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Background: Four voltage-gated potassium currents, I(to,f) (K(V)4.2), I(to,s) (K(V)1.4), I(K,slow) (K(V)1.5+K(V)2.1), and I(SS) (TASK1), govern murine ventricular repolarization. Although the accessory subunit KChIP2 influences I(to,f) expression, in preliminary experiments we found that action potential duration (APD) is maintained in KChIP2 knockout mice.Objective: We tested the role of KChIP2 in regulating APD and studied the underlying ionic currents.Methods: We used microelectrode techniques, whole-cell patch clamp studies, and real-time polymerase chain reaction amplification to characterize ventricular repolarization and its determinants in wild-type and KChIP2(-/-) mice.Results: Despite comparable baseline action potentials, APD was more markedly prolonged by 4-aminopyridine (4-AP) in KChIP2(-/-) preparations. Peak K(+) current densities were similar in wild-type and KChIP2(-/-) cells (mean +/- SEM I(P): 28.3 +/- 2 (n = 27) vs. 29.2 +/- 2 pA/pF (n = 24), respectively; P > .05). Heteropodatoxin-2 (HpTx-2, 1 microM) had no effect on current amplitude in KChIP2(-/-) myocytes. The current fractions sensitive to 4-AP (50 microM and 1 mM) were larger in KChIP2(-/-) than wild-type (P < .05). Real-time polymerase chain reaction showed absence of KChIP2 and increased K(V)1.5 expression in KChIP2(-/-) ventricular myocardium.Conclusion: KChIP2 deficiency eliminated HpTx-2-sensitive I(to,f), but had little impact on total APD, secondary to upregulation of 4-AP-sensitive I(K,slow) in association with increased K(V)1.5 expression. There is increased sensitivity to 4-AP-mediated APD prolongation in KChIP2(-/-). Thus, KChIP2 seems important for murine repolarization in circumstances of reduced repolarization reserve. [ABSTRACT FROM AUTHOR]

This article reviews work over the past three decades that is related to the contribution of the pacemaker current, I f , 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 The latter studies include previously unpublished work making use of mice in which both the type 1 and type 8 Ca-sensitive adenylyl cyclase isoforms were knocked out.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., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The His-Purkinje system is a network of bundles and fibres comprised of specialised cells that allow for coordinated, synchronous activation of the ventricles. Although the histology and physiology of the His-Purkinje system have been studied for more than a century, its role in ventricular arrhythmias has recently been discovered with the ongoing elucidation of the mechanisms leading to both benign and life-threatening arrhythmias. Studies of Purkinje-cell electrophysiology show multiple mechanisms responsible for ventricular arrhythmias, including enhanced automaticity, triggered activity and reentry. The variation in functional properties of Purkinje cells in different areas of the His-Purkinje system underlie the propensity for reentry within Purkinje fibres in structurally normal and abnormal hearts. Catheter ablation is an effective therapy in nearly all forms of reentrant arrhythmias involving Purkinje tissue. However, identifying those at risk of developing fascicular arrhythmias is not yet possible. Future research is needed to understand the precise molecular and functional changes resulting in these arrhythmias., Competing Interests: Disclosure: The authors have no conflicts of interest to declare., (Copyright © 2021, Radcliffe Cardiology.)

