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1.  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.
doi:10.1016/j.yjmcc.2008.08.001
PMCID: PMC4332524  PMID: 18778712
Arrhythmias; Purkinje cells; Ion channels; Ca2+ waves; Ventricular cells
2.  Nonuniform Ca2+ transients in arrhythmogenic Purkinje cells that survive in the infarcted canine heart 
Cardiovascular research  2003;57(3):681-693.
Objective and methods
In this study, we investigated whether Ca2+ transients are altered in Purkinje cell aggregates dispersed from the subendocardium overlying the infarcted zone of the left ventricle (IZPCs) 48 h after coronary artery occlusion. To do so, we combined epifluorescent imaging with microelectrode recordings of IZPCs and normal canine Purkinje cell aggregates (NZPCs).
Results
NZPCs respond to an action potential (AP) by a small Ca2+ transient at the cell surface immediately after the AP upstroke followed by a large [Ca2+ ] transient, which propagates to the cell core. In addition, focal Ca2+ waves can originate spontaneously later during the AP or during the diastolic interval (Circ Res 2000;86:448-55) and then propagate throughout the aggregate as ‘cell-wide Ca2+ waves’. Electrically-evoked Ca2+ transients in IZPCs arose significantly faster than those in NZPCs, and showed substantial spatiotemporal nonuniformity within an IZPC aggregate as well as between IZPC aggregates. IZPCs showed, hitherto undetected, low amplitude, micro Ca2+ transients (extent ≤5 μm) at a fivefold higher incidence than in NZPCs. Micro Ca2+ transients appeared to meander over distances ≤100 μm and reduced the local Ca2+ transient of the next paced beat. Micro Ca2+ transients nearly always preceded the cell-wide Ca2+ waves, which occurred more frequently in IZPCs than in NZPCs and caused non-driven electrical activity of the Purkinje aggregate.
Conclusions
Micro Ca2+ transients preceded cell-wide Ca2+ waves so often that it is probable that micro Ca2+ transients induced cell-wide Ca2+ waves. Cell-wide Ca2+ waves, in turn, clearly elicited spontaneous APs. We propose that the high incidence of micro Ca2+ transients in IZPCs is a fundamental element of the abnormal Ca2+ handling of diseased Purkinje cells, underlying arrhythmias originating in the subendocardial Purkinje network post myocardial infarction.
PMCID: PMC4332527  PMID: 12618230
Arrhythmia (mechanisms); Calcium (cellular); Impulse formation; Infarction; Membrane potential; Purkinje fiber
3.  Aged atria: electrical remodeling conducive to atrial fibrillation 
The incidence of atrial fibrillation (AF) increases with age. Alterations in structure and function of atrial ion channels associated with aging provide the substrate for AF. In this review we provide an overview of current knowledge regarding these age-related changes in atria, focusing on intrinsic ion channel function, impulse initiation and conduction. Studies on the action potentials (APs) of atria have shown that the AP contour is altered with age and the dispersion of AP parameters is increased with age. However, studies using human tissues are not completely consistent with experimental animal studies, since specimens from humans have been obtained from hearts with concomitant cardiovascular diseases and/or that are under the influence of pharmacologic agents. Ionic current studies show that while there are no age-related changes in sodium currents in atrial tissue, the calcium current is reduced and the transient outward and sustained potassium currents are increased in aged cells. While sinoatrial node firing is reduced with age, enhanced impulse initiation may occur in aged atrial cells, for example in the pulmonary veins and coronary sinus. Fibrous tissue is increased in aged atria, which is associated with an increased likelihood of abnormal electrical conduction. Thus, age-related AF involves alterations in the substrate as well as in the passive properties of aged atria.
doi:10.1007/s10840-008-9358-3
PMCID: PMC4332532  PMID: 19280327
Age; Atria; Ion channels; Calcium; Atrial fibrillation
4.  Calcium and Arrhythmogenesis 
Physiological reviews  2007;87(2):457-506.
