Abnormal propagation of calcium waves and ultrastructural remodeling in recessive catecholaminergic polymorphic ventricular tachycardia.

RATIONALE: The recessive form of catecholaminergic polymorphic ventricular tachycardia is caused by mutations in the cardiac calsequestrin-2 gene; this variant of catecholaminergic polymorphic ventricular tachycardia is less well characterized than the autosomal-dominant form caused by mutations in the ryanodine receptor-2 gene. OBJECTIVE: We characterized the intracellular Ca²âº homeostasis, electrophysiological properties, and ultrastructural features of the Ca²âº release units in the homozygous calsequestrin 2-R33Q knock-in mouse model (R33Q) R33Q knock-in mouse model. METHODS AND RESULTS: We studied isolated R33Q and wild-type ventricular myocytes and observed properties not previously identified in a catecholaminergic polymorphic ventricular tachycardia model. As compared with wild-type cells, R33Q myocytes (1) show spontaneous Ca²âº waves unable to propagate as cell-wide waves; (2) show smaller Ca²âºsparks with shortened coupling intervals, suggesting a reduced refractoriness of Ca²âº release events; (3) have a reduction of the area of membrane contact, of the junctions between junctional sarcoplasmic reticulum and T tubules (couplons), and of junctional sarcoplasmic reticulum volume; (4) have a propensity to develop phase 2 to 4 afterdepolarizations that can elicit triggered beats; and (5) involve viral gene transfer with wild-type cardiac calsequestrin-2 that is able to normalize structural abnormalities and to restore cell-wide calcium wave propagation. CONCLUSIONS: Our data show that homozygous cardiac calsequestrin-2-R33Q myocytes develop spontaneous Ca²âº release events with a broad range of intervals coupled to preceding beats, leading to the formation of early and delayed afterdepolarizations. They also display a major disruption of the Ca²âº release unit architecture that leads to fragmentation of spontaneous Ca²âº waves. We propose that these 2 substrates in R33Q myocytes synergize to provide a new arrhythmogenic mechanism for catecholaminergic polymorphic ventricular tachycardia.

Molecular mechanisms underlying cardiac protein phosphatase 2A regulation in heart.

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.

Extracellular Matrix Instability and Chronic Inflammation Underlie Maladaptive Right Ventricular Pressure Overload Remodeling and Failure in Male Mice.

Background: Right ventricular dysfunction (RVD) portends increased death risk for heart failure (HF) and pulmonary arterial hypertension (PAH) patients, regardless of left ventricular function or etiology. In both, RVD arises from the chronic RV pressure overload, and represents advanced cardiopulmonary disease. RV remodeling responses and survival rates of HF and PAH patients, however, differ by sex. Men develop more severe RVD and die at younger ages than do women. Mechanistic details of this sexual dimorphism in RV remodeling are incompletely understood. We sought to elucidate the cardiac pathophysiology underlying the sex-specific RV remodeling phenotypes, RV failure (RVF) versus compensated RVD. Methods: We subjected male (M-) and female (F-) adult mice to moderate pulmonary artery banding (PAB) for 9wks. Mice underwent serial echocardiography, cardiac MRI, RV pressure-volume loop recordings, histologic and molecular analyses. Results: M-PAB developed severe RVD with RVF, increased RV collagen deposition and degradation, extracellular matrix (ECM) instability, and activation and recruitment of macrophages. Despite the same severity and chronicity of RV pressure overload, F-PAB had more stable ECM, lacked chronic inflammation, and developed mild RVD without RVF. Conclusions: ECM destabilization and chronic activation of recruited macrophages are associated with maladaptive RV remodeling and RVF in male PAB mice. Adaptive RV remodeling of female PAB mice lacked these histopathologic changes. Our findings suggest that these two pathophysiologic processes likely contribute to the sexual dimorphism of RV pressure overload remodeling. Further mechanistic studies are needed to assess their pathogenic roles and potential as targets for RVD therapy and RVF prevention. CLINICAL PERSPECTIVE: What is new?: In a mouse model of pure PH, males but not females showed an association between ECM instability, chronic inflammation with activation of recruited macrophages, and severe RV dysfunction and failure.What are the clinical implications?: In male HF and PH patients, enhancing ECM stability and countering the recruitment and activation of macrophages may help preserve RV function such that RVF can be prevented or delayed. Further preclinical mechanistic studies are needed to assess the therapeutic potential of such approaches. RESEARCH PERSPECTIVE: What new question does this study raise? What question should be addressed next?: What mechanisms regulate RV ECM stability and macrophage recruitment and activation in response to chronic RV pressure overload? Are these regulatory mechanisms dependent upon or independent of sex hormone signaling?