Cardiac excitation-contraction (E-C) coupling is influenced by (at least) three dynamic systems that couple and feedback to one another (see Abstract Figure). Here we review the mechanical effects on cardiomyocytes that include mechano-electro-transduction (commonly referred to as mechano-electric coupling, MEC) and mechano-chemo-transduction (MCT) mechanisms at cell and molecular levels which couple to Ca 2+ -electro and E-C coupling reviewed elsewhere. These feedback loops from muscle contraction and mechano-transduction to the Ca 2+ homeodynamics and to the electrical excitation are essential for understanding the E-C coupling dynamic system and arrhythmogenesis in mechanically loaded hearts. This white paper comprises two parts, each reflecting key aspects from the 2018 UC Davis symposium: MEC (how mechanical load influences electrical dynamics) and MCT (how mechanical load alters cell signalling and Ca 2+ dynamics). Of course, such separation is artificial since Ca 2+ dynamics profoundly affect ion channels and electrogenic transporters and vice versa. In time, these dynamic systems and their interactions must become fully integrated, and that should be a goal for a comprehensive understanding of how mechanical load influences cell signalling, Ca 2+ homeodynamics and electrical dynamics. In this white paper we emphasize current understanding, consensus, controversies and the pressing issues for future investigations. Space constraints make it impossible to cover all relevant articles in the field, so we will focus on the topics discussed at the symposium., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Much has been written about arrhythmias in structurally normal hearts. In this review, we focus on rapid ventricular arrhythmias that occur in hearts having a pathogenic genetic variant that has been found in families in which arrhythmias occur. We discuss these mutations in terms of their effect on cardiac cell electrical function and initiation of arrhythmias. We also focus on Purkinje cells, their anatomic networks, and their molecular signatures as the sites of origin of arrhythmias. We discuss therapeutic options for treatment of these potentially life-threatening arrhythmias. Although all Purkinje-based arrhythmias are not included (eg, conduction block rhythms), syndromes discussed include idiopathic ventricular fibrillation, catecholaminergic polymorphic ventricular tachycardia, long QT syndrome, Andersen-Tawil syndrome, and Brugada syndrome., (Copyright © 2019 Heart Rhythm Society. All rights reserved.)

Background: The apamin-sensitive small-conductance calcium-activated K (SK) current I KAS modulates automaticity of the sinus node. I KAS blockade by apamin causes sinus bradycardia., Objective: The purpose of this study was to test the hypothesis that I KAS modulates ventricular automaticity., Methods: We tested the effects of apamin (100 nM) on ventricular escape rhythms in Langendorff-perfused rabbit ventricles with atrioventricular block (protocol 1) and on recorded transmembrane action potential of pseudotendons of superfused right ventricular endocardial preparations (protocol 2)., Results: All preparations exhibited spontaneous ventricular escape rhythms. In protocol 1, apamin decreased the atrial rate from 186.2 ± 18.0 bpm to 163.8 ± 18.7 bpm (N = 6; P = .006) but accelerated the ventricular escape rate from 51.5 ± 10.7 bpm to 98.2 ± 25.4 bpm (P = .031). Three preparations exhibited bursts of nonsustained ventricular tachycardia and pauses, resulting in repeated burst termination pattern. In protocol 2, apamin increased the ventricular escape rate from 70.2 ± 13.1 bpm to 110.1 ± 2.2 bpm (P = .035). Spontaneous phase 4 depolarization was recorded from the pseudotendons in 6 of 10 preparations at baseline and in 3 in the presence of apamin. There were no changes of phase 4 slope (18.37 ± 3.55 mV/s vs 18.93 ± 3.26 mV/s, N = 3; P = .231, ), but the threshold of phase 0 activation (mV) reduced from -67.97 ± 1.53 to -75.26 ± 0.28 (P = .034). Addition of JTV-519, a ryanodine receptor 2 stabilizer, in 5 preparations reduced escape rate back to baseline., Conclusion: Contrary to its bradycardic effect in the sinus node, I KAS blockade by apamin accelerates ventricular automaticity and causes repeated nonsustained ventricular tachycardia in normal ventricles. ryanodine receptor 2 blockade reversed the apamin effects on ventricular automaticity., (Copyright © 2018 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Ankyrin polypeptides are intracellular proteins responsible for targeting cardiac membrane proteins. Here, the authors demonstrate that ankyrin-G plays an unexpected role in normal compensatory physiological remodeling in response to myocardial stress and aging; the authors implicate disruption of ankyrin-G in human heart failure. Mechanistically, the authors illustrate that ankyrin-G serves as a key nodal protein required for cardiac myofilament integration with the intercalated disc. Their data define novel in vivo mechanistic roles for ankyrin-G, implicate ankyrin-G as necessary for compensatory cardiac physiological remodeling under stress, and implicate disruption of ankyrin-G in the development and progression of human heart failure.

