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The Calcium/Calmodulin/Kinase System and Arrhythmogenic Afterdepolarizations in Bradycardia-Related Acquired Long-QT Syndrome
Abstract
Sustained bradycardia is associated with long-QT syndrome in human beings and causes spontaneous torsades de pointes in rabbits with chronic atrioventricular block (CAVB), at least partly by downregulating delayed-rectifier K(+)-current to cause action potential (AP) prolongation. We addressed the importance of altered Ca(2+) handling, studying underlying mechanisms and consequences.
We measured ventricular cardiomyocyte [Ca(2+)](i) (Indo1-AM), L-type Ca(2+)-current (I(CaL)) and APs (whole-cell perforated-patch), and Ca(2+)-handling protein expression (immunoblot). CAVB increased AP duration, cell shortening, systolic [Ca(2+)](i) transients, and caffeine-induced [Ca(2+)](i) release, and CAVB cells showed spontaneous early afterdepolarizations (EADs). I(CaL) density was unaffected by CAVB, but inactivation was shifted to more positive voltages, increasing the activation-inactivation overlap zone for I(CaL) window current. Ca(2+)-calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation was enhanced in CAVB, indicating CaMKII activation. CAVB also enhanced CaMKII-dependent phospholamban-phosphorylation and accelerated [Ca(2+)](i)-transient decay, consistent with phosphorylation-induced reductions in phospholamban inhibition of sarcoplasmic reticulum (SR) Ca(2+)-ATPase as a contributor to enhanced SR Ca(2+) loading. The CaMKII-inhibitor KN93 reversed CAVB-induced changes in caffeine-releasable [Ca(2+)](i) and I(CaL) inactivation voltage and suppressed CAVB-induced EADs. Similarly, the calmodulin inhibitor W7 suppressed CAVB-induced I(CaL) inactivation voltage shifts and EADs, and a specific CaMKII inhibitory peptide prevented I(CaL) inactivation voltage shifts. The SR Ca(2+)-uptake inhibitor thapsigargin and the SR Ca(2+) release inhibitor ryanodine also suppressed CAVB-induced EADs, consistent with an important role for SR Ca(2+) loading and release in arrhythmogenesis. AP-duration changes reached a maximum after 1 week of bradypacing, but peak alterations in CaMKII and [Ca(2+)](i) required 2 weeks, paralleling the EAD time course.
CAVB-induced remodeling enhances [Ca(2+)](i) load and activates the Ca(2+)-calmodulin-CaMKII system, producing [Ca(2+)](i)-handling abnormalities that contribute importantly to CAVB-induced arrhythmogenic afterdepolarizations.
... These EADs and DADs were first described in Purkinje fibres [35,36], a widely used experimental preparation in early electrophysiological studies. Similar afterdepolarizations have been recorded in ventricular (figure 3c adapted from [37]) and atrial myocytes. Extracellular potassium ions and I K1 are particularly important in determining whether the membrane potential is closer to the plateau or the resting potential [46,63,64]. ...
... the conduction system to initiate EADs. It was said that 'Salvos of EADs produced undulating ECG patterns associated with TdP (torsades de pointes arrhythmias), which progressed to VT.' AP prolongation involving downregulation of delayed rectifier K + currents has also been investigated in rabbit hearts with chronic AV block [37]. Ventricular myocytes from this model showed EADs associated with the prolonged APs (figure 3). ...
... Blocking SR Ca 2+ uptake with thapsigargin and SR Ca 2+ release with ryanodine both suppressed the EADs. Interestingly, the EADs were also suppressed by the calmodulin inhibitor W7 and the CaMKII inhibitor, KN-93 [37]. Possible effects of the CaMKII pathway to promote persistence of L-type Ca 2+ currents were considered, but there was also evidence for increased Ca 2+ loading of the SR. ...
Rhythms of electrical activity in all regions of the heart can be influenced by a variety of intracellular membrane bound organelles. This is true both for normal pacemaker activity and for abnormal rhythms including those caused by early and delayed afterdepolarizations under pathological conditions. The influence of the sarcoplasmic reticulum (SR) on cardiac electrical activity is widely recognized, but other intracellular organelles including lysosomes and mitochondria also contribute. Intracellular organelles can provide a timing mechanism (such as an SR clock driven by cyclic uptake and release of Ca ²⁺ , with an important influence of intraluminal Ca ²⁺ ), and/or can act as a Ca ²⁺ store involved in signalling mechanisms. Ca ²⁺ plays many diverse roles including carrying electric current, driving electrogenic sodium–calcium exchange (NCX) particularly when Ca ²⁺ is extruded across the surface membrane causing depolarization, and activation of enzymes which target organelles and surface membrane proteins. Heart function is also influenced by Ca ²⁺ mobilizing agents (cADP-ribose, nicotinic acid adenine dinucleotide phosphate and inositol trisphosphate) acting on intracellular organelles. Lysosomal Ca ²⁺ release exerts its effects via calcium/calmodulin-dependent protein kinase II to promote SR Ca ²⁺ uptake, and contributes to arrhythmias resulting from excessive beta-adrenoceptor stimulation. A separate arrhythmogenic mechanism involves lysosomes, mitochondria and SR. Interacting intracellular organelles, therefore, have profound effects on heart rhythms and NCX plays a central role.
This article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’.
... Furthermore, the bradycardia resulting from AVB leads to cardiac remodeling that can cause malignant Torsades de Pointes (TdP) ventricular tachyarrhythmias (VTs). 8 Because of the potential contribution of TTCCs to automaticity and their presence throughout the conduction system, 7 we investigated the role of TTCCs in the response to AVB, specifically the generation of ERs and prevention of life-threatening VTs, with the use of targeted Cav3.1 deletion and a mouse model of AVB. ...
... These include downregulation of delayed rectifier K + currents and Ca 2+ /calmodulin kinase II-mediated changes in LTCCs. 8,[22][23][24] VT susceptibility early after AVB in the mouse is associated with downregulation of Kv4.2 mRNA and protein and a parallel decrease in the fast-inactivating transient outward current. 10 The ionic changes post-AVB prolong the action potential, providing time for LTCCs to recover from inactivation and reactivate, leading to membrane depolarization that can form early afterdepolarizations that trigger TdP. 25 Enhanced Ca 2+ release and Na + /Ca 2+ exchange also contribute to the development of early afterdepolarizations and TdP. ...
... 10 The ionic changes post-AVB prolong the action potential, providing time for LTCCs to recover from inactivation and reactivate, leading to membrane depolarization that can form early afterdepolarizations that trigger TdP. 25 Enhanced Ca 2+ release and Na + /Ca 2+ exchange also contribute to the development of early afterdepolarizations and TdP. 8,26 Because AVB-induced bradycardia promotes early afterdepolarization-related ventricular arrhythmia formation, anything that worsens bradycardia (like the absence of TTCCs) would be expected to enhance arrhythmogenesis. Consistent with greater action potential prolongation, ventricular effective refractory period, the most reliable in vivo method to evaluate ventricular repolarization independent of rate, was greater in Cav3.1 −/− versus WT mice 24 hours post-AVB ( Figure 1D). ...
-When complete atrioventricular-block (AVB) occurs, infranodal escape-rhythms (ERs) are essential to prevent bradycardic death. The role of T-type Ca(2+)-channels (TTCCs) in pacemaking outside the sinus node is unknown. We investigated the role of TTCCs in ERs and bradycardia-related ventricular tachyarrhythmias following AVB in mice.
-Adult male mice lacking Cav3.1 (Cav3.1-/-) and wild-type (WT)-controls implanted with ECG telemetry-devices underwent radiofrequency atrioventricular-node ablation to produce AVB. Pre-ablation, Cav3.1-/- mice showed sinus bradycardia (mean±SEM RR-intervals 148±3 ms versus 128±2 ms WT, P<0.001). Immediately post-AVB, Cav3.1-/- mice had slower ERs (RR-intervals 650±75 ms, versus 402±26 ms in WT, P<0.01), but a preserved heart-rate response to isoproterenol. Over the next 24 hours, mortality was markedly greater in Cav3.1-/- mice (19/31, 61%) versus WT (8/26, 31%; P<0.05), and Torsades de Pointes occurred more frequently (73% Cav3.1-/- versus 35% WT, P<0.05). ERs improved in both groups over the next 4 weeks, but remained significantly-slower in Cav3.1-/-. At 4 weeks post-AVB, ventricular tachycardia was more frequent in Cav3.1-/- than in WT mice (746±116 versus 214±78 episodes/24 hours, P<0.01). Ventricular-function remodeling was similar in Cav3.1-/- and WT, except for smaller post-AVB fractional-shortening increase in Cav3.1-/-. Expression-changes were seen post AVB for a variety of genes: these tended to be greater in Cav3.1-/- mice, and overexpression of fetal and profibrotic genes occurred only in Cav3.1-/-.
-This study suggests that TTCCs play an important role in infranodal escape automaticity. Loss of TTCCs worsens bradycardia-related mortality, increases bradycardia-associated adverse remodeling and enhances the risk of malignant ventricular tachyarrhythmias complicating AVB.
... In several subsequent papers, the Ca 2+ window current was considered to play a central role in cardiac arrhythmogenesis, specifically EADs. For instance, it has been shown in cardiac myocytes with chronic atrioventricular block (cAVB) that Ca 2+ inactivation current was shifted to more positive membrane potentials, resulting in a larger window current area in cAVB associated to EADs (Antoons et al., 2007;Qi et al., 2009). Although not specifically mentioned, the shift to the right and the larger Ca 2+ window current observed in cAVB was associated with a higher incomplete inactivation of I CaL (Antoons et al., 2007), increasing what is known as late Ca 2+ current or pedestal, which reflects the incomplete inactivation of the channel (Cohen and Lederer, 1987). ...