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 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 (Na(v)1.5) by Ca(2+)/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 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 the human arrhythmia susceptibility variants (A572D and Q573E) alter CaMKII-dependent regulation of Na(v)1.5, 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 Na(v)1.5 channel activity through direct effects on channel posttranslational modification. We propose that the CaMKII phosphorylation motif in the Na(v)1.5 DI-DII cytoplasmic loop is a critical nodal point for proarrhythmic changes to Na(v)1.5 in congenital and acquired cardiac disease.

Hierarchical network relaxation of a dynamic cross-linked polyolefin elastomer for advanced reversible shape memory effect.

Reversible shape-memory polymers (RSMPs) are highly desired for soft actuators due to the repeatability of deformation. Herein, a polyolefin elastomer vitrimer (POEV) was prepared by constructing a dynamic cross-linked network based on boronic ester bonds. POEV showed varied network relaxation in a wide temperature range due to hierarchical network relaxation, and then the entropy decreased and the relaxation of POEV chains was facilely controlled by temperature. The controllable relaxation of POEV by programming the temperature enabled the actuation domain with a reduction in entropy and the skeleton domain with a relatively high entropy can be built in POEV, greatly affecting the reversible shape memory effects (RSMEs). The topological rearrangement resulted from the activated exchange of dynamic covalent bonds, which enables POEV with good shape reconfigurability, and allows for complicated 3D shapes and shape-shifting on demand. More interestingly, combining the decreasing entropy of POEV chains and fully topological rearrangement tailored by temperature, hybrid aligned carbon nanotubes (CNTs) can be constructed in POEV via a two-stage training. Then, the aligned CNTs can enhance the elasticity and act as a hybrid skeleton for RSMEs, avoiding the negative impact of CNTs on the reversible actuation strain. The hierarchical network relaxation facilitates combining all these unusual properties in one shape memory network synergistically, paving new avenues for realizing smart materials with advanced RSME.

Configurational Entropy Regulation in Polyolefin Elastomer/Paraffin Wax Vitrimers by Thermally Responsive Liquid-Solid Transition for Force Storage.

The work output of shape memory polymers during shape shifting is desired for practical application as actuators. Herein, a polyolefin elastomer (POE) and paraffin wax (PW) are co-cross-linked by dynamic boronic ester bonds to enhance the network elasticity and the stress transfer between the two phases, endowing high force storage capacity to the prepared vitrimers. Depending on the phase of PW, one-way force storage is realized by programming at a low temperature (25 °C), owing to which solid PW can promote the locking of POE chains in a low-entropy state, while reversible force storage can be realized by programming at a high temperature (75 °C), owing to which the relaxation of chains facilitated by liquid PW can promote the construction of a stable structure. Based on one-way force storage, a weight-lifting machine with a weight of 20 mg prestrained at 25 °C can lift a 100 g weight, showing a lifting ratio of no less than 5000, with a high work output of 0.98 J/g. A high-temperature alarm can be triggered at varied temperatures (43-56 °C) through controlled force release by adjusting the PW content and programmed prestrains. Based on the reversible force storage, crawling robots and artificial muscles with a work output of 0.025 J/g are demonstrated. The dynamic cross-linking network also confers mold-free self-healing capability to POE/PW vitrimers, and the repair efficiency is enhanced compared with the POE vitrimer due to the improved POE chain motion by liquid PW. The realized one-way and reversible force storage and self-healing by POE/PW vitrimers pave the way for the application of SMPs in the fields of soft robotic actuators.