There has always been an appreciation of the role of Purkinje fibers in the fast conduction of the normal cardiac impulse. Here, we briefly update our knowledge of this important set of cardiac cells. We discuss the anatomy of a Purkinje fiber strand, the importance of longitudinal conduction within a strand, circus movement within a strand, conduction, and excitability properties of Purkinjes. At the cell level, we discuss the important components of the ion channel makeup in the nonremodeled Purkinjes of healthy hearts. Finally, we discuss the role of the Purkinjes in forming the heritable arrhythmogenic substrates such as long QT, heritable conduction slowing, CPVT, sQT, and Brugada syndromes.

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 Na v 1.5 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 I Na density is decreased in the epicardial border zone cells of the 48 h infarcted heart (IZ 48h ). Immunostaining studies reveal that in post MI cells the cell surface staining of Na v 1.5 was reduced and Na v 1.5 distribution changed. However, intense co-staining of Na v 1.5 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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

For arrhythmia triggers that are secondary to dysfunctional intracellular Ca 2+ cycling, there are few, if any, agents that specifically target the Ca 2+ handling machinery. However, several candidates have been proposed in the literature. Here we review the idea that these agents or their derivatives will prove invaluable in clinical applications in the future., (Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Fascicular ventricular arrhythmias represent a spectrum of ventricular tachycardias dependent on the specialized conduction system. Although they are more common in structurally abnormal hearts, there is an increasing body of literature describing their role in normal hearts. In this review, the authors present data from both basic and clinical research that explore the current understanding of idiopathic fascicular ventricular arrhythmias. Evaluation of the cellular electrophysiology of the Purkinje cells shows clear evidence of enhanced automaticity and triggered activity as potential mechanisms of arrhythmias. Perhaps more importantly, heterogeneity in conduction system velocity and refractoriness of the left ventricular conduction system in animal models are in line with clinical descriptions of re-entrant fascicular arrhythmias in humans. Further advances in our understanding of the conduction system will help bridge the current gap between basic science and clinical fascicular arrhythmias., (Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Arrhythmia subcellular mechanisms are constantly being explored. Recent knowledge has shown that travelling Ca(2+) waves in cardiac cells are critical for delayed afterdepolarisations and in some cases, early afterdepolarisations. In this review, we comment on the properties of cardiac Ca(2+) waves and abnormal Ca(2+) releases in terms of properties used to describe electrical waves; propagation, excitability and refractoriness.

In February 2014, a group of scientists convened as part of the University of California Davis Cardiovascular Symposium to bring together experimental and mathematical modelling perspectives and discuss points of consensus and controversy on the topic of sodium in the heart. This paper summarizes the topics of presentation and discussion from the symposium, with a focus on the role of aberrant sodium channels and abnormal sodium homeostasis in cardiac arrhythmias and pharmacotherapy from the subcellular scale to the whole heart. Two following papers focus on Na(+) channel structure, function and regulation, and Na(+)/Ca(2+) exchange and Na(+)/K(+) ATPase. The UC Davis Cardiovascular Symposium is a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The focus on Na(+) in the 2014 symposium stemmed from the multitude of recent studies that point to the importance of maintaining Na(+) homeostasis in the heart, as disruption of homeostatic processes are increasingly identified in cardiac disease states. Understanding how disruption in cardiac Na(+)-based processes leads to derangement in multiple cardiac components at the level of the cell and to then connect these perturbations to emergent behaviour in the heart to cause disease is a critical area of research. The ubiquity of disruption of Na(+) channels and Na(+) homeostasis in cardiac disorders of excitability and mechanics emphasizes the importance of a fundamental understanding of the associated mechanisms and disease processes to ultimately reveal new targets for human therapy., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

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.