... As stated above, several compounds that decrease I CaL window area, like Ca 2+ channel blockers, may either diminish or suppress EADs (Cohen and Lederer, 1987). Calmodulin and Ca-calmodulin-dependent protein kinase (CaMKII) inhibitors were also able to suppress EADs that originate in oxidative stress or chronic atrioventricular blocks (Xie et al., 2009;Qi et al., 2009). However, the relevant finding of this work (which also provides its translational strength) is the demonstration that it is possible to avoid EADs Figure 1. ...
This commentary is on the paper by Angelini et al. Here, we set the original paper in the context of triggered arrhythmias, particularly early after depolarizations (EADs), emphasizing the importance of pharmacologically inhibiting late Ca2+ current to prevent EADs without affecting myocardial contractility.
... Various underlying mechanisms have been proposed, including CaMKIIδ-dependent phosphorylation of the L-type Ca 2+ channel and a resulting increase in channel open probability. 5,6 Thus, enhanced CaMKIIδ activity is linked to Ca 2+ -dependent arrhythmogenesis by multiple mechanisms. ...
... Although CaMKIIδ-dependent activation of RyRs and L-type Ca 2+ channels promotes the generation of DADs and EADs, it is important to point out that these effects can be codependent and that the presently observed actions of SN may reflect these overlap-ping roles. For example, although CaMKIIδ-dependent phosphorylation of the L-type Ca 2+ channel increases the likelihood of EADs, 5,6 increased L-type current also loads the SR and promotes spontaneous RyR2 Ca 2+ release (DADs). 1 Similarly, although increased RyR2 open probability promotes Ca 2+ waves/DADs during diastole, Ca 2+ release during action potential repolarization can promote EADs. 26 Protection against EADs and DADs by SN may also involve additional CaMKIIδregulated proteins and include inhibition of late Na + current and various K + channels. ...
Background:
Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia.
Methods:
Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes.
Results:
SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current.
Conclusions:
SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.
... CaMKII has been shown to promote EADs in ventricular cardiomyocytes (Qi et al., 2009), which can produce ectopic (triggered) activity. CaMKII-dependent phosphorylation of I Ca,L slows I Ca,L inactivation, increasing the I Ca,L window current that plays a major role in the generation of EADs (Qi et al., 2009). ...
... CaMKII has been shown to promote EADs in ventricular cardiomyocytes (Qi et al., 2009), which can produce ectopic (triggered) activity. CaMKII-dependent phosphorylation of I Ca,L slows I Ca,L inactivation, increasing the I Ca,L window current that plays a major role in the generation of EADs (Qi et al., 2009). In addition, the APD-prolonging effects of CaMKII-dependent phosphorylation of I Na , increasing I Na,late , could further promote the occurrence of EADs and ectopic activity (Wagner et al., 2011). ...
Atrial fibrillation (AF) is the most frequently encountered clinical arrhythmia and is associated with increased morbidity and mortality. Ectopic activity and reentry are considered major arrhythmogenic mechanisms contributing to the initiation and maintenance of AF. In addition, AF is self-reinforcing through progressive electrical and structural remodeling which stabilize the arrhythmia and make it more difficult to treat. Recent research has suggested an important role for Ca(2+)-dysregulation in AF. Ca(2+)-handling abnormalities may promote ectopic activity, conduction abnormalities facilitating reentry, and AF-related remodeling. In this review article, we summarize the Ca(2+)-handling derangements occurring in AF and discuss their impact on fundamental arrhythmogenic mechanisms. We focus in particular on the role of the multifunctional Ca(2+)/calmodulin-dependent protein kinase type-II (CaMKII), which acts as a major link between Ca(2+)-dysregulation and arrhythmogenesis. CaMKII expression and activity are increased in AF and promote arrhythmogenesis through phosphorylation of various targets involved in cardiac electrophysiology and excitation-contraction coupling. We discuss the implications for potential novel therapeutic strategies for AF based on CaMKII and Ca(2+)-handling abnormalities.
... However, inappropriate SR Ca 2+ release is likely causative in a variety of cardiac arrhythmias. 3 Augmentation of I Ca,L has been proposed as a major contributing factor to arrhythmogenesis, 4,5 and blocking I Ca,L suppresses ventricular arrhythmogenesis. 6 Thus, I Ca,L is a focal point in the regulation of cardiac action potential (AP), excitation-contraction coupling, and arrhythmogenesis. ...
... 13 Enhanced LTCC function can induce activation of CaMKII, which in turn prolongs QT interval and creates a substrate for bradycardiainduced arrhythmias. 3 Rad À/À mice display elevated phosphorylated-CaMKII 14 and increased heart growth in response to pressure overload, 14 but there are no reports of the effect of deletion of Rad on myocardial function. Cardiac arrhythmias are comorbid with cardiac hypertrophy. ...
The small GTPase Rad is a negative regulator of voltage-dependent L-type calcium channel current (ICaL); however, the effects of Rad ablation on cardiomyocyte function are unknown. The objective of this study is to test the hypothesis that Rad-depletion causes positive inotropic effects without inducing cardiac hypertrophy.
Ventricular myocytes from adult Rad(-/-) mice were isolated and evaluated by patch-clamp recordings for ICa,L and action potentials, Ca(2+) transients, and sarcomere shortening. Maximum ICaL is elevated in Rad(-/-) (maximal conductance 0.35±0.04 picoSiemens/picoFarad (pS/pF) wild-type; 0.61±0.14 pS/pF Rad(-/-)), decay kinetics are faster, and ICa,L activates at lower voltages (activation midpoint -7.2±0.6 wild-type; -11.7±0.9 Rad(-/-)) mimicking effects of β-adrenergic receptor stimulation. Diastolic and twitch calcium are elevated in Rad(-/-) (F340/380: 1.03 diastolic and 0.35 twitch for wild-type; 1.47 diastolic and 0.736 twitch for Rad(-/-)) and sarcomere shortening is enhanced (4.31% wild-type; 14.13% Rad(-/-)) at lower pacing frequencies. Consequentially, frequency-dependence of Ca(2+) transients is less in Rad(-/-), and the frequency dependence of relaxation is also blunted. In isolated working hearts, similar results were obtained; chiefly, +dP/dt was elevated at baseline and developed pressure was relatively nonresponsive to acute β-adrenergic receptor stimulation. In single cells, at subphysiological frequencies, nonstimulated calmodulin-dependent protein kinase-sensitive calcium release is observed. Remarkably, Rad(-/-) hearts did not show hypertrophic growth despite elevated levels of diastolic calcium.
This study demonstrates that the depletion of Rad GTPase is equivalent to sympathomimetic β-adrenergic receptor, without stimulating cardiac hypertrophy. Thus, targeting Rad GTPase is a novel potential therapeutic target for Ca(2+)-homeostasis-driven positive inotropic support of the heart.
... Our results indicate that RyR Ca 2+ leak influence gating properties of the LTCCs. One intriguing but remarkable aspect of our findings is that the changes in the voltage-dependence of LTCC activation and inactivation were not paralleled by changes in the kinetic properties of I Ca (Figure 2A & B), similar to that observed by others [33]. Whereas this might indicate that recovery from the inactivated state was impeded, this interpretation will be difficult to reconcile with the maintenance of I Ca peak density at more depolarized voltages (Figure 1D). ...
... Thus, diastolic Ca 2+ leak would promote DADs [21], while would be protective from EADs by reducing I W Ca . Our results indeed echo a prevention of EAD induced Torsades de pointes by W7 through a decrease of I W Ca [33,44]. ...
Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.
... Our results indicate that RyR Ca 2+ leak influence gating properties of the LTCCs. One intriguing but remarkable aspect of our findings is that the changes in the voltage-dependence of LTCC activation and inactivation were not paralleled by changes in the kinetic properties of I Ca (Figure 2A & B), similar to that observed by others [33]. Whereas this might indicate that recovery from the inactivated state was impeded, this interpretation will be difficult to reconcile with the maintenance of I Ca peak density at more depolarized voltages ( Figure 1D). ...
... Thus, diastolic Ca 2+ leak would promote DADs [21], while would be protective from EADs by reducing I W Ca . Our results indeed echo a prevention of EAD induced Torsades de pointes by W7 through a decrease of I W Ca [33,44]. ...
... Torsades de Pointes (TdP) is a polymorphic type of VT also associated to acquired QT prolongation and maintained bradycardia that potentially leads to SCD (Jackman et al., 1988). Several studies carried out in rabbits and dogs prone to spontaneous TdP as a consequence of the chronic atrioventricular block (AVB) showed important alterations in the control of Ca 2+ (Sipido et al., 2000; Antoons et al., 2007; Qi et al., 2009). For example, AVB in dogs resulted in an increase in the SR Ca 2+ content which improved Ca 2+ release from SR as Ca 2+ transients (Sipido et al., 2000). ...
... Although, the overall density-voltage relationship of I CaL is unchanged, a depolarizing shift in the f ∞ curve resulted in an increased window current (Antoons et al., 2007). The CaM activation of CaMKII has been proposed to underlie this effect, as well the induced EADs (Qi et al., 2009). ...
... The molecular mechanisms of EAD remain mysterious. Some studies suggest that EAD might result from the Ca 2+ influx from L-type Ca 2+ channels or spontaneous Ca 2+ release from the endoplasmic reticulum (ER) [86]. Considering the important role and active involvement of aging and oxidative stress in the development of AF and TRPM2 in these two conditions, TRPM2-mediated Ca 2+ influx or Ca 2+ release from lysosome might also contribute to the Ca 2+ overload during EAD and the triggering of AF. ...