Triggered activity in cardiac muscle and intracellular Ca2+ have been linked in the past. However, today not only are there a number of cellular proteins that show clear Ca2+ dependence but also there are a number of arrhythmias whose mechanism appears to be linked to Ca2+-dependent processes. Thus we present a systematic review of the mechanisms of Ca2+ transport (forward excitation-contraction coupling) in the ventricular cell as well as what is known for other cardiac cell types. Second, we review the molecular nature of the proteins that are involved in this process as well as the functional consequences of both normal and abnormal Ca2+ cycling (e.g., Ca2+ waves). Finally, we review what we understand to be the role of Ca2+ cycling in various forms of arrhythmias, that is, those associated with inherited mutations and those that are acquired and resulting from reentrant excitation and/or abnormal impulse generation (e.g., triggered activity). Further solving the nature of these intricate and dynamic interactions promises to be an important area of research for a better recognition and understanding of the nature of Ca2+ and arrhythmias. Our solutions will provide a more complete understanding of the molecular basis for the targeted control of cellular calcium in the treatment and prevention of such.
doi:10.1152/physrev.00011.2006
PMCID: PMC4332537  PMID: 17429038
5.  Dynamic remodeling of K+ and Ca2+ currents in cells that survived in the epicardial border zone of canine healed infarcted heart 
Action potentials (APs) of the epicardial border zone (EBZ) cells from the day 5 infarcted heart continue to be altered by day 14 postocclusion, namely, they shortened. However, by 2 mo, EBZ APs appear “normal,” yet conduction of wave fronts remains abnormal. We hypothesize that the changes in transmembrane APs are due to a change in the distribution of ion channels in either density or function. Thus we focused on the changes in Ca2+ and K+ currents in cells isolated from the 14-day (IZ14d) and 2-mo (IZ2m) EBZ and compared them with those occurring in cells from the same hearts but remote (Rem) from the EBZ. Whole cell voltage-clamp techniques were used to measure and compare Ca2+ and K+ currents in cells from the different groups. Ca2+ current densities remain reduced in cells of the 14-day and 2-mo infarcted heart and the kinetic changes previously identified in the 5-day heart begin to, but do not recover to, cells from noninfarcted epicardium (NZ) values. Importantly, ICa,L in both the EBZ and Rem regions still show a slowed recovery from inactivation. Furthermore, during the remodeling process, there is an increased expression of T-type Ca2+ currents, but only regionally, and only within a specific time window postmyocardial infarction (MI). Regional heterogeneity in β-adrenergic responsiveness of ICa,L exists between EBZ and remote cells of the 14-day hearts, but this regional heterogeneity is gone in the healed infarcted heart. In IZ14d, the transient outward K+ current (Ito) begins to reemerge and is accompanied by an upregulated tetraethylammonium-sensitive outward current. By 2-mo post-occlusion, Ito and sustained outward K+ current have completed the reverse remodeling process. During the healing process post-MI, canine epicardial cells downregulate the fast Ito but compensate by upregulating a K+ current that in normal cells is minimally functional. For recovering ICa,L of the 14-day and 2-mo EBZ cells, voltage-dependent processes appear to be reset, such that ICa,L “window” current occurs at hyperpolarized potentials. Thus dynamic changes in both Ca2+ and K+ currents contribute to the altered AP observed in 14-day fibers and may account for return of APs of 2 mo EBZ fibers.
doi:10.1152/ajpheart.00082.2004
PMCID: PMC4289133  PMID: 15087289
myocardial infarction
6.  2APB- and JTV519(K201)-sensitive micro Ca2+ waves in arrhythmogenic Purkinje cells that survive in infarcted canine heart 
Objectives/Background
Studies from several laboratories have implicated intracellular Ca2+ dynamics in the modulation of electrical activity. We have reported that abnormal Ca2+ wave activity is the underlying cause of afterdepolarization-induced electrical activity in subendocardial Purkinje cells that survive in the 48-hour infarcted canine heart. These cells form the focus of arrhythmias at this time postcoronary artery occlusion.
Methods
We studied the effects of agonists and antagonists on the abnormal Ca2+ release activity of Purkinje cell aggregates dispersed from the subendocardium 48 hours postcoronary artery occlusion (IZPCs). Studies were completed using epifluorescent microscopy of Fluo-3 loaded Purkinje cells.
Results
Similar to our previous report, highly frequent traveling micro Ca2+ transients(μCaiTs) and cell-wide Ca2+ waves were seen in IZPCs in the absence of any drug. Isoproterenol (ISO) increased μCaiTs and cell-wide Ca2+ waves in Purkinje cells dispersed from the normal heart (NZPCs). In IZPCs, ISO increased cell-wide wave frequency but had no effect on the already highly frequent micro Ca2+ wave transient activity, suggesting that ISO lowers the threshold of cell-wide generators responding to micro Ca2+ transients. Drugs that block inward sodium or calcium currents (verapamil, tetrodotoxin) had no effect on Ca2+ activity in Purkinje cells. Antagonists of intracellular Ca2+ release channels [ryanodine, JTV519(K201)] greatly suppressed spontaneous Ca2+ release events in IZPCs. 2APB, an agent that blocks IP3 receptors, greatly reduced the frequency of Ca2+events in IZPCs.