Rationale: Cardiac function is dependent on the coordinate activities of membrane ion channels, transporters, pumps, and hormone receptors to tune the membrane electrochemical gradient dynamically in response to acute and chronic stress. Although our knowledge of membrane proteins has rapidly advanced during the past decade, our understanding of the subcellular pathways governing the trafficking and localization of integral membrane proteins is limited and essentially unstudied in vivo. In the heart, to our knowledge, there are no in vivo mechanistic studies that directly link endosome-based machinery with cardiac physiology., Objective: To define the in vivo roles of endosome-based cellular machinery for cardiac membrane protein trafficking, myocyte excitability, and cardiac physiology., Methods and Results: We identify the endosome-based Eps15 homology domain 3 (EHD3) pathway as essential for cardiac physiology. EHD3-deficient hearts display structural and functional defects including bradycardia and rate variability, conduction block, and blunted response to adrenergic stimulation. Mechanistically, EHD3 is critical for membrane protein trafficking, because EHD3-deficient myocytes display reduced expression/localization of Na/Ca exchanger and L-type Ca channel type 1.2 with a parallel reduction in Na/Ca exchanger-mediated membrane current and Cav1.2-mediated membrane current. Functionally, EHD3-deficient myocytes show increased sarcoplasmic reticulum [Ca], increased spark frequency, and reduced expression/localization of ankyrin-B, a binding partner for EHD3 and Na/Ca exchanger. Finally, we show that in vivo EHD3-deficient defects are attributable to cardiac-specific roles of EHD3 because mice with cardiac-selective EHD3 deficiency demonstrate both structural and electric phenotypes., Conclusions: These data provide new insight into the critical role of endosome-based pathways in membrane protein targeting and cardiac physiology. EHD3 is a critical component of protein trafficking in heart and is essential for the proper membrane targeting of select cellular proteins that maintain excitability., (© 2014 American Heart Association, Inc.)

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.

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 Ca²⁺ homeostasis, electrophysiological properties, and ultrastructural features of the Ca²⁺ 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 Ca²⁺ waves unable to propagate as cell-wide waves; (2) show smaller Ca²⁺sparks with shortened coupling intervals, suggesting a reduced refractoriness of Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ release unit architecture that leads to fragmentation of spontaneous Ca²⁺ waves. We propose that these 2 substrates in R33Q myocytes synergize to provide a new arrhythmogenic mechanism for catecholaminergic polymorphic ventricular tachycardia.

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.

Aims: The transient outward potassium current (I(to)) 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 I(to) 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 H(2)O(2) (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 I(to) with 2 mM 4-aminopyridine. In addition to enhancing the L-type calcium current (I(Ca,L)) and the late sodium current, H(2)O(2) also increased the conductance, slowed inactivation, and accelerated recovery from the inactivation of I(to). Computer simulations showed that I(to) 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 I(to) conductance and the inactivation time constant. The underlying mechanism is that I(to) lowers the AP plateau voltage into the range at which the time-dependent potassium current (namely I(Ks)) activation is further slowed and I(Ca,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, I(to), with a proper conductance and inactivation speed, potentiates EADs by setting the AP plateau into the voltage range where I(Ca,L) reactivation is facilitated and I(Ks) activation is slowed.