Ischemic stroke causes a heavy health burden worldwide, with over 10 million new cases every year. Despite the high prevalence and mortality rate of ischemic stroke, the underlying molecular mechanisms for the common etiological factors of ischemic stroke and ischemic stroke itself remain unclear, which results in insufficient preventive strategies and ineffective treatments for this devastating disease. In this review, we demonstrate that transient receptor potential cation channel, subfamily M, member 2 (TRPM2), a non-selective ion channel activated by oxidative stress, is actively involved in all the important steps in the etiology and pathology of ischemic stroke. TRPM2 could be a promising target in screening more effective prophylactic strategies and therapeutic medications for ischemic stroke.
... Roscovitine selectively reduces late I Ca,L in rabbit ventricular myocytes without affecting peak current Changes in the I Ca,L properties that increase the window current ( Fig. 1) have been associated with an increased susceptibility to EADs and arrhythmia (e.g., LQT; January and Riddle, 1989;Antoons et al., 2007;Qi et al., 2009;Madhvani et al., 2011;Qu and Chung, 2012;Madhvani et al., 2015;Kettlewell et al., 2019;Liu et al., 2019). To evaluate the ability of roscovitine to reduce I Ca,L window current by limiting the late I Ca,L , we first studied its action on native I Ca,L in rabbit ventricular myocytes exposed to oxidative stress (600 µM H 2 O 2 ) to enhance late I Ca,L . ...
Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation–contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.
... The cellular and molecular bases of arrhythmogenic ventricular remodeling in CAVB rabbits have been characterized to some extent previously. [12][13][14] CAVB rabbit cardiomyocytes show APD prolongation with concurrent early and delayed afterdepolarization (EAD/DAD). Downregulation of subunits encoding rapid and slow delayed-rectifier K 1 -current components underlies APD prolongation. ...
Background
Electrical storm (ES) is a life-threatening emergency in patients at high risk of ventricular tachycardia/fibrillation (VT/VF), but the pathophysiology and molecular basis are poorly understood.
Objective
To explore the electrophysiological substrate for experimental ES.
Methods
A model was created by inducing chronic complete atrioventricular-block in defibrillator-implanted rabbits, which recapitulates QT-prolongation, Torsades-des-Pointes (TdP) and VF-episodes.
Results
Optical mapping revealed island-like regions with action potential duration (APD) prolongation in the left ventricle (LV), leading to increased spatial APD-dispersion, in rabbits with ES (defined as ≥3 VF-episodes/24-h). The maximum APD and its dispersion correlated with the total number of VF-episodes in-vivo. TdP was initiated by an ectopic beat that failed to enter the island and formed a reentrant wave, and perpetuated by rotors whose centers swirled in the periphery of the island. Epinephrine exacerbated the island by prolonging APD and enhancing APD-dispersion, which was less evident after late Na⁺-current (INa-L) blockade with 10 μM ranolazine. Non-sustained VT in a non-ES rabbit heart with homogeneous APD prolongation resulted from multiple foci with an electrocardiographic morphology different from TdP driven by drifting rotors in ES-rabbit hearts. The neuronal Na⁺-channel subunit NaV1.8 was upregulated in ES-rabbit LV-tissues and expressed within myocardium corresponding to the island location in optically mapped ES-rabbit hearts. The NaV1.8-blocker A-803467 (10 mg/kg, i.v.) attenuated QT-prolongation and suppressed epinephrine-evoked TdP.
Conclusion
A tissue-island with enhanced refractoriness contributes to the generation of drifting rotors that underlies ES in this model. NaV1.8-mediated INa-L merits further investigation as a contributor to the substrate for ES.
... Therefore, given that arrhythmia episodes in human LQT3 patients are prevalent during slower heart rates and assuming that spontaneous Ca 2+ release is a key element of trigger [58], there is a good chance that SERCa activity is increased, in stark contrast to mice. Indeed, Xiao Yan Qi et al. [66] showed enhanced SERCa activity and PLB phosphorylation due to enhanced activity of CaMKII in rabbit hearts with slowed heart rate induced by AV block 2 weeks after the procedure. At the cellular level, bradycardia was accompanied by AP prolongation resulting in enhanced LTCCmediated Ca 2+ influx, increased SR Ca 2+ load, increased Ca 2+ transient amplitude, increased contraction, and importantly, arrhythmogenic EADs at very slow pacing rates in the absence of β-adrenergic stimulation. ...
Sudden cardiac death due to malignant ventricular arrhythmias remains the major cause of mortality in the postindustrial world. Defective intracellular Ca ²⁺ homeostasis has been well established as a key contributing factor to the enhanced propensity for arrhythmia in acquired cardiac disease, such as heart failure or diabetic cardiomyopathy. More recent advances provide a strong basis to the emerging view that hereditary cardiac arrhythmia syndromes are accompanied by maladaptive remodeling of Ca ²⁺ homeostasis which substantially increases arrhythmic risk. This brief review will focus on functional changes in elements of Ca ²⁺ handling machinery in cardiomyocytes that occur secondary to genetic mutations associated with catecholaminergic polymorphic ventricular tachycardia, and long QT syndrome.
... On the one hand, CaMKII expression and activity increases were observed in various species with atrial tachycardia and fibrillation, such as human with chronic AF Voigt et al., 2012), goat with long-standing AF (Greiser et al., 2009) and canine with pacing-induced atrial tachycardia remodeling (Wakili et al., 2010), showing the role of AF in promoting CaMKII overexpression and hyperactivities. On the other hand, CaMKII abnormities were considered to promote ectopic activities such as early afterdepolarizations (EAD) (Qi et al., 2009) and delayed afterdepolarizations (DAD) (Dobrev et al., 2011), and to improve reentry generations by increasing the repolarization dispersion (Yue et al., 2011) or slowing the conduction velocity of the electrical wave (Wagner et al., 2006;Wang et al., 2018), all of which were the key mechanisms related to AF. In addition, AF was found to relate to a higher level of oxidative stress and CaMKII oxidation also increased in AF patients. ...
This simulation study aims to investigate how the Calcium/calmodulin-dependent protein kinase II (CaMKII) overexpression and oxidation would influence the cardiac electrophysiological behavior and its arrhythmogenic mechanism in atria. A new-built CaMKII oxidation module and a refitted CaMKII overexpression module were integrated into a mouse atrial cell model for analyzing cardiac electrophysiological variations in action potential (AP) characteristics and intracellular Ca ²⁺ cycling under different conditions. Simulation results showed that CaMKII overexpression significantly increased the phosphorylation level of its downstream target proteins, resulting in prolonged AP and smaller calcium transient amplitude, and impaired the Ca ²⁺ cycling stability. These effects were exacerbated by extra reactive oxygen species, which oxidized CaMKII and led to continuous high CaMKII activation in both systolic and diastolic phases. Intracellular Ca ²⁺ depletion and sustained delayed afterdepolarizations (DADs) were observed under co-existing CaMKII overexpression and oxidation, which could be effectively reversed by clamping the phosphorylation level of ryanodine receptor (RyR). We also found that the stability of RyR release highly depended on a delicate balance between the level of RyR phosphorylation and sarcoplasmic reticulum Ca ²⁺ concentration, which was closely related to the genesis of DADs. We concluded that the CaMKII overexpression and oxidation have a synergistic role in increasing the activity of CaMKII, and the unstable RyR may be the key downstream target in the CaMKII arrhythmogenic mechanism. Our simulation provides detailed mechanistic insights into the arrhythmogenic effect of CaMKII overexpression and oxidation, which suggests CaMKII as a promising target in the therapy of atrial fibrillation.
... I NCX augmentation provided a depolarization component to counterbalance the repolarization reserve and prolong the APD. The elevated and prolonged AP in phase 3 gradually caused reactivation of I CaL (Figure 5(c)), which ultimately induced action potential depolarization and produced EAD ( Figure 5(a)), consistent with previous studies [57][58][59]. ...
Atrial fibrillation is a common cardiac arrhythmia with an increasing incidence rate. Particularly for the aging population, understanding the underlying mechanisms of atrial arrhythmia is important in designing clinical treatment. Recently, experiments have shown that atrial arrhythmia is associated with oxidative stress. In this study, an atrial cell model including oxidative-dependent Ca2+/calmodulin- (CaM-) dependent protein kinase II (CaMKII) activation was developed to explore the intrinsic mechanisms of atrial arrhythmia induced by oxidative stress. The simulation results showed that oxidative stress caused early afterdepolarizations (EADs) of action potentials by altering the dynamics of transmembrane currents and intracellular calcium cycling. Oxidative stress gradually elevated the concentration of calcium ions in the cytoplasm by enhancing the L-type Ca2+ current and sarcoplasmic reticulum (SR) calcium release. Owing to increased intracellular calcium concentration, the inward Na+/Ca2+ exchange current was elevated which slowed down the repolarization of the action potential. Thus, the action potential was prolonged and the L-type Ca2+ current was reactivated, resulting in the genesis of EAD. Furthermore, based on the atrial single-cell model, a two-dimensional (2D) ideal tissue model was developed to explore the effect of oxidative stress on the electrical excitation wave conduction in 2D tissue. Simulation results demonstrated that, under oxidative stress conditions, EAD hindered the conduction of electrical excitation and caused an unstable spiral wave, which could disrupt normal cardiac rhythm and cause atrial arrhythmia. This study showed the effects of excess reactive oxygen species on calcium cycling and action potential in atrial myocytes and provided insights regarding atrial arrhythmia induced by oxidative stress.
... Although several mechanisms can lead to afterdepolarizations, many of these relate to abnormalities in calcium handling. There are two types of afterdepolarization termed "early" or "delayed" afterdepolarizations. Early afterdepolarizations (EADs; Figure 1B), occur within the action potential (phases 2-3) and may arise from reactivation of I Ca(L) or spontaneous calcium release (Qi et al., 2009) driving reverse mode I NCX as reviewed by Weiss et al. (2010). Delayed afterdepolarizations (DADs; Figure 1C), occurring after the action potential (within phase 4) arise from spontaneous calcium release from the SR in the form of a calcium wave (Venetucci et al., 2008). ...