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.

Self-Sensing Actuators Based on a Stiffness Variable Reversible Shape Memory Polymer Enabled by a Phase Change Material.

Soft actuators with integrated mechanical and actuation properties and self-sensing ability are still a challenge. Herein, a stiffness variable polyolefin elastomer (POE) with a reversible shape memory effect is prepared by introducing a typical phase change material, i.e., paraffin wax (PW). It is found that the variable stiffness of POE induced by PW can balance the reversible strain and load-bearing capability of actuators. Especially, carbon nanotubes (CNTs) are concentrated in a thin surface layer by spraying and hot pressing in the soft state of POE/PW blends, providing signal transductions for the strain and temperature perception for actuators. Taking advantage of tunable reversible deformation and mechanical transformation of the POE/PW actuator, different biomimetic robotics, including grippers with high load-bearing capability (weight-lifting ratio > 146), walking robots that can sense angles of joints, and high-temperature warning robots are demonstrated. A scheme combining the variable stiffness and electrical properties provides a versatile strategy to integrate actuation performance and self-sensing ability, inspiring the development of multifunctional composite designs for soft robotics.

Rad regulation of CaV1.2 channels controls cardiac fight-or-flight response.

Fight-or-flight responses involve ß-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for ß-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of ß-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel ß-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

Complex and rate-dependent beat-to-beat variations in Ca2+ transients of canine Purkinje cells.

Purkinje fibers play an essential role in transmitting electrical impulses through the heart, but they may also serve as triggers for arrhythmias linked to defective intracellular calcium (Ca(2+)) regulation. Although prior studies have extensively characterized spontaneous Ca(2+) release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca(2+) transients. Therefore we explored the behaviors of Ca(2+) transients at pacing rates ranging from 0.125 to 3 Hz in single canine Purkinje cells loaded with fluo3 and imaged with a confocal microscope. The experiments uncovered the following novel aspects of Ca(2+) regulation in Purkinje cells: 1) the cells exhibit a negative Ca(2+)-frequency relationship (at 2.5 Hz, Ca(2+) transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca(2+) release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca(2+) load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca(2+) transients show considerable beat-to-beat variability, with greater variability occurring at higher pacing rates. Analysis of beat-to-beat variability suggests that it can be accounted for by stochastic triggering of local Ca(2+) release events. Consistent with this hypothesis, an increase in triggering probability caused a decrease in the relative variability. These results offer new insight into how Ca(2+) release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias.

Autonomic modulation of sinoatrial node: Role of pacemaker current and calcium sensitive adenylyl cyclase isoforms.

This article reviews work over the past three decades that is related to the contribution of the pacemaker current, If, to basal and autonomically regulated spontaneous rate in the sinoatrial node. It also addresses how the actions of the pacemaker current relate to those of Ca homeostasis with respect to basal and autonomically regulated rhythm. In this regard, it explores the relative contributions of Ca-sensitive and Ca-insensitive isoforms of adenylyl cyclase to sinoatrial node automaticity. The latter studies include previously unpublished work making use of mice in which both the type 1 and type 8 Ca-sensitive adenylyl cyclase isoforms were knocked out. These studies indicate that the pacemaker current and the L-type Ca current are distinctly influenced by Ca-sensitive and insensitive adenylyl cyclase isoforms.

Combustion Synthesis of Nanostructured ZrC: Formation Process and Influencing Factors.

ZrC was produced by the combustion synthesis technology using Cu, Zr, and graphite as the starting element powders. The synthesis mechanism of ZrC was investigated by the combustion wave quenching experiment. Furthermore, the effects of sizes of C and Cu on the combustion synthesis behavior and products were also explored. Results revealed that ZrC was fabricated through the displacement reaction between C and Cu-Zr liquid. The Cu size hardly affected the combustion temperature and resultant products, indicating that the preparation cost of ZrC could be decreased by employing coarse Cu powders. With increasing C size, the burning temperature and ceramic particle size reduced. Graphite with size of 2.6 µm was used as the C source, and only ZrC nanoparticles and Cu were obtained. The products could be employed to prepare nano-sized ZrC/Cu composites without the elimination of by-products.