In the ischemic myocardium, extracellular potassium ([K(+)](o)) increases to ≥20 mmol/l. To determine how lethal arrhythmias occur during ischemia, we investigated whether the increased spatial pattern of [K(+)](o), i.e., a regional or a global increase, affects the incidence of arrhythmias. Force, sarcomere length, membrane potential, and nonuniform intracellular Ca(2+) ([Ca(2+)](i)) were measured in rat ventricular trabeculae. A "regional" or "global" increase in [K(+)](o) was produced by exposing a restricted region of muscle to a jet of 30 mmol/l KCl or by superfusing trabeculae with a solution containing 30 mmol/l KCl, respectively. The increase in [Ca(2+)](i) (Ca(CW)) during Ca(2+) waves was measured (24°C, 3.0 mmol/l [Ca(2+)](o)). A regional increase in [K(+)](o) caused nonuniform [Ca(2+)](i) and contraction. In the presence of isoproterenol, the regional increase in [K(+)](o) induced sustained arrhythmias in 10 of 14 trabeculae, whereas the global increase did not induce such arrhythmias. During sustained arrhythmias, Ca(2+) surged within the jet-exposed region. In the absence of isoproterenol, the regional increase in [K(+)](o) increased Ca(CW), whereas the global increase decreased it. This increase in Ca(CW) with the regional increase in [K(+)](o) was not suppressed by 100 μmol/l streptomycin, whereas it was suppressed by 1) a combination of 10 μmol/l cilnidipine and 3 μmol/l SEA0400; 2) 20 mmol/l 2,3-butanedione monoxime; and 3) 10 μmol/l blebbistatin. A regional but not a global increase in [K(+)](o) induces sustained arrhythmias, probably due to nonuniform excitation-contraction coupling. The same mechanism may underlie arrhythmias during ischemia.

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.

Rationale: Flecainide prevents arrhythmias in catecholaminergic polymorphic ventricular tachycardia, but the antiarrhythmic mechanism remains unresolved. It is possible for flecainide to directly affect the cardiac ryanodine receptor (RyR2); however, an extracellular site of action is suggested because of the hydrophilic nature of flecainide., Objective: To investigate the mechanism for the antiarrhythmic action of flecainide in a RyR2(R4496C+/-) knock-in mouse model of catecholaminergic polymorphic ventricular tachycardia., Methods and Results: Flecainide prevented catecholamine-induced sustained ventricular tachycardia in RyR2(R4496C+/-) mice. Cellular studies were performed with isolated RyR2(R4496C+/-) myocytes. Isoproterenol caused the appearance of spontaneous Ca(2+) transients, which were unaffected by flecainide (6 μmol/L). Flecainide did not affect Ca(2+) transient amplitude, decay, or sarcoplasmic reticulum Ca(2+) content. Moreover, it did not affect the frequency of spontaneous Ca(2+) sparks in permeabilized myocytes. In contrast, flecainide effectively prevented triggered activity induced by isoproterenol. The threshold for action potential induction was increased significantly (P<0.01), which suggests a primary extracellular antiarrhythmic effect mediated by Na(+) channel blockade., Conclusions: Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in RyR2(R4496C+/-) mice; however, at variance with previous reports, we observed minimal effects on intracellular Ca(2+) homeostasis. Our data suggest that the antiarrhythmic activity of the drug is caused by reduction of Na(+) channel availability and by an increase in the threshold for triggered activity.

Background: The border zone of healing myocardial infarcts is an arrhythmogenic substrate, partly the result of 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 the EBZ, nor did it decrease the induction of sustained ventricular tachycardia by programmed stimulation, although it did decrease the EBZ effective refractory period., 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.

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

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 nonuniform muscle contraction, we investigated the effect of nonuniformity on sustainability and frequency of triggered arrhythmias., Methods and Results: Force, membrane potential, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in 51 rat ventricular trabeculae. Nonuniform contraction was produced by exposing a restricted region of muscle to a jet of 20 mmol/L 2,3-butanedione monoxime (BDM) or 20 mumol/L blebbistatin. Sustained arrhythmias (>10 seconds) could be induced by stimulus trains for 7.5 seconds only with the BDM or blebbistatin jet (100 nmol/L isoproterenol, 1.0 mmol/L [Ca(2+)](o), 24 degrees C). During sustained arrhythmias, Ca(2+) surges preceded synchronous increases in [Ca(2+)](i), whereas the stoppage of the BDM jet made the Ca(2+) surges unclear and arrested sustained arrhythmias (n=6). With 200 nmol/L isoproterenol, 2.5 mmol/L [Ca(2+)](o), and the BDM jet, lengthening or shortening of the muscle during sustained arrhythmias accelerated or decelerated their cycle in both the absence (n=10) and presence (n=10) of 100 mumol/L streptomycin, 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 mumol/L SCH00013, a Ca(2+) sensitizer of myofilaments (n=10)., Conclusions: These results suggest that nonuniformity of muscle contraction is an important determinant of the sustainability and frequency of triggered arrhythmias caused by the surge of Ca(2+) dissociated from myofilaments in cardiac muscle.

