactivation curve was shifted positively in Aged cells. Finally, composite I(to) and I(sus) currents of Aged cells were more sensitive to tetraethylammonium chloride (TEA), a specific inhibitor of some types of K(+) currents. In the presence of TEA (5 mM), I(to) in Aged cells was significantly greater than that in Adult cells. CONCLUSIONS Ionic currents differ in Aged versus Adult right atrial cells, such that a reduced Ca(2+) current and augmented outward currents could contribute significantly to the altered AP contour of the Aged RA cell. Adrenergic stimulation appears to restore Ba(2+) currents in Aged cells. Finally, an augmented TEA sensitive current plays a role in changes of I(sus) in Aged right atrial cells.