Conclusions
In arrhythmogenic Purkinje cells that survive in the infarcted heart, agents that block or inhibit intracellular Ca2+ release channel activity reduced Ca2+ waves and could be antiarrhythmic.
doi:10.1016/j.hrthm.2004.03.068
PMCID: PMC4289136  PMID: 15851156
Purkinje; Action potentials; Ca2+ waves; Cai transients; Automaticity; Myocardial infarction
7.  Ca2+ Sparks and Waves in Canine Purkinje Cells 
Circulation research  2005;97(1):35-43.
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-μm 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 μm 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.
doi:10.1161/01.RES.0000173375.26489.fe
PMCID: PMC4289137  PMID: 15947247
Purkinje; Ca2+ sparks; Ca2+ waves; Ca transients; automaticity
8.  Remodeling in Cells From Different Regions of the Reentrant Circuit During Ventricular Tachycardia 
Circulation  2005;112(16):2386-2396.
Background
Anisotropic reentrant excitation occurs in the remodeled substrate of the epicardial border zone (EBZ) of the 5-day infarcted canine heart. Reentry is stabilized because of the formation of functional lines of block. We hypothesized that regional differences of ionic currents in cells of the EBZ form these lines of block. Therefore, we first mapped reentrant circuits of sustained tachycardias, then dispersed cells (infarct zone cells, IZs) from the central common pathway of the circuit (IZc) as well as from the other side of the line of block (outer pathway, IZo) for study.
Methods and Results
We mapped reentrant circuits in the EBZ of infarcted hearts during sustained ventricular tachycardias (>30 seconds, n=17 episodes, cycle lengths=218±7.9 ms). INa density was reduced in both IZc and IZo, and the kinetic properties of IZc INa were markedly altered versus IZo. Structural remodeling of the sodium channel protein Nav1.5 occurred in IZs, with cell surface localization differing from normal cells. Both IZc and IZo have similar but reduced ICaL, whereas IZc showed changes in Ca2+ current kinetics with an acceleration of current decay. Computer simulations of the 2D EBZ showed that incorporating only differences between INa in IZc and IZo prevented stability of the reentrant circuit. Incorporating only differences between ICaL in the IZc and IZo cells also prevented stability of the circuit. However, incorporating both INa and ICaL current differences stabilized the simulated reentrant circuit, and lines of block formed between the 2 distinct regions.
Conclusions
Despite differences in INa and ICaL properties in cells of the center and outer pathways of a reentrant circuit, the resulting changes in effective refractory periods tend to stabilize reentry in this remodeled substrate.
doi:10.1161/CIRCULATIONAHA.105.534784
PMCID: PMC4289139  PMID: 16203911
myocardial infarction; ion channels; reentry; arrhythmia; electrophysiology
9.  Ca2+ Transients and Ca2+ Waves in Purkinje Cells Role in Action Potential Initiation 
Circulation research  2000;86(4):448-455.
Purkinje cells contain sarcoplasmic reticulum (SR) directly under the surface membrane, are devoid of t-tubuli, and are packed with myofibrils surrounded by central SR. Several studies have reported that electrical excitation induces abiphasic Ca2+ transient in Purkinje fiber bundles. We determined the nature of the biphasic Ca2+ transient in aggregates of Purkinje cells. Aggregates (n=12) were dispersed from the subendocardial Purkinje fiber network of normal canine left ventricle, loaded with Fluo-3/AM, and studied in normal Tyrode's solution (24°C). Membrane action potentials were recorded with fine-tipped microelectrodes, and spatial and temporal changes in [Ca2+]i were obtained from fluorescent images with an epifluorescent microscope (×20; Nikon). Electrical stimulation elicited an action potential as well as a sudden increase in fluorescence (L0) compared with resting levels. This was followed by a further increase in fluorescence (L1) along the edges of the cells. Fluorescence then progressed toward the Purkinje cell core (velocity of propagation 180 to 313 μm/s). In 62% of the aggregates, initial fluorescent changes of L0 were followed by focally arising Ca2+ waves (L2), which propagated at 158± 14 μm/s (n= 13). Spontaneous Ca2+ waves (L2*) propagated like L2 (164±10 μm/s) occurred between stimuli and caused slow membrane depolarization; 28% of L2* elicited action potentials. Both spontaneous Ca2+ wave propagation and resulting membrane depolarization were thapsigargin sensitive. Early afterdepolarizations were not accompanied by Ca2+ waves. Action potentials in Purkinje aggregates induced a rapid rise of Ca2+ through ICaL and release from a subsarcolemmal compartment (L0). Ca2+ release during L0 either induced further Ca2+ release, which propagated toward the cell core (L1), or initiated Ca2+ release from small regions and caused L2 Ca2+ waves, which propagated throughout the aggregate. Spontaneous Ca2+ waves (L2*) induce action potentials.