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 beta-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 Ca(2+) 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.

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.

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.

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.

Unlabelled: Ionic channels and gap junctions are remodeled in cells from the 5-day epicardial border zone (EBZ) of the healing canine infarct. The main objective of the study was to determine the effect of gap junctional conductance (Gj) remodeling and Cx43 redistribution to the lateral membrane on conduction velocity (theta) and anisotropic ratio, and how gap junctional remodeling is modulated by the extracellular space. We first implemented subcellular monodomain and two-domain computer models of normal epicardium (NZ) to understand how extracellular space modulates the relationship between Gj and theta in NZ. We found that the extracellular space flattens the Gj-theta relationship, thus theta becomes less sensitive to changes in Gj. We then investigated the functional consequences of Gj remodeling and Cx43 distribution in subcellular computer models of cells of the outer pathway (IZo) and central pathway (IZc) of reentrant circuits. In IZo cells, side-to-side (transverse) Gj is 10% the value in NZ cells. Such Gj remodeling causes a 45% decrease in transverse theta (theta(T)). Inclusion of an extracellular space reduces the decrease in theta(T) to 31%. In IZc cells, Cx43 redistribution along the lateral membrane results in a 29% increase in theta(T). That increase in theta(T) is a consequence of the decrease in access resistance to the Cx43 plaques that occur with the Cx43 redistribution. Extracellular space reduces the increase in theta(T) to 10%., In Conclusion: 1), The extracellular space included in normal epicardial simulations flattens the Gj-theta relationship with theta becoming less sensitive to changes in Gj. 2), The extracellular space attenuates the effects of gap junction epicardial border zone remodeling (i.e., Gj reduction and Cx43 lateralization) on theta(T).

Background: Pharmacologic restoration of the trafficking defects of HERG missense mutations has been documented. However, whether correction of HERG nonsense mutations is possible is unknown., Objective: The purpose of this study was to investigate the effect of aminoglycoside antibiotics on the expression of nonsense mutants expected to produce truncated HERG channels., Methods: HERG channel and mutant currents were recorded by whole-cell patch clamp techniques. Pharmacologic rescue was applied by culturing the cells in 400 microg/mL G-418 or gentamicin for 24 hours., Results: Current densities were significantly reduced in cells expressing R1014X and W927X mutants compared to those of cells expressing wild-type (WT) HERG. R863X and E698X mutants failed to generate any typical HERG currents. Mean peak tail current density of R1014X mutant was significantly lower than that of WT (3.9 +/- 1.4 pA/pF, n = 8, vs 47.8 +/- 6.3 pA/pF, n = 12, P <.05) and increased to 12.7 +/- 3.3 pA/pF (n = 7, P <.05) and 18.3 +/- 3.7 pA/pF (n = 8, P <.05) after G-418 and gentamicin treatment. The voltage dependence of activation of R1014X was also restored after drug treatment. Furthermore, expression of full-length proteins for R1014X induced by drugs was detected by western blot and confocal imaging. Similar results were observed in W927X. For R863X and E698X, however, gentamicin treatment had no effect. In the cells cotransfected with WT/R1014X, gentamicin and G-418 demonstrated different results: gentamicin, but not G-418, increased the current density by 2.2-fold (n = 12, P <.05)., Conclusion: The study findings provide proof of principle that interventions designed to read through premature stop mutations may at least partially reverse the LQT2 phenotype in vitro.