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.

In vitro characterization of HCN channel kinetics and frequency dependence in myocytes predicts biological pacemaker functionality.

The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1-5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers.

WIPI1 is a conserved mediator of right ventricular failure.

Right ventricular dysfunction is highly prevalent across cardiopulmonary diseases and independently predicts death in both heart failure (HF) and pulmonary hypertension (PH). Progression towards right ventricular failure (RVF) can occur in spite of optimal medical treatment of HF or PH, highlighting current insufficient understanding of RVF molecular pathophysiology. To identify molecular mechanisms that may distinctly underlie RVF, we investigated the cardiac ventricular transcriptome of advanced HF patients, with and without RVF. Using an integrated systems genomic and functional biology approach, we identified an RVF-specific gene module, for which WIPI1 served as a hub and HSPB6 and MAP4 as drivers, and confirmed the ventricular specificity of Wipi1, Hspb6, and Map4 transcriptional changes in adult murine models of pressure overload induced RV- versus LV- failure. We uncovered a shift towards non-canonical autophagy in the failing RV that correlated with RV-specific Wipi1 upregulation. In vitro siRNA silencing of Wipi1 in neonatal rat ventricular myocytes limited non-canonical autophagy and blunted aldosterone-induced mitochondrial superoxide levels. Our findings suggest that Wipi1 regulates mitochondrial oxidative signaling and non-canonical autophagy in cardiac myocytes. Together with our human transcriptomic analysis and corroborating studies in an RVF mouse model, these data render Wipi1 a potential target for RV-directed HF therapy.

Electro and Light-Active Actuators Based on Reversible Shape-Memory Polymer Composites with Segregated Conductive Networks.

Reversible shape-memory polymers (RSMPs) show great potential in actuating applications because of its repeatability among many other advantages. Indeed, in many cases, multiresponsive RSMPs are more expected, and the strategy to introduce functional fillers without deteriorating the reversible deformation performance is of great importance. Here, a facile strategy to balance the electro, photothermal performance, and molecular chain mobility is reported. Segregated conductive networks of carbon nanotubes (S-CNTs) are constructed in the poly(ethylene-co-octene) (POE) matrix at a relatively low filler loading, which renders the composite good electrical, photothermal, and actuating properties. A low percolation threshold of 0.25 vol % is achieved. The electrical conductivity is up to 0.046 S·cm-1 for the POE/S-CNT composites with 2 vol % CNT, and the absorption of light (760 nm) is above 90%. These characteristics guarantee that the actuator can be driven at low voltage (≤36 V) and suitable light intensity (250 mW·cm-2) with a good actuating performance. An electric gripper and a light-active crawling robot demonstrate the potential applications in multiresponsive robots. This work introduces a facile strategy to fabricate multiresponsive RSMPs by designing CNT network structures in polymer composites and holds great potential to enlarge the applications of RSMPs in many areas including artificial muscles and bionic robots.

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.

Wide long lasting perinuclear Ca2+ release events generated by an interaction between ryanodine and IP3 receptors in canine Purkinje cells.

The purpose of this study was to determine whether IP(3)Rs contribute to the generation of wide long lasting perinuclear Ca(2+) release events in canine Purkinje cells. Spontaneous Ca(2+) release events (elevations of basal [Ca(2+)] equivalent to F/F(0) 3.4SD over F(0)) were imaged using Fluo-4AM and 2D confocal microscope. Only cells free of Ca(2+) waves were analyzed. Subsarcolemmal region (SSL) was defined as 5 microm from cell edges. Core was the remaining cell. The majority of events (94%, 0.0035+/-0.0007 events (ev)/microm(2)/s, N=34 cells) were detected within a single frame (typical events, TE). However, a subpopulation (6.0%, 0.00022+/-0.00005 ev/microm(2)/s, N=41 cells: wide long lasting events, WLE) lasted for several frames, showed a greater spatial extent (51.0+/-3.9 vs. TE 9.0+/-0.3 microm(2), P<0.01) and higher amplitude (F/F(0) 1.38+/-0.02 vs. TE 1.20+/-0.003, P<0.01). WLE event rate was increased by phenylephrine (10 microM, P<0.01), inhibited by 2APB and U73122 (P<0.05), and abolished by tetracaine (1 mM) and ryanodine (100 microM). While SSL WLEs were scattered randomly, Core WLEs (n=69 events) were predominantly distributed longitudinally 18.2+/-1.6 microm from the center of nuclei. Immunocytochemistry showed that IP(3)R1s were located not only at SSL region but also near both ends of nucleus overlapping with RyRs. In Purkinje cells, wide long lasting Ca(2+) release events occur in SSL and in specific perinuclear regions. They are likely due to RyRs and IP(3)R1s evoked Ca(2+) release and may play a role in Ca(2+) dependent nuclear processes.