Atrial fibrillation (AF) is commonly associated with heart failure. A bidirectional relationship exists between the two—AF exacerbates heart failure causing a significant increase in heart failure symptoms, admissions to hospital and cardiovascular death, while pathological remodeling of the atria as a result of heart failure increases the risk of AF. A comprehensive understanding of the pathophysiology of AF is essential if we are to break this vicious circle. In this review, the latest evidence will be presented showing a fundamental role for calcium in both the induction and maintenance of AF. After outlining atrial electrophysiology and calcium handling, the role of calcium-dependent afterdepolarizations and atrial repolarization alternans in triggering AF will be considered. The atrial response to rapid stimulation will be discussed, including the short-term protection from calcium overload in the form of calcium signaling silencing and the eventual progression to diastolic calcium leak causing afterdepolarizations and the development of an electrical substrate that perpetuates AF. The role of calcium in the bidirectional relationship between heart failure and AF will then be covered. The effects of heart failure on atrial calcium handling that promote AF will be reviewed, including effects on both atrial myocytes and the pulmonary veins, before the aspects of AF which exacerbate heart failure are discussed. Finally, the limitations of human and animal studies will be explored allowing contextualization of what are sometimes discordant results.
... A possible explanation is bradycardia-induced AP prolongation. Qi et al. demonstrated AP prolongation and ion current remodeling in the rabbit heart after 1-2 weeks of AV block-induced ventricular bradycardia [29]. Conversely, APD decreases with increasing beating frequencies [4,11]. ...
Engineered heart tissue (EHT) from rat cells is a useful tool to study ventricular biology and cardiac drug safety. Since atrial and ventricular cells differ significantly, EHT and other 3D cell culture formats generated from ventricular cells have been of limited value to study atrial biology. To date, reliable in vitro models that reflect atrial physiology are lacking. Therefore, we established a novel EHT model using rat atrial cells (atrial EHT, aEHT) to assess atrial physiology, contractility and drug response. The tissue constructs were characterized with regard to gene expression, histology, electrophysiology, and the response to atrial-specific drugs. We observed typical functional properties of atrial tissue in our model such as more regular spontaneous beating with lower force, shorter action potential duration, and faster contraction and relaxation compared to ventricular EHT (vEHT). The expression of atrial-specific genes and proteins was high, whereas ventricle-specific transcripts were virtually absent. The atrial-selective drug carbachol had a strong negative inotropic and chronotropic effect on aEHT only. Taken together, the results demonstrate the feasibility of aEHT as a novel atrial 3D model and as a benchmark for tissue engineering with human induced pluripotent stem cell-derived atrial-like cardiomyocytes. Atrial EHT faithfully recapitulates atrial physiology and shall be useful to study atrial molecular physiology in health and disease as well as drug response.
... Dilation and hypertrophy allow for maintenance of cardiac output in the face of severe bradycardia. The cellular basis for these electrophysiologic and structural changes have been studied extensively in several animal models, [5][6][7][8] but miRNA involvement has yet to be investigated. ...
Background:
Complete atrioventricular block (CAVB) causes arrhythmogenic remodeling and increases the risk of Torsades de Pointes (TdP) arrhythmias. MicroRNAs (miRNAs) are key regulators of gene-expression that contribute to cardiac remodeling.
Objective:
To assess miRNA changes after complete atrioventricular block (CAVB) and identify novel candidates potentially involved in arrhythmogenic cardiac remodeling.
Methods:
CAVB was induced in mice via His bundle ablation. Expression of microRNAs was evaluated by pan-miRNA microarray with qPCR confirmation, on samples obtained 24 hours and 4 weeks post-CAVB. MiRNA target-prediction algorithms were used to identify potential target genes. Targets confirmed by luciferase assays in HEK293 cells were followed up with overexpression studies in neonatal rat ventricular myocytes (NRVMs) to evaluate regulation using RT-qPCR, Western blots, cell shortening measurements and fura-2 Ca2+ fluorescence imaging.
Results:
Of >400 miRNAs assayed, only miRNA-135a was altered at 24 hours, downregulated 78% (p<0.001). Algorithms predicted miRNA-135a regulation of the sodium-calcium exchanger type-1 (NCX1). miRNA-135a transfection suppressed NCX1 3'UTR reporter activity by 42% (p<0.001), mRNA expression by 34% (p<0.001) and protein levels by 45% (p<0.001), versus noncoding miRNA control. miR-135a overexpression reduced spontaneous beating frequency of NRVMs by 63% (p<0.001), while slowing decay (by 56%, p<0.05) of caffeine-induced Ca2+ transients. miR-135a also suppressed the Ca2+-loading effects of ouabain and ouabain-induced spontaneous Ca2+-release events (SCREs).
Conclusions:
NCX1 is negatively regulated by miR-135a, a microRNA that is downregulated in the heart following CAVB in mice. By controlling NCX1 expression, miR-135a modulates cardiomyocyte automaticity, Ca2+-extrusion and arrhythmogenic Ca2+-loading/SCREs. miR-135a may therefore contribute to pro-arrhythmic remodeling following CAVB.
... Because of the attenuation of LA arrhythmogenic activity through Ca 2+ regulation, we predict that EGCG might inhibit Ca 2+ /calmodulindependent protein kinase. Previous studies showed that KN93, an inhibitor of multifunctional Ca 2+ /calmodulin-dependent protein kinase, decreased early afterdepolarizations in rabbit heart [41]. We found KN93 had a partial effect on the suppression of ISO-induced LA burst firing, and EGCG could suppress most ISO-induced LA burst firing. ...
Background:
Atrial fibrillation (AF) is the commonest sustained arrhythmia, and increases the risk of stroke, heart failure, and mortality. Calcium (Ca(2+)) overload and oxidative stress are thought to participate in the pathogenesis of AF. Epigallocatechin-3-gallate (EGCG) has an antioxidative effect and been shown to be beneficial in promoting cardiovascular health. However, it is not clear if EGCG directly modulates the electrophysiological characteristics and Ca(2+) homeostasis of the left atrium (LA).
Methods and results:
Conventional microelectrodes, whole-cell patch-clamp, and Fluo-3 fluorometric ratio technique were performed using the isolated rabbit LA preparations or isolated single LA cardiomyocytes before and after EGCG treatment. EGCG (0.01, 0.1, 1, and 10μM) which concentration-dependently decreased the APD20 by 13±8%, 25±5%, 31±6%, and 37±5%, APD50 by 9±8%, 22±6%, 32±7%, and 40±4%, and APD90 by 2±12%, 9±8%, 24±10%, and 34±5% in LA preparations. EGCG (0.1μM) decreased the late sodium (Na(+)) current, L-type Ca(2+) current, nickel-sensitive Na(+)-Ca(2+) exchanger current, and transient outward current, but did not change the Na(+) current and ultra-rapid delayed rectifier potassium current in LA cardiomyocytes. EGCG decreased intracellular Ca(2+) transient and sarcoplasmic reticulum Ca(2+) content in LA cardiomyocytes. Furthermore, EGCG decreased isoproterenol (ISO, 1μM)-induced burst firing. KT5823 (1μM) or KN93 (1μM) decreased the incidences of ISO-induced LA burst firing, which became lower with EGCG treatment. H89 (10μM) and KN92 (1μM) did not suppress the incidence of ISO-induced LA burst firing. However, EGCG decreased the incidences of ISO-induced LA burst firing in the presence of H89 or KN92.
Conclusion:
EGCG directly regulates LA electrophysiological characteristics and Ca(2+) homeostasis, and suppresses ISO-induced atrial arrhythmogenesis through inhibiting Ca(2+)/calmodulin or cGMP-dependent protein kinases.
... Ryanodine gene defects are hypothesized as responsible for both catecholaminergic polymorphic ventricular tachycardia and exercise induced QT-prolongation 14 . The calcium/calmodulin/kinase system may be involved in arrhythmogenic after-depolarizations of bradycardia-related acquired long-QT syndrome 15 . Putative pathogenic catecholaminergic polymorphic ventricular tachycardia -causing mutations in ryanodine receptor were detected in 6% of unrelated, genotype-negative long QT syndrome referrals 16 . ...
We report the case of a 38-year old woman with history of syncope and polymorphic ventricular tachycardia; tachycardia was inducible at exercise stress test, not at electrophysiologic study. Phases of QT prolongation were found at ambulatory electrocardiogram monitoring. The woman came to our attention for periodic control of implantable loop recorder. Rest electrocardiogram at admission unexpectedly showed sinus bradycardia, junctional rhythm and ventricular premature beats. Furthermore, loop recorder control revealed a short run of bidirectional tachycardia, not associated with syncope. Final diagnosis was catecholaminergic polymorphic ventricular tachycardia and the patient was implanted with an ICD. We therefore report an unusual case of bidirectional ventricular tachycardia associated with sinus node dysfunction and junctional escape rhythm. We hypothesize that a diffuse dysfunction of cardiac conduction system, presumably based on diffuse disorder of calcium handling, may be responsible for both sinus node failure and ventricular tachycardia.
... Both RyR2 and the less abundant IP3R1 are calcium release channels and SERCA2 is a calcium uptake channel located on the sarcoplasmic reticulum. Dysfunctional or abnormal expressions of RyR and SERCA lead to intracellular calcium overload and diastolic calcium leak from sarcoplasmic reticulum, resulting in cardiac arrhythmia or heart failure [27][28][29][30]. It has been recently shown that the Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and is related to its propensity to develop arrhythmias [31]. ...
Background:
Atrial fibrillation (AF) causes atrial remodeling, and the left atrium (LA) is the favored substrate for maintaining AF. It remains unclear if AF remodels both atria differently and contributes to LA arrhythmogenesis and thrombogenesis. Therefore, we wished to characterize the transcript profiles in the LA and right atrium (RA) in sinus rhythm (SR) and AF respectively.