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.

Background: Triggered Purkinje ectopy can lead to the initiation of serious ventricular arrhythmias in post-myocardial infarction patients. In the canine model, Purkinje cells from the subendocardial border of the healing infarcted heart can initiate ventricular arrhythmias. Intracellular Ca(2+) 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 Ca(2+) events in specific subcellular regions of normal Purkinje cells with those Purkinje cells from the subendocardium of the 48-hour infarcted canine heart (IZPCs). The Ca(2+) event rate was higher in the subsarcolemmal region of IZPCs when compared with normal Purkinje cells; IZPC amplitudes were higher, yet the spatial extents of these events were similar. The amplitude of caffeine-releasable Ca(2+) in either the subsarcolemmal or core regions of IZPCs did not differ from normal Purkinje cells, suggesting that Ca(2+) overload was not related to the frequency change. In permeabilized Purkinje cells from both groups, the event rate was related to free [Ca(2+)] in both subsarcolemmal and core, but in IZPCs, this event rate was significantly increased at each free Ca(2+), suggesting an enhanced sensitivity to Ca(2+) release. Furthermore, decays of wide long lasting Ca(2+) release events in IZPC's core were significantly accelerated compared with those in normal Purkinje cells. JTV519 (K201) suppressed IZPC cell wide Ca(2+) waves as well as normalized the enhanced event rate and its response to free Ca(2+)., Conclusions: Increased spontaneous Ca(2+) release events in IZPCs are due to uniform regionally increased Ca(2+) release channel sensitivity to Ca(2+) without a change in sarcoplasmic reticulum content. In addition, Ca(2+) reuptake in IZPCs is accelerated. These properties would lower the threshold of Ca(2+) release channels, setting the stage for the highly frequent arrhythmogenic cell wide Ca(2+) waves observed in IZPCs.

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.

Ca(2+)/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 Ca(2+) transients and action potentials characteristic of the BZ. Our simulations show that CaMKII hyperactivity contributes to abnormal Ca(2+) homeostasis and reduced action potential upstroke velocity due to effects on I(Na) 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.

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.

Starling's Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starling's Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.

Starling's law and the end-systolic pressure-volume relationship (ESPVR) reflect the effect of sarcomere length (SL) on the development of stress (sigma) and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to that of skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL -[Ca2+](free) relationships (sigma-SL-Ca relationships) at submaximal [Ca2+] in intact and skinned trabeculae were similar, although the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-Ca relationship by using a kinetic model assuming that the rates of Tn-C Ca2+ binding and/or cross-bridge (XB) cycling are determined by either the SL, [Ca2+], or sigma. We analyzed the correlation between the model results and steady-state sigma measurements at varied SL at [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that the dominant feedback mechanism is SL-, sigma-, or [Ca2+]-dependent, and that the feedback mechanism regulates Tn-C Ca2+ affinity, XB kinetics, or the unitary XB force. The analysis strongly suggests that the feedback of the number of strong XBs to cardiac Tn-C Ca2+ affinity is the dominant mechanism regulating XB recruitment. Using this concept in a model of twitch-sigma accurately reproduced the sigma-SL-Ca relationship and the time courses of twitch sigma and the intracellular [Ca2+]i. The foregoing concept has equally important repercussions for the nonuniformly contracting heart, in which arrhythmogenic Ca2+ waves arise from weakened areas in the cardiac muscle. These Ca2+ waves can reversibly be induced with nonuniform excitation-contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by the sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). Modeling of the response of the cardiac twitch to rapid force changes using the feedback concept uniquely predicts the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. These results are consistent with the hypothesis that a force feedback to Ca2+ binding by Tn-C is responsible for Starling's law and the ESPVR in the uniform myocardium and leads to a surge of Ca2+ released by the myofilaments during relaxation in the nonuniform myocardium, which initiates arrhythmogenic propagating Ca2+ release by the SR.