PMCID: PMC4289140  PMID: 10700450
Purkinje fibers; action potentials; Ca2+; electrophysiology; automaticity
10.  SAP97 and Cortactin Remodeling in Arrhythmogenic Purkinje Cells 
PLoS ONE  2014;9(9):e106830.
Because structural remodeling of several proteins, including ion channels, may underlie the abnormal action potentials of Purkinje cells (PCs) that survive in the 48 hr infarcted zone of the canine heart (IZPCs), we sought to determine the subcellular structure and function of the KV1.5 (KCNA5) protein in single IZPCs. Clustering of the Kv1.5 subunit in axons is regulated by a synapse-associated protein, SAP97, and is linked to an actin-binding protein, cortactin, and an intercellular adhesion molecule, N-cadherin. To understand the functional remodeling of the Kv1.5 channel and its regulation in IZPCs, Kv1.5 currents in PCs were measured as the currents blocked by 10 µM RSD1379 using patch-clamp techniques. Immunocytochemistry and confocal imaging were used for both single and aggregated IZPCs vs normal PCs (NZPCs) to determine the relationship of Kv1.5 with SAP-97, cortactin and N-cadherin. In IZPCs, both the sarcolemma (SL) and intercalated disk (ID) Kv1.5 protein are abundant, and the amount of cytosolic Kv1.5 protein is greatly increased. SAP-97 is also increased at IDs and has notable cytosolic localization suggesting that SAP-97 may regulate the functional expression and stabilization of Kv1.5 channels in IZPCs. Cortactin, which is located with N-cadherin at IDs in NZPCs, remains at IDs but begins to dissociate from N-cadherin, often forming ring structures and colocalizing with Kv1.5 within IZPCs. At the same time, cortactin/Kv1.5 colocalization is increased at the ID, suggesting an ongoing active process of membrane trafficking of the channel protein. Finally, the Kv1.5 current, measured as the RSD1379-sensitive current, at +40 mV did not differ between NZPCs (0.81±0.24 pA/pF, n = 14) and IZPCs (0.83±0.21 pA/pF, n = 13, NS). In conclusion, the subcellular structural remodeling of Kv1.5, SAP97 and cortactin maintained and normalized the function of the Kv1.5 channel in Purkinje cells that survived myocardial infarction.
doi:10.1371/journal.pone.0106830
PMCID: PMC4153673  PMID: 25184222
11.  CaMKII-Based Regulation of Voltage-Gated Na+ Channel in Cardiac Disease 
Circulation  2012;126(17):10.1161/CIRCULATIONAHA.112.105320.
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 (Nav1.5) 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 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 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 Nav1.5 channel activity through direct effects on channel post-translational modification. We propose that the CaMKII phosphorylation motif in the Nav1.5 DI-DII cytoplasmic loop is a critical nodal point for pro-arrhythmic changes to Nav1.5 in congenital and acquired cardiac disease.
doi:10.1161/CIRCULATIONAHA.112.105320
PMCID: PMC3811023  PMID: 23008441
arrhythmia (mechanisms); calmodulin dependent protein kinase II; heart failure; ion channels; long-QT syndrome; myocardial infarction
12.  Ankyrin-G Participates in INa Remodeling in Myocytes from the Border Zones of Infarcted Canine Heart 
PLoS ONE  2013;8(10):e78087.
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 assembly and function are linked to ankyrin-G, gap, and mechanical junction proteins. In this study our objective is to expound the status of the cardiac Na channel, its interacting protein ankyrinG and the mechanical and gap junction proteins at two different times post infarction when arrhythmias are known to occur; that is, 48 hr and 5 day post coronary occlusion. Previous studies have shown the origins of arrhythmic events come from the subendocardial Purkinje and epicardial border zone. Our Purkinje cell (Pcell) voltage clamp study shows that INa and its kinetic parameters do not differ between Pcells from the subendocardium of the 48hr infarcted heart (IZPCs) and control non-infarcted Pcells (NZPCs). Immunostaining studies revealed that disturbances of Nav1.5 protein location with ankyrin-G are modest in 48 hr IZPCs. Therefore, Na current remodeling does not contribute to the abnormal conduction in the subendocardial border zone 48 hr post myocardial infarction as previously defined. In addition, immunohistochemical data show that Cx40/Cx43 co-localize at the intercalated disc (IDs) of control NZPCs but separate in IZPCs. At the same time, Purkinje cell desmoplakin and desmoglein2 immunostaining become diffuse while plakophilin2 and plakoglobin increase in abundance at IDs. In the epicardial border zone 5 days post myocardial infarction, immunoblot and immunocytochemical analyses showed that ankyrin-G protein expression is increased and re-localized to submembrane cell regions at a time when Nav1.5 function is decreased. Thus, Nav1.5 and ankyrin-G remodeling occur later after myocardial infarction compared to that of gap and mechanical junctional proteins. Gap and mechanical junctional proteins remodel in IZPCs early, perhaps to help maintain Nav1.5 subcellular location position and preserve its function soon after myocardial infarction.