Methods:
Paired LA and RA appendages acquired from patients receiving cardiac surgery were used for ion-channel- and whole-exome-based transcriptome analysis. The ultrastructure was evaluated by immunohistochemistry.
Results:
Twenty-two and twenty ion-channels and transporters were differentially expressed between the LA and RA in AF and SR, respectively. Among these, 15 genes were differentially expressed in parallel between AF and SR. AF was associated with increased LA/RA expression ratio in 9 ion channel-related genes, including genes related to calcium handling. In microarray, AF was associated with a differential LA/RA gene expression ratio in 309 genes, and was involved in atherosclerosis-related signaling. AF was associated with the upregulation of thrombogenesis-related genes in the LA appendage, including P2Y12, CD 36 and ApoE. Immunohistochemistry showed higher expressions of collagen-1, oxidative stress and TGF-β1 in the RA compared to the LA.
Conclusions:
AF was associated with differential LA-to-RA gene expression related to specific ion channels and pathways as well as upregulation of thrombogenesis-related genes in the LA appendage. Targeting the molecular mechanisms underlying the LA-to-RA difference and AF-related remodeling in the LA appendage may help provide new therapeutic options in treating AF and preventing thromboembolism in AF.
... Numerous studies have reported increased cardiac calcineurin activity and NFAT nuclear translocation in human failing and hypertrophied hearts as well as in arrhythmia animal models. 30,31 It has been shown in canine atrial myocytes that NFAT is activated by rapid electrical stimulation mimicking the condition of atrial tachycardia, which mediates tachypacing-induced Cav1.2 downregulation and explains the electric mechanisms of AF begets AF in respect of decreased L-type calcium current in AF. 21 The same group proposed in another study that the atrial fibroblasts were also activated in an AF-promoting dog model, in which NFAT in the fibroblasts were activated as well and also played a profibrotic role in AF. 32 Consistent with the previous studies, we observed higher expression of nuclear NFATc3 and increased oxidative stress in patients with recurrent AF after the maze procedure, indicating that NFAT activation is likely associated more advanced atrial remodeling as well as oxidative stress, which may make it more difficult for rhythm control to be achieved. ...
Objective:
It is difficult to achieve rhythm control in patients with long-standing persistent atrial fibrillation (AF). The radiofrequency maze procedure is an effective means in curing AF with a variable recurrence rate depending on patient characteristics and AF duration. In these patients, the characteristics of the atrial substrate have not been well investigated. Because the inflammatory process has been shown to be important in the pathogenesis of AF, we sought to characterize the proinflammatory gene expression in left atria obtained from patients with AF undergoing mitral valve surgery combined with the maze procedure to distinguish the changes associated with AF and its recurrence after the surgical ablation.
Methods:
Left atrial appendages from 35 patients receiving mitral valve surgery were used for study. Ten patients had sinus rhythm (SR) and 25 patients had persistent AF for more than 1 year and underwent the maze procedure. Among the AF patients, 13 patients remained in SR (AF-SR) and 12 patients had recurrent AF during the 1-year clinical follow-up (AF-AF). The nCounter Human Inflammation Array (NanoString Technologies, Seattle, Wash) was used for evaluating proinflammatory gene expression. Quantitative polymerase chain reaction, Western blot, and immunohistochemistry were applied for studying messenger RNA and protein expression.
Results:
Of 144 expressed proinflammatory genes, the inflammation array analysis revealed that 32 genes were differentially expressed between AF (including AF-SR and AF-AF) and SR. Thirteen genes were differentially expressed between AF-SR and AF-AF. The array and quantitative polymerase chain reaction produced parallel results in analyzing the expression of particular genes. Concordant with the gene expression difference between AF and SR patients, rapid pacing increased the expressions of SHC1, RHOA, PDGFA, and TRAF2 in HL-1 myocytes, implicating a causative effect of tachyarrhythmia on these genes. Compared with AF-SR, AF-AF expressed more intense oxidative stress, upregulations of collagen, transforming growth factor beta 1, and intranuclear nuclear factor of activated T-cells. Regression analysis showed that increased left atrial diameter was associated with the expression of RHOA and STAT1.
Conclusions:
Differential expression profiles of proflammatory genes were presented between SR and AF and between maintained SR and recurrent AF after the maze procedure. The identified inflammatory molecules associated with AF and failed surgical ablation may provide clues for developing new potential therapeutic targets to improve AF rhythm control.
... Such a mechanism could explain why CaMKII inhibition suppresses EADs in rabbits with chronic heart block (prone to TdP). 57 Isolated cell experiments were used to study the mechanism of EAD generation. In a related paper by Horvath et al., 58 experiments on myocytes with drug-induced LQT3 (gain of function of the late Na + current) demonstrated that EADs are preceded by a rise of Ca i . ...
Release of Ca²⁺ ions from sarcoplasmic reticulum (SR) into myocyte cytoplasm and their binding to troponin C is the final signal form myocardial contraction. Synchronous contraction of ventricular myocytes is necessary for efficient cardiac pumping function. This requires both shuttling of Ca²⁺ between SR and cytoplasm in individual myocytes, and organ-level synchronization of this process by means of electrical coupling among ventricular myocytes. Abnormal Ca²⁺ release from SR causes arrhythmias in the setting of CPVT (catecholaminergic polymorphic ventricular tachycardia) and digoxin toxicity.
Recent optical mapping data indicate that abnormal Ca²⁺ handling causes arrhythmias in models of both repolarization impairment and profound bradycardia. The mechanisms involve dynamic spatial heterogeneity of myocardial Ca²⁺ handling preceding arrhythmia onset, cell-synchronous systolic secondary Ca²⁺ elevation (SSCE), as well as more complex abnormalities of intracellular Ca²⁺ handling detected by subcellular optical mapping in Langendorff-perfused hearts. The regional heterogeneities in Ca²⁺ handling cause action potential (AP) heterogeneities through sodium–calcium exchange (NCX) activation and eventually overwhelm electrical coupling of the tissue.
Divergent Ca²⁺ dynamics among different myocardial regions leads to temporal instability of AP duration and – on the patient level – in T wave lability. Although T-wave alternans has been linked to cardiac arrhythmias, non-alternans lability is observed in pre-clinical models of the long QT syndrome (LQTS) and CPVT, and in LQTS patients. Analysis of T wave lability may provide a real-time window on the abnormal Ca²⁺ dynamics causing specific arrhythmias such as Torsade de Pointes (TdP).
... Nearly 20 years ago it was shown that blockade of Ca 2+ release using ryanodine was able to reduce the occurrence of TdP in the CAVB model when the hearts were challenged with an I Kr blocker together with abrupt changes in pacing frequency [42]. More recently it was also shown that either thapsigargin or ryanodine was able to suppress CAVB-induced EADs in a rabbit model of CAVB, although BVR was not recorded [43]. These agents all affect global Ca 2+ release and therefore it would not be possible to use these as a therapeutic regimen, however perhaps more specific targeting of Ca 2+ homeostasis would be an option. ...
Beat-to-beat variability of ventricular repolarization (BVR) has been proposed as a strong predictor of Torsades de Pointes (TdP). BVR is also observed at the myocyte level, and a number of studies have shown the importance of calcium handling in influencing this parameter. The chronic AV block (CAVB) dog is a model of TdP arrhythmia in cardiac hypertrophy, and myocytes from these animals show extensive remodeling, including of Ca(2+) handling. This remodeling process also leads to increased BVR. We aimed to determine the role that (local) Ca(2+) handling plays in BVR. In isolated LV myocytes an exponential relationship was observed between BVR magnitude and action potential duration (APD) at baseline. Inhibition of Ca(2+) release from sarcoplasmic reticulum (SR) with thapsigargin resulted in a reduction of [Ca(2+)]i, and of both BVR and APD. Increasing ICaL in the presence of thapsigargin restored APD but BVR remained low. In contrast, increasing ICaL with preserved Ca(2+) release increased both APD and BVR. Inhibition of Ca(2+) release with caffeine, as with thapsigargin, reduced BVR despite maintained APD. Simultaneous inhibition of Na(+)/Ca(2+) exchange and ICaL decreased APD and BVR to similar degrees, whilst increasing diastolic Ca(2+). Buffering of Ca(2+) transients with BAPTA reduced BVR for a given APD to a greater extent than buffering with EGTA, suggesting subsarcolemmal Ca(2+) transients modulated BVR to a larger extent than the cytosolic Ca(2+) transient. In conclusion, BVR in hypertrophied dog myocytes, at any APD, is strongly dependent on SR Ca(2+) release, which may act through modulation of the l-type Ca(2+) current in a subsarcolemmal microdomain.
... 17 Increased CaMKII autophoshorylation was observed in rabbit chronic atrioventricular block models of left ventricular dysfunction, acquired long QT syndrome and incessant ventricular tachycardia. 120,121 In addition, CaMKII inhibition prevents the development of structural heart disease or arrhythmias upon myocardial infarction, 122 increased ROS formation, 15 pressure overload, 123 or pacing-induced incessant VT. 121 Calcineurin overexpressing mice with increased CaMKII activity show contractile dysfunction and arrhythmias. 124 Also here, CaMKII inhibition improved contractile function and suppressed arrhythmias. ...
Despite improvements in the therapy of underlying heart disease, sudden cardiac death is a major cause of death worldwide. Disturbed Na and Ca handling is known to be a major predisposing factor for life-threatening tachyarrhythmias. In cardiomyocytes, many ion channels and transporters, including voltage-gated Na and Ca channels, cardiac ryanodine receptors, Na/Ca-exchanger, and SR Ca-ATPase are involved in this regulation. We have learned a lot about the pathophysiological relevance of disturbed ion channel function from monogenetic disorders. Changes in the gating of a single ion channel and the activity of an ion pump suffice to dramatically increase the propensity for arrhythmias even in structurally normal hearts. Nevertheless, patients with heart failure with acquired dysfunction in many ion channels and transporters exhibit profound dysregulation of Na and Ca handling and Ca/calmodulin-dependent protein kinase and are especially prone to arrhythmias. A deeper understanding of the underlying arrhythmic principles is mandatory if we are to improve their outcome. This review addresses basic tachyarrhythmic mechanisms, the underlying ionic mechanisms and the consequences for ion homeostasis, and the situation in complex diseases like heart failure.