Early electrical remodeling in rabbit pulmonary vein results from trafficking of intracellular SK2 channels to membrane sites.

OBJECTIVE: Atrial fibrillation is often initiated by bursts of ectopic activity arising in the pulmonary veins. We have previously shown that a 3-h intermittent burst pacing protocol (BPP), mimicking ectopic pulmonary vein foci, shortens action potential duration (APD) locally at the pulmonary vein-atrial interface (PV) while having no effect elsewhere in rabbit atrium. This shortening is Ca(2+) dependent and is prevented by apamin, which blocks small conductance Ca(2+)-activated K(+) channels (SK(Ca)). The present study investigates the ionic and molecular mechanisms whereby two apamin-sensitive SK(Ca) channels, SK2 and SK3, might contribute to the regional APD changes. METHODS: Microelectrode and patch clamp techniques were used to record APDs and apamin-sensitive currents in isolated rabbit left atria and cells dispersed from PV and Bachmann's bundle (BB) regions. SK2 and SK3 mRNA and protein levels were quantified, and immunofluorescence was used to observe channel protein distribution. RESULTS: There was a direct relationship between APD shortening and apamin-sensitive current in burst-paced but not sham-paced PV. Moreover, apamin-sensitive current density increased in PV but not BB after BPP. SK2 mRNA, protein, and current were increased in PV after BPP, while SK2 immunostaining shifted from a perinuclear pattern in sham atria to predominance at sites near or at the PV membrane. CONCLUSIONS: BPP-induced acceleration of repolarization in PV results from SK2 channel trafficking to the membrane, leading to increased apamin-sensitive outward current. This is the first indication of involvement of Ca(2+)-activated K(+) currents in atrial remodeling and provides a possible basis for evolution of an arrhythmogenic substrate.

Microtubular remodeling and decreased expression of Nav1.5 with enhanced EHD4 in cells from the infarcted heart.

Cardiac Na+ channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 cytoskeletal- and endosomal-based membrane trafficking and function are linked to tubulin, microtubular (MT) networks, and Eps15 homology domain containing proteins like EHD4. AIM: Our objective is to understand the relation of tubulin and EHD4 to Nav1.5 channel protein remodeling observed in border zone cells (IZs) when arrhythmias are known to occur; that is, 3-h, 48-h and 5-day post coronary occlusion. MATERIALS METHODS FINDINGS: Our voltage clamp and immunostaining data show that INa density is decreased in the epicardial border zone cells of the 48 h infarcted heart (IZ48h). Immunostaining studies reveal that in post MI cells the cell surface staining of Nav1.5 was reduced and Nav1.5 distribution changed. However, intense co-staining of Nav1.5 and tubulin occurs in core planes and the perinuclear areas in post MI cells. At the same time, there were marked changes in the subcellular location of the EHD4 protein. EHD4 is co-localized with tubulin protein in discrete intracellular "highway" structures. SIGNIFICANCE: The distribution and expression of the three proteins are altered dynamically in post MI cells. In sum, our work illustrates the spatiotemporal complexity of remodeling mechanisms in the post-infarct myocyte. It will be important in future experiments to further explore direct links between MT, EHD proteins, and cell proteins involved in forward trafficking.