doi:10.1371/journal.pone.0078087
PMCID: PMC3796465  PMID: 24155982
13.  Role of the transient outward potassium current in the genesis of early afterdepolarizations in cardiac cells 
Cardiovascular Research  2012;95(3):308-316.
Aims
The transient outward potassium current (Ito) plays important roles in action potential (AP) morphology and dynamics; however, its role in the genesis of early afterdepolarizations (EADs) is not well understood. We aimed to study the effects and mechanisms of Ito on EAD genesis in cardiac cells using combined experimental and computational approaches.
Methods and results
We first carried out patch-clamp experiments in isolated rabbit ventricular myocytes exposed to H2O2 (0.2 or 1 mM), in which EADs were induced at a slow pacing rate. EADs were eliminated by either increasing the pacing rate or blocking Ito with 2 mM 4-aminopyridine. In addition to enhancing the L-type calcium current (ICa,L) and the late sodium current, H2O2 also increased the conductance, slowed inactivation, and accelerated recovery from the inactivation of Ito. Computer simulations showed that Ito promoted EADs under the condition of reduced repolarization reserve, consistent with the experimental observations. However, EADs were only promoted in the intermediate ranges of the Ito conductance and the inactivation time constant. The underlying mechanism is that Ito lowers the AP plateau voltage into the range at which the time-dependent potassium current (namely IKs) activation is further slowed and ICa,L is available for reactivation, leading to voltage oscillations to manifest EADs. Further experimental studies in cardiac cells of other species validated the theoretical predictions.
Conclusion
In cardiac cells, Ito, with a proper conductance and inactivation speed, potentiates EADs by setting the AP plateau into the voltage range where ICa,L reactivation is facilitated and IKs activation is slowed.
doi:10.1093/cvr/cvs183
PMCID: PMC3400356  PMID: 22660482
Transient outward current; Early afterdepolarization; Cardiac arrhythmias; Computer model; Dynamic mechanisms
14.  SkM1 and Cx32 improve conduction in canine myocardial infarcts yet only SkM1 is antiarrhythmic 
Cardiovascular Research  2012;94(3):450-459.
Aims
Reentry accounts for most life-threatening arrhythmias, complicating myocardial infarction, and therapies that consistently prevent reentry from occurring are lacking. In this study, we compare antiarrhythmic effects of gene transfer of green fluorescent protein (GFP; sham), the skeletal muscle sodium channel (SkM1), the liver-specific connexin (Cx32), and SkM1/Cx32 in the subacute canine infarct.
Methods and results
Immediately after ligation of the left anterior descending artery, viral constructs were implanted in the epicardial border zone (EBZ). Five to 7 days later, efficient restoration of impulse propagation (narrow QRS and local electrogram duration) occurred in SkM1, Cx32, and SkM1/Cx32 groups (P< 0.05 vs. GFP). Programmed electrical stimulation from the EBZ induced sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) in 15/22 GFP dogs vs. 2/12 SkM1, 6/14 Cx32, and 8/10 SkM1/Cx32 (P< 0.05 SkM1 vs. GFP). GFP, SkM1, and SkM1/Cx32 had predominantly polymorphic VT/VF, whereas in Cx32 dogs, monomorphic VT predominated (P< 0.05 for Cx32 vs. GFP). Tetrazolium red staining showed significantly larger infarcts in Cx32- vs. GFP-treated animals (P< 0.05).