© 2015 American Heart Association, Inc.
... A reduction of I Kr or I Ks can also occur as a result of mutations in several forms of the long-QT syndrome [45,46]. Arrhythmias in that cases are also associated with the onset of EADs [3,24,[47][48][49]. It has also been suggested that I NaCa is important for the onset of EADs, especially during adrenergic stimulation [10]. ...
Sudden cardiac death is often caused by cardiac arrhythmias. Recently, special attention has been given to a certain arrhythmogenic condition, the long-QT syndrome, which occurs as a result of genetic mutations or drug toxicity. The underlying mechanisms of arrhythmias, caused by the long-QT syndrome, are not fully understood. However, arrhythmias are often connected to special excitations of cardiac cells, called early afterdepolarizations (EADs), which are depolarizations during the repolarizing phase of the action potential. So far, EADs have been studied mainly in isolated cardiac cells. However, the question on how EADs at the single-cell level can result in fibrillation at the tissue level, especially in human cell models, has not been widely studied yet. In this paper, we study wave patterns that result from single-cell EAD dynamics in a mathematical model for human ventricular cardiac tissue. We induce EADs by modeling experimental conditions which have been shown to evoke EADs at a single-cell level: by an increase of L-type Ca currents and a decrease of the delayed rectifier potassium currents. We show that, at the tissue level and depending on these parameters, three types of abnormal wave patterns emerge. We classify them into two types of spiral fibrillation and one type of oscillatory dynamics. Moreover, we find that the emergent wave patterns can be driven by calcium or sodium currents and we find phase waves in the oscillatory excitation regime. From our simulations we predict that arrhythmias caused by EADs can occur during normal wave propagation and do not require tissue heterogeneities. Experimental verification of our results is possible for experiments at the cell-culture level, where EADs can be induced by an increase of the L-type calcium conductance and by the application of I blockers, and the properties of the emergent patterns can be studied by optical mapping of the voltage and calcium.
... The left ventricular tissue was then isolated and flash frozen in liquid nitrogen. Protein preparations were performed using methods as described previously [25] and the protein contents of CaMKII (antiphospho-CaMKII, 1:1000, Abcam, Cambridge, UK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, Beyotime, Haimen, China) in total protein preparations were analyzed by standard Western blotting. ...
Aim:
To investigate whether resveratrol suppressed oxidative stress-induced arrhythmogenic activity and Ca2+ overload in ventricular myocytes and to explore the underlying mechanisms.
Methods:
Hydrogen peroxide (H2O2, 200 μmol/L)) was used to induce oxidative stress in rabbit ventricular myocytes. Cell shortening and calcium transients were simultaneously recorded to detect arrhythmogenic activity and to measure intracellular Ca2+ ([Ca2+]i). Ca2+/calmodulin-dependent protein kinases II (CaMKII) activity was measured using a CaMKII kit or Western blotting analysis. Voltage-activated Na+ and Ca2+ currents were examined using whole-cell recording in myocytes.
Results:
H2O2 markedly prolonged Ca2+ transient duration (CaTD), and induced early afterdepolarization (EAD)-like and delayed afterdepolarization (DAD)-like arrhythmogenic activity in myocytes paced at 0.16 Hz or 0.5 Hz. Application of resveratrol (30 or 50 μmol/L) dose-dependently suppressed H2O2-induced EAD-like arrhythmogenic activity and attenuated CaTD prolongation. Co-treatment with resveratrol (50 μmol/L) effectively prevented both EAD-like and DAD-like arrhythmogenic activity induced by H2O2. In addition, resveratrol markedly blunted H2O2-induced diastolic [Ca2+]i accumulation and prevented the myocytes from developing hypercontracture. In whole-cell recording studies, H2O2 significantly enhanced the late Na+ current (INa,L) and L-type Ca2+ current (ICa,L) in myocytes, which were dramatically suppressed or prevented by resveratrol. Furthermore, H2O2-induced ROS production and CaMKII activation were significantly prevented by resveratrol.
Conclusion:
Resveratrol protects ventricular myocytes against oxidative stress-induced arrhythmogenic activity and Ca2+ overload through inhibition of INa,L/ICa,L, reduction of ROS generation, and prevention of CaMKII activation.
... Work with larger animals has extended these findings to other forms of acquired heart disease. Models of long-QT-associated arrhythmia induced by AV nodal ablation have been developed in both the rabbit [62,63] and dog [64], and CaMKII appears to play a role in the arrhythmiasusceptibility in both species. Recently, a series of studies have shown that CaMKII is also involved in the etiology of atrial fibrillation (AF), which is the most common clinical arrhythmia [65]. ...
Introduction:
Calcium-calmodulin-dependent protein kinase II (CaMKII) has emerged as a central mediator of cardiac stress responses which may serve several critical roles in the regulation of cardiac rhythm, cardiac contractility and growth. Sustained and excessive activation of CaMKII during cardiac disease has, however, been linked to arrhythmias, and maladaptive cardiac remodeling, eventually leading to heart failure (HF) and sudden cardiac death.
Areas covered:
In the current review, the authors describe the unique structural and biochemical properties of CaMKII and focus on its physiological effects in cardiomyocytes. Furthermore, they provide evidence for a role of CaMKII in cardiac pathologies, including arrhythmogenesis, myocardial ischemia and HF development. The authors conclude by discussing the potential for CaMKII as a target for inhibition in heart disease.
Expert opinion:
CaMKII provides a promising nodal point for intervention that may allow simultaneous prevention of HF progression and development of arrhythmias. For future studies and drug development there is a strong rationale for the development of more specific CaMKII inhibitors. In addition, an improved understanding of the differential roles of CaMKII subtypes is required.
... The mechanism by which a decreased LVEF results in QT prolongation is unknown, but may involve ion-channel remodelling and/or intracellular calcium transport. 32 LVEF is frequently used to prognosticate patients who have suffered a myocardial infarction or who have dilated cardiomyopathy. It is generally accepted that patients with an impaired systolic function have a higher risk for sudden cardiac death, presumably due to malignant ventricular tachydysrhythmias. ...
QT prolongation on the surface ECG is associated with sudden cardiac death. The cause of QT prolongation in ischaemic heart disease (IHD) patients remains unknown, but may be due to a complex interplay between genetic factors and impaired systolic and/or diastolic function through as yet unexplained mechanisms. It was hypothesised that QT prolongation before elective coronary angiography is associated with an increased mortality at six months.
Complete records of 321 patients who underwent coronary angiography were examined for QT interval corrected for heart rate (QTc), left ventricular ejection fraction (LVEF), left ventricular end-diastolic pressure (LVEDP) and known ischaemic heart disease risk factors. Patients were designated long QTc (LQTc) when they had prolonged QTc intervals or normal QTc (NQTc) when the QTc interval was normal. Patients with atrial fibrillation, bundle branch blocks, no ECG in the 24 hours before angiography, or a creatinine level > 200 µmol/l were excluded. Survival was determined telephonically at six months.
Twenty-eight per cent of the total population had LQTc. During follow up, 15 patients (4.7%) died suddenly, 73% of whom had a LQTc. LQTc was significantly associated with mortality (LQTc 12% vs NQTc 1.7%; p < 0.01), and with lower but normal LVEF (LQTc 52.9 ± 15.4% vs NQTc 61.6 ± 13.6%; p < 0.01), higher LVEDP at LVEF > 45% (LQTc 19.2 ± 9.0 mmHg vs NQTc 15.95 ± 7.5 mmHg; p < 0.05), hypercholesterolaemia and a negative family history of IHD.
In patients with sinus rhythm and normal QRS width, QTc prolongation before coronary angiography predicted increased mortality at six months. QTc also associated strongly with left ventricular systolic and diastolic dysfunction, hypercholesterolaemia and a negative family history of IHD.
... 72 CaMKII inhibition has also been shown to be effective in rabbit chronic atrioventricular block models of acquired long-QT and electrical storm. 61,165 The antiarrhythmic benefit of CaMKII inhibition also has been explored in ischemic heart disease. Importantly, transgenic inhibition of CaMKII prevents structural remodeling after myocardial infarction. ...
Understanding relationships between heart failure and arrhythmias, important causes of suffering and sudden death, remains an unmet goal for biomedical researchers and physicians. Evidence assembled over the past decade supports a view that activation of the multifunctional Ca(2+) and calmodulin-dependent protein kinase II (CaMKII) favors myocardial dysfunction and cell membrane electrical instability. CaMKII activation follows increases in intracellular Ca(2+) or oxidation, upstream signals with the capacity to transition CaMKII into a Ca(2+) and calmodulin-independent constitutively active enzyme. Constitutively active CaMKII appears poised to participate in disease pathways by catalyzing the phosphorylation of classes of protein targets important for excitation-contraction coupling and cell survival, including ion channels and Ca(2+) homeostatic proteins, and transcription factors that drive hypertrophic and inflammatory gene expression. This rich diversity of downstream targets helps to explain the potential for CaMKII to simultaneously affect mechanical and electrical properties of heart muscle cells. Proof-of-concept studies from a growing number of investigators show that CaMKII inhibition is beneficial for improving myocardial performance and for reducing arrhythmias. We review the molecular physiology of CaMKII and discuss CaMKII actions at key cellular targets and results of animal models of myocardial hypertrophy, dysfunction, and arrhythmias that suggest CaMKII inhibition may benefit myocardial function while reducing arrhythmias.