Conclusion
Whereas SkM1 gene transfer reduces the incidence of inducible VT/VF, Cx32 therapy to improve gap junctional conductance results in larger infarct size, a different VT morphology, and no antiarrhythmic efficacy.
doi:10.1093/cvr/cvs107
PMCID: PMC3410408  PMID: 22374989
Myocardial infarction; Arrhythmias; Na+ channels; Connexins; Gene therapy
15.  Diverse phenotypes of outward currents in cells that have survived in the 5-day-infarcted heart 
We have shown reduced density and altered kinetics in slowly activating K+ currents (IKs) in epicardial border zone (EBZ) cells (IZs) of the 5-day-infarcted canine heart (Jiang M, Cabo, C, Yao J-A, Boyden PA, and Tseng G-N. Cardiovasc Res 48: 34–43, 2000). β-Adrenergic stimulation with isoproterenol increases IKs in normal cells (NZs). In this study, we used a voltage-clamp protocol with an external solution to isolate IKs from contaminating currents to determine the effects of 1 µM isoproterenol on IKs in IZs and NZs. Under our recording conditions, 10 µM azimilide-sensitive currents were stimulated with isoproterenol to compare responsiveness of IKs to isoproterenol in the two cell groups. IKs tail density was reduced 67% in IZs (group I, n = 26) compared with NZs (n = 24, P < 0.05). Isoproterenol-stimulated azimilide-sensitive tail currents were increased 1.72 ± 0.2-fold in NZs and 2.2 ± 0.3-fold in IZs (P > 0.05). In 33% of IZs (group II, n = 13), native currents showed no tail currents; however, isoproterenol-stimulated azimilide-sensitive currents were voltage dependent, fast activating, and large in amplitude compared with group I IZs, similar to “lone” KCNQ1 currents. Using short clamp pulses, we also found an increase in sustained currents sensitive to tetraethylammonium chloride (TEA) and no change in C-9356-sensitive currents in IZs with little or no transient outward current. In some IZs where IKs is downregulated, the effect of isoproterenol on IKs was similar to that on IKs in NZs. In others, the existence of lone KCNQ1-type currents, which are sensitive to β-adrenergic stimulation, is consistent with our findings of an increased KCNQ1-to-KCNE1 mRNA ratio (Jiang et al.). Accompanying altered IKs in IZs are an enhanced TEA-sensitive current and a normal C-9356-sensitive current.
doi:10.1152/ajpheart.00180.2005
PMCID: PMC3410671  PMID: 15821036
epicardial border zone; delayed rectifier; tetraethylammonium; C-9356; isoproterenol
16.  Characterization of Gap Junction Remodeling in Epicardial Border Zone of Healing Canine Infarcts and Electrophysiological Effects of Partial Reversal by Rotigaptide 
Background
The border zone of healing myocardial infarcts is an arrhythmogenic substrate partly due to structural and functional remodeling of the ventricular gap junction protein, Connexin43 (Cx43). Cx43 in arrhythmogenic substrates is a potential target for antiarrhythmic therapy.
Methods and Results
We characterized Cx43 remodeling in the epicardial border zone (EBZ) of healing canine infarcts, 5 days after coronary occlusion and examined whether the gap junction specific agent, Rotigaptide, could reverse it. Cx43 remodeling in the EBZ was characterized by a decrease in Cx43 protein, lateralization and increased Cx43 phosphorylation at serine (S) 368. Rotigaptide partially reversed the loss of Cx43 but did not affect the increase in S368 phosphorylation nor did it reverse Cx43 lateralization. Rotigaptide did not prevent conduction slowing in EBZ nor did it decrease the induction of sustained ventricular tachycardia (SMVT) by programmed stimulation, although it did decrease the EBZ effective refractory period (ERP).
Conclusions
We conclude that partial reversal of Cx43 remodeling in healing infarct border zone may not be sufficient to restore normal conduction or prevent arrhythmias.
doi:10.1161/CIRCEP.110.959312
PMCID: PMC3116056  PMID: 21493965
myocardial infarction; arrhythmias; gap junctions; remodeling
17.  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 (Ca2+) regulation. Although prior studies have extensively characterized spontaneous Ca2+ release in nondriven Purkinje cells, little attention has been paid to rate-dependent changes in Ca2+ transients. Therefore we explored the behaviors of Ca2+ 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 Ca2+ regulation in Purkinje cells: 1) the cells exhibit a negative Ca2+-frequency relationship (at 2.5 Hz, Ca2+ transient amplitude was 66 ± 6% smaller than that at 0.125 Hz); 2) sarcoplasmic reticulum (SR) Ca2+ release occurs as a propagating wave at very low rates but is localized near the cell membrane at higher rates; 3) SR Ca2+ load declines modestly (10 ± 5%) with an increase in pacing rate from 0.125 Hz to 2.5 Hz; 4) Ca2+ 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 Ca2+ 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 Ca2+ release is normally regulated in Purkinje cells and provide clues regarding how disruptions in this regulation may lead to deleterious consequences such as arrhythmias.
doi:10.1016/j.yjmcc.2010.12.023
PMCID: PMC3059096  PMID: 21232541
Ca2+ transients; pacing rate; conduction system; Ca2+ sparks; Ca2+ waves
18.  EH domain proteins regulate cardiac membrane protein targeting 
Circulation research  2010;107(1):84-95.