... 19,20 I CaL recovery from inactivation, enhanced NCX and CaMKII phosphorylation of Ca 2+ channels all contribute to EAD occurrence. 21 Detailed role of Ca 21 -related processes in re-entrant mechanisms underlying atrial fibrillation ...
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, and its prevalence is increasing with the ageing of the population. Presently available treatment options are far from optimal and new insights into underlying mechanisms are needed to improve therapy. A variety of recent lines of research are converging to reveal important and relatively underappreciated multidimensional roles of cellular Ca(2+) content, distribution, and handling in AF pathophysiology. The objective of the present paper is to review the participation of changes in cell Ca(2+) and related processes in the mechanisms that lead to AF initiation and maintenance, and to consider the relevance of new knowledge in this area to therapeutic innovation. We first review the involvement of Ca(2+)-related functions in the principal arrhythmia mechanisms underlying AF: focal ectopic activity due to afterdepolarizations and re-entrant mechanisms. The detailed molecular pathophysiology of focal ectopic and re-entrant activity is then discussed in relationship to the participation of cell Ca(2+) changes and related Ca(2+)-handling and Ca(2+)-sensitive signalling systems. We then go on to consider the participation of Ca(2+)-related functions in electrical and structural remodelling processes leading to the AF substrate. Finally, we consider the implications for development of new arrhythmia management approaches and future research and development.
... By constructing steady-state activation and inactivation curves, we found that H 2 O 2 produced an ∼−5 mV shift of the steady state half-activation potential (V 1/2,act ), accompanied by an ∼+5 mV shift in the steady state half-inactivation potential (V 1/2,inact ) (Fig. 2D). Effectively, H 2 O 2 increased the height of the I Ca,L window current region, changes which are expected to promote EAD formation (Antoons et al. 2007b;Qi et al. 2009;Tran et al. 2009). ...
Sudden cardiac death (SCD) due to ventricular fibrillation (VF) is a major world-wide health problem. A common trigger of VF involves abnormal repolarization of the cardiac action potential causing early after depolarizations (EADs). Here we used a hybrid biological-computational approach to investigate the dependence of EADs on the biophysical properties of the L-type Ca2+ current (I-Ca,I-L) and to explore how modifications of these properties could be designed to suppress EADs. EADs were induced in isolated rabbit ventricular myocytes by exposure to 600 mu mol l(-1) H2O2 (oxidative stress) or lowering the external [K+] from 5.4 to 2.0-2.7 mmol l(-1) (hypokalaemia). The role of I-Ca,I-L in EAD formation was directly assessed using the dynamic clamp technique: the paced myocyte's V-m was input to a myocyte model with tunable biophysical parameters, which computed a virtual I-Ca,I-L, which was injected into themyocyte in real time. This virtual current replaced the endogenous I-Ca,I-L, which was suppressed with nifedipine. Injecting a current with the biophysical properties of the native I-Ca,I-L restored EAD occurrence in myocytes challenged by H2O2 or hypokalaemia. A mere 5 mV depolarizing shift in the voltage dependence of activation or a hyperpolarizing shift in the steady-state inactivation curve completely abolished EADs in myocytes while maintaining a normal Ca-i transient. We propose thatmodifying the biophysical properties of I-Ca,I-L has potential as a powerful therapeutic strategy for suppressing EADs and EAD-mediated arrhythmias.
Calmodulin kinase II (CaMKII) is expressed in tissues throughout the body with essential roles in a wide variety of cellular functions from synaptic transmission in neurons to solute absorption in the epithelium to excitation–contraction coupling in cardiac myocytes. Importantly, CaMKII activity is sensitive to a variety of physiologically relevant stimuli, including intracellular Ca2+ and reactive oxygen species. In cardiomyocytes, CaMKII targets a host of intracellular substrates, including ion channels, Ca2+ cycling proteins, and transcription factors to regulate cardiac contractility, pacemaking, and electrical conduction. This chapter discusses the multiple roles of CaMKII in the heart and its emergence as an important determinant of the heart’s response to both acute and chronic stress relevant to normal physiology as well as disease.
Key points:
At the cellular level cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and intracellular Ca2+ release amplitude and represents a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricle are poorly understood. We investigated if pharmacological modulation of SK channels affects the development of cardiac alternans in normal ventricular cells and in cells with drug-induced long QT syndrome (LQTS). While SK channel blockers have only a minor effect on AP morphology, their activation leads to AP shortening and abolishes or reduces the degree of pacing induced Ca2+ and AP alternans. AP shortening contributed to protection against alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest SK activation as a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention for patients with LQTS.
Abstract:
At the cellular level cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and Ca2+ transient (CaT) amplitude and represents a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricle are poorly understood. We tested the hypothesis that in single rabbit ventricular myocytes pharmacological modulation of SK channels plays a causative role for the development of pacing-induced CaT and AP duration (APD) alternans. SK channel blockers (apamin, UCL1684) had only a minor effect on AP repolarization. However, SK channel activation by NS309 resulted in significant APD shortening, demonstrating that functional SK channels are well expressed in ventricular myocytes. Effects of NS309 were prevented or reversed by apamin and UCL1684, indicating that NS309 acted on SK channels. SK channel activation abolished or reduced the degree of pacing induced CaT and APD alternans. Inhibition of KV 7.1 (with HMR1556) and KV 11.1 (with E4031) channels was used to mimic conditions of long QT syndromes type-1 and type-2, respectively. Both HMR1556 and E4031 enhanced CaT alternans that was prevented by SK channel activation. In AP voltage-clamped cells the SK channel activator had no effect on CaT alternans, confirming that suppression of CaT alternans was caused by APD shortening. APD shortening contributes to protection from alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest SK activation as a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy for patients with LQT syndrome. Abstract figure legend SK channel activation shortens ventricular action potentials and abolishes CaT and APD alternans. A: (a) Activation of SK channels by NS309 (2 μM) leads to APD shortening. (b) APD70 recorded from an individual ventricular myocyte during the course of an experiment where APD shortening caused by NS309 was eliminated by application of apamin (100 nM) demonstrating that effect of NS309 is elicited due to activation of SK channels. B: AP and CaT traces simultaneously recorded from the same current-clamped ventricular myocyte in control, in presence of SK channel activator NS309 (2 μM) and after washout of NS309. Activation of SK channels abolishes CaT and APD alternans. C: Activation of SK channels reduced risk of alternans in ventricular cells with drug-induced LQTS. (a) CaT alternans observed in control and in the presence of KV 7.1 channel blocker HMR1556 (1 μM), subsequently were abolished by NS309 application in the presence of HMR1556. (b) Mean and individual cell CaT alternans ratios recorded from field stimulated ventricular myocytes in control, in the presence of HMR1556, followed by simultaneous application of HMR1556 and NS309. This article is protected by copyright. All rights reserved.
The rabbit has been and continues to be a vital resource for biomedical research. This chapter focuses on the role of rabbits in toxicologic research and related biomedical studies. First, the rabbit is placed in context compared to other animal models; aspects where rabbits closely match human physiology and/or anatomy are highlighted. Basic rabbit physiological and anatomic features are discussed. This is followed by a review of the different classes of studies in which rabbits are a commonly used species (e.g., medical device, regenerative medicine, development and reproductive toxicity studies). Finally, key species-specific microscopic findings will be highlighted for the rabbit, as well as unique tissues/structures that could be misidentified.
Several false-positive results in the human ether-à-gogo-related gene test suggest that blockers of the rapid component of delayed rectifier K⁺ current (IKr) do not necessarily produce drug-induced arrhythmias. Specifically, the occurrence of early afterdepolarization (EAD) differs among IKr blockers, even if the prolonged action potential duration is in the same range. To predict EAD in drug-induced arrhythmias, we proposed a prediction method based on the mechanisms underlying the difference in frequency of EAD among nonselective IKr blockers. The mechanisms were elucidated by examining how different blockade kinetics of L-type Ca²⁺ current (ICaL) affect the frequency of EAD, using mathematical models of human ventricular myocytes. Addition of voltage-independent ICaL blockade resulted in the suppression of EAD. However, when voltage-dependent ICaL blockade kinetics of amiodarone, bepridil, and terfenadine were incorporated into ICaL in the model, bepridil and terfenadine induced EAD more than the voltage-independent ICaL blockade, while amiodarone suppressed EAD more effectively. Opposite effects were accounted for by the difference in ICaL blockade at negatively polarized potential. EAD occurrence was found to be associated with ICaL blockade measured at -20 mV. These results suggest that voltage dependence of ICaL blockade may be useful in predicting the different risks of nonselective IKr blockers.
Acquired long QT syndrome is an arrhythmogenic syndrome which is associated with life-threatening ventricular tachyarrhythmias termed as torsades de pointes and sudden death as the consequence of QT prolongation after exposure to secondary factors, e.g., drugs; electrolyte abnormalities including hypokalemia, hypomagnesemia, or hypocalcemia; and marked bradycardia. The clinical findings of QT prolongation on electrocardiogram (ECG) and torsades de pointes have been considered to appear unexpectedly after exposure to secondary factors. Prolonged QT interval generally becomes normalized after removal of secondary factors, although the QT interval may sometimes remain mildly prolonged even without secondary factors. We need to take account of a latent genetic background that plays a role in this syndrome because significant QT prolongation could be revealed only in some of patients who have been exposed to the same secondary factors. When we manage those with a potential risk for such a syndrome, we should not miss any subtle alert from patients in the presence of secondary factors. What we have to keep in mind is that keeping alert for this syndrome is critically important not only to cardiologists but also to all of other physicians for the prevention of unexpected sudden.