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 employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four 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 co-expressed 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.
doi:10.1161/CIRCRESAHA.110.216713
PMCID: PMC2901408  PMID: 20489164
trafficking; ion channel; ankyrin; EHD proteins; cytoskeleton; arrhythmia
19.  Effect of Non-Uniform Muscle Contraction on Sustainability and Frequency of Triggered Arrhythmias in Rat Cardiac Muscle 
Circulation  2010;121(25):2711-2717.
Background
Arrhythmias are benign or lethal depending on their sustainability and frequency. To determine why lethal arrhythmias are prone to occur in diseased hearts, usually characterized by non-uniform muscle contraction, we investigated the effect of non-uniformity on sustainability and frequency of triggered arrhythmias.
Methods and Results
Force, membrane potential, and intracellular Ca2+ concentration ([Ca2+]i) were measured in 51 rat ventricular trabeculae. Non-uniform contraction was produced by exposing a restricted region of muscle to a jet of 20 mmol/L 2,3-butanedione monoxime (BDM) or 20 μmol/L blebbistatin. Sustained arrhythmias (>10 s) could be induced by stimulus trains for 7.5 s only with the BDM or blebbistatin jet (100 nmol/L isoproterenol, 1.0 mmol/L [Ca2+]o, 24°C). During sustained arrhythmias, Ca2+ surges preceded synchronous increases in [Ca2+]i, while the stoppage of the BDM jet made the Ca2+ surges unclear and arrested sustained arrhythmias (n = 6). With 200 nmol/L isoproterenol, 2.5 mmol/L [Ca2+]o, and the BDM jet, lengthening or shortening of the muscle during sustained arrhythmias accelerated or decelerated their cycle both in the absence (n = 10) and the presence of 100 μmol/L streptomycin (n = 10), a stretch-activated channel blocker, respectively. The maximum rate of force relaxation correlated inversely with the change in cycle lengths (n = 14, P<0.01). Sustained arrhythmias with the BDM jet were significantly accelerated by 30 μmol/L SCH00013, a Ca2+ sensitizer of myofilaments (n = 10).
Conclusion
These results suggest that non-uniformity of muscle contraction is an important determinant of the sustainability and frequency of triggered arrhythmias due to the surge of Ca2+ dissociated from myofilaments in cardiac muscle.
doi:10.1161/CIRCULATIONAHA.109.907717
PMCID: PMC2909439  PMID: 20547931
triggered activity; contraction; calcium; non-uniform myocardium
20.  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 β-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.
doi:10.1161/CIRCEP.109.928820
PMCID: PMC3068837  PMID: 20110552
cell adhesion molecules; electrophysiology; genetics; Purkinje fiber
21.  Stem Cell Therapy is Proarrhythmic 
Circulation  2009;119(13):1814-1823.
doi:10.1161/CIRCULATIONAHA.108.779900
PMCID: PMC2739413  PMID: 19349334
stem cells; cell therapy; arrhythmias; heart disease
22.  Regulation of the ankyrin-B-based targeting pathway following myocardial infarction 
Cardiovascular Research  2008;81(4):742-749.
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 β2-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.
doi:10.1093/cvr/cvn348
PMCID: PMC2642599  PMID: 19074823
Arrhythmia (mechanisms); Infarction; Remodelling; Signal transduction; Cytoskeleton
23.  Oxidized Calmodulin Kinase II Regulates Conduction Following Myocardial Infarction: A Computational Analysis 
PLoS Computational Biology  2009;5(12):e1000583.
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.
Author Summary
Calmodulin kinase II (CaMKII) is a multifunctional serine/threonine kinase that regulates diverse functions in heart. Recently, a novel pathway for CaMKII activation was discovered where oxidation of the kinase at specific methionine residues produces persistent activity. This alternative oxidation-dependent pathway has important implications for heart disease where oxidative stress is increased (e.g., heart failure and following myocardial infarction). We hypothesized that myocardial infarction caused by occlusion of a coronary artery would increase levels of oxidized CaMKII. Moreover, we hypothesized that oxidative CaMKII activation represents an important mechanistic link between increased oxidative stress and life-threatening heart rhythm disturbances (arrhythmias) in heart disease. We report a dramatic increase in levels of oxidized CaMKII following myocardial infarction in the canine. Based on these experimental data, we developed a novel mathematical model of CaMKII activity to study the role of oxidation-dependent CaMKII activation in regulating cardiac cell excitability. Our findings identify a novel role for oxidation-dependent CaMKII activation following myocardial infarction and provide a mechanistic link between oxidative stress and lethal cardiac arrhythmias in heart disease.
doi:10.1371/journal.pcbi.1000583
PMCID: PMC2778128  PMID: 19997488
24.  Function of Ca2+ release Channels in Purkinje cells that survive in the Infarcted Canine Heart; a Mechanism for triggered Purkinje Ectopy 
Background
Triggered Purkinje ectopy can lead to the initiation of serious ventricular arrhythmias in post myocardial infarction (MI) patients. In the canine model, Purkinje cells from the subendocardial border of the healing infarcted heart can initiate ventricular arrhythmias. Intracellular Ca2+ abnormalities underlie these arrhythmias yet the subcellular reasons for these abnormalities remain unknown.
Methods and Results
Using 2D confocal microscopy, we directly quantify and compare typical spontaneous Ca2+ events in specific subcellular regions of normal Purkinje cells (NZPCs) with those Purkinje cells from the subendocardium of the 48hr infarcted canine heart (IZPCs). The Ca2+ event rate was higher in subsarcolemmal region (SSL) of IZPCs when compared to NZPCs, IZPC amplitudes were higher yet the spatial extents of these events were similar. The amplitude of Caffeine releasable Ca2+ in either the SSL or Core regions of IZPCs did not differ from NZPCs suggesting that Ca2+ overload was not related to the frequency change. In permeabilized Purkinje cells from both groups, the event rate was related to free [Ca2+] in both SSL and Core but in IZPCs this event rate was significantly increased at each free Ca2+ suggesting an enhanced sensitivity to Ca2+ release. Furthermore, decays of wide long lasting Ca2+ release events in IZPC's Core were significantly accelerated compared to those in NZPCs. JTV519 (K201) suppressed IZPC cell wide Ca2+ waves as well as normalized the enhanced event rate and its response to free Ca2+.
Conclusions
Increased spontaneous Ca2+ release events in IZPCs are due to uniform regionally increased Ca2+ release channel sensitivity to Ca2+ without a change in SR content. In addition, Ca2+ reuptake in IZPCs is accelerated. These properties would lower the threshold of Ca2+ release channels, setting the stage for the highly frequent arrhythmogenic cell wide Ca2+ waves observed in IZPCs.
doi:10.1161/CIRCEP.107.758110
PMCID: PMC2727694  PMID: 19753099
myocardial infarction; calcium; arrhythmias; Purkinje cells; Ca2+ waves
25.  Role of activated CaMKII in abnormal calcium homeostasis and INa remodeling after myocardial infarction: Insights from mathematical modeling 
Ca2+/calmodulin-dependent protein kinase II is a multifunctional serine/threonine kinase with diverse cardiac roles including regulation of excitation contraction, transcription, and apoptosis. Dynamic regulation of CaMKII activity occurs in cardiac disease and is linked to specific disease phenotypes through its effects on ion channels, transporters, transcription and cell death pathways. Recent mathematical models of the cardiomyocyte have incorporated limited elements of CaMKII signaling to advance our understanding of how CaMKII regulates cardiac contractility and excitability. Given the importance of CaMKII in cardiac disease, it is imperative that computer models evolve to capture the dynamic range of CaMKII activity. In this study, using mathematical modeling combined with biochemical and imaging techniques, we test the hypothesis that CaMKII signaling in the canine infarct border zone (BZ) contributes to impaired calcium homeostasis and electrical remodeling. We report that the level of CaMKII autophosphorylation is significantly increased in the BZ region. Computer simulations using an updated mathematical model of CaMKII signaling reproduce abnormal Ca2+ transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca2+ homeostasis and reduced action potential upstroke velocity due to effects on INa gating kinetics. In conclusion, we present a new mathematical tool for studying effects of CaMKII signaling on cardiac excitability and contractility over a dynamic range of kinase activities. Our experimental and theoretical findings establish abnormal CaMKII signaling as an important component of remodeling in the canine BZ.
doi:10.1016/j.yjmcc.2008.06.007
PMCID: PMC2587155  PMID: 18639555
Calcium/calmodulin-dependent protein kinase II; myocardial infarction; calcium handling; mathematical modeling; arrhythmia

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