Objective To investigate the effect of the calmodulin kinase II Inhibitor KN-93 on L-typecalcium current (I Ca,L) and intracellular calcium concentration ([ Ca 2+] 1) in hypertrophic cardiac myocytes. Methods Forty-eight female New Zealand white rabbits were randomized (random number) into four groups (12 animals in each group): the sham operation group (sham group), the left ventricular hypertrophy group (LVH group), the myocardial hypertrophy + KN-93 group (KN-93 group), and the myocardial hypertrophy + KN-92 group (KN-92 group). Myocardial hypertrophy in the rabbits was established by coarctation of the abdominal aorta. In the sham group, the abdominal aorta was dissociated without coarctation. Eight weeks after coarctation, single ventricular myocytes were isolated by enzymatic dissociation, and I Ca,L was recorded using perforated-patch recording ( PPR) techniques. [ Ca 2+] 1 was measured using single-cell calcium imaging with the fluorescence calcium indicator dye fura-2/AM. Results Cardiac hypertrophy was successfully established after 8 weeks of coarctation of the abdominal aorta. The peak I Ca,L in the LVH group and the sham group was (1.38 ± 0.3) nA and (0.87 ± 0.1) nA at 0 mV, respectively (P < 0.01, n = 12) . There was no significant difference in I Ca,L, L density between the LVH group and the sham group [(6.7 ± 1.0) pA/pF vs. (6.3 ± 0.7) pA/pF; P ≥ O.05, n = 12]. The addition of either KN-92 (0.5 μmol/L) or KN-93 (0.5 μmol/L) to the perfusing solution caused a modest steady-state inhibition of peak ICaL (9.4% ± 2.8%, KN-92; 10.5% ± 3%, KN-93) (P≥0.05, n = 12) at 0 mV. However, at a higher concentration ( 1 μmol/L), KN-93 more potently inhibited peak I Ca,L (40% ± 4.9%) compared to KN-92 (13.4% ± 3.7%; P < 0.01, n = 12). Resting [Ca 2+] 1, levels in fura-2-loaded myocytes isolated from the sham, LVH, KN-92, and KN-93 groups were (98 ± 12.3) nmol/L, (154 ± 26.2) nmol/L, (147 ± 29.6) nmol/L, and (108 ± 21.2) nmol/L, respectively. Conclusions The CaMK II specific inhibitor, KN-93 , can effectively block I Ca,L and reduce intracellular calcium overload in hypertrophic cardiac myocytes. This action may account for the antiarrhythmic effect of KN-93 in hypertrophic ventricular myocardium.
Rationale:
Loss-of-function mutations in human ether go-go (HERG) potassium channels underlie long QT syndrome type 2 (LQT2) and are associated with fatal ventricular tachyarrhythmia. Previously, most studies focused on plasma membrane-related pathways involved in arrhythmogenesis in long QT syndrome, whereas proarrhythmic changes in intracellular Ca(2+) handling remained unexplored.
Objective:
We investigated the remodeling of Ca(2+) homeostasis in ventricular cardiomyocytes derived from transgenic rabbit model of LQT2 to determine whether these changes contribute to triggered activity in the form of early after depolarizations (EADs).
Methods and results:
Confocal Ca(2+) imaging revealed decrease in amplitude of Ca(2+) transients and sarcoplasmic reticulum Ca(2+) content in LQT2 myocytes. Experiments using sarcoplasmic reticulum-entrapped Ca(2+) indicator demonstrated enhanced ryanodine receptor (RyR)-mediated sarcoplasmic reticulum Ca(2+) leak in LQT2 cells. Western blot analyses showed increased phosphorylation of RyR in LQT2 myocytes versus controls. Coimmunoprecipitation experiments demonstrated loss of protein phosphatases type 1 and type 2 from the RyR complex. Stimulation of LQT2 cells with β-adrenergic agonist isoproterenol resulted in prolongation of the plateau of action potentials accompanied by aberrant Ca(2+) releases and EADs, which were abolished by inhibition of Ca(2+)/calmodulin-dependent protein kinase type 2. Computer simulations showed that late aberrant Ca(2+) releases caused by RyR hyperactivity promote EADs and underlie the enhanced triggered activity through increased forward mode of Na(+)/Ca(2+) exchanger type 1.
Conclusions:
Hyperactive, hyperphosphorylated RyRs because of reduced local phosphatase activity enhance triggered activity in LQT2 syndrome. EADs are promoted by aberrant RyR-mediated Ca(2+) releases that are present despite a reduction of sarcoplasmic reticulum content. Those releases increase forward mode Na(+)/Ca(2+) exchanger type 1, thereby slowing repolarization and enabling L-type Ca(2+) current reactivation.
Sudden cardiac death occurs due to a limited number of pathological events. The heart can beat too fast or too slow to maintain adequate cardiac output or the heart can rupture. Here we survey recent evidence that excessive activation of calcium calmodulin-dependent protein kinase II by three core neurohumoral pathways or by oxidant stress can lead to sudden cardiac death due to sinus node dysfunction and bradycardia, ventricular tachycardia or fibrillation, and cardiac rupture.
This article reviews the manner in which age and gender affect electrocardiographic measurements of repolarization and therefore the QT interval. The authors detail the effects of age and gender on clinical outcomes in long QT syndrome and review how these modulations drive treatment recommendations. They also discuss basic research that has recently begun to shed light on these clinical phenomena and may give insight into potential future treatments.
The contribution of transient outward current (Ito) to changes in ventricular action potential (AP) repolarization induced by acidosis is unresolved as is the indirect effect of these changes on calcium handling. To address this issue we measured intracellular pH (pHi), Ito, L-type calcium current (ICa,L), and calcium transients (CaT) in rabbit ventricular myocytes. Intracellular acidosis (pHi 6.75 with pHo 7.4) reduced Ito by approximately fifty percent in myocytes with both high (epicardial) and low (papillary muscle) Ito densities with little effect on steady-state inactivation and activation. Of the two candidate alpha subunits underlying Ito, hKv4.3 and hKv1.4, only hKv4.3 current was reduced by intracellular acidosis. Extracellular acidosis (pHo 6.5) shifted Ito inactivation towards less negative potentials but had negligible effect on peak current at +60 mV when initiated from -80 mV. The effects of low pHi-induced inhibition of Ito on AP repolarization were much greater in epicardial than papillary muscle myocytes and included slowing of phase 1, attenuation of the notch, and elevation of the plateau. Low pHi increased AP duration in both cell types with the greatest lengthening occurring in epicardial myocytes. The changes in epicardial AP repolarization induced by intracellular acidosis reduced peak ICa,L, increased net calcium influx via ICa,L, and increased CaT amplitude. Summary: In contrast to low pHo, intracellular acidosis has a marked inhibitory effect on ventricular Ito, perhaps mediated by Kv4.3. By altering the trajectory of the AP repolarization, low pHi has a significant indirect effect on calcium handling, especially evident in epicardial cells.
The implantable cardioverter-defibrillator significantly improves survival in patients with malignant ventricular arrhythmias but does not target the underlying pathological substrate responsible for arrhythmic events. A significant proportion of defibrillator recipients experience multiple ventricular tachycardia/fibrillation episodes over a short period of time, termed electrical storm (ES). The current therapeutic strategy for ES is complex and unsatisfactory because simultaneous administration of several medications and additional invasive procedures are often required to control ES. Moreover, this treatment does not favorably influence the long-term outcome. Clearly, improved ES therapies are necessary and desirable, but a lack of understanding of the pathophysiological mechanisms underlying ES has hindered the development of more effective, rationally based therapeutic approaches. This paper reviews emerging experimental and clinical findings that provide insights into the pathophysiology of ES and discusses mechanism-based innovative therapeutic strategies.
Cardiac arrhythmias are a major epidemiological and public health problem and contribute significantly to sudden cardiac death, heart failure, stroke, suffering, debilitation, and healthcare expenses. In the United States alone, sudden cardiac death is estimated to kill 250 000 to 400 000 people annually.1 Most sudden death is due to cardiac arrhythmias,2 with ventricular tachycardia and fibrillation as the most commonly (≈80%) recorded rhythms in out-of-hospital cardiac arrests.3 In patients with structural heart disease, mostly resulting from a history of myocardial infarction, arrhythmias are the main cause of death.4 Atrial fibrillation (AF) and sinus node dysfunction (SND) are the most common sustained arrhythmias. AF affects ≈2.3 million patients in the United States,5 and because the prevalence of AF increases with age, it is predicted to increase by 2.5-fold by 2050.6 Patients with AF have approximately twice the mortality rate of patients in sinus rhythm,6 and the incidence of stroke is increased by 2- to 7-fold.7 AF is a costly disease and causes a public health burden estimated at $6.0 to $26.0 billion annually in the United States.8 SND is associated with increased sudden cardiac death, particularly in patients with heart failure, and a large portion (≈40%) of mortality in hospitalized patients with heart failure may be secondary to SND.9 SND is the indication for ≈60% of the 180 000 pacemakers implanted in the United States each year, a procedure that in 2004 accounted for ≈$2 billion in expenses.10,11 The negative impact of arrhythmias on human health and medical economics is a major motivating factor for establishing new and effective therapeutic approaches.
Cardiac arrhythmias are the result of cell membrane hyperexcitability (the cause of automatic and triggered tachyarrhythmias), defective impulse formation (the cause of SND), or reduction in …
Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related. The aim of this study was to determine the effects of the calmodulin-dependent protein kinase (CaMK) II inhibitor, KN-93, on L-type calcium current (I(Ca, L)) and early after-depolarizations (EADs) in hypertrophic cardiomyocytes. A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation (LVH group). The control group (sham group) received a sham operation, in which the abdominal aortic was dissected but not coarcted. Eight weeks later, the degree of left ventricular hypertrophy (LVH) was evaluated using echocardiography. Individual cardiomyocyte was isolated through collagenase digestion. Action potentials (APs) and I(Ca, L) were recorded using the perforated patch clamp technique. APs were recorded under current clamp conditions and I(Ca, L) was recorded under voltage clamp conditions. The incidence of EADs and I(ca, L) in the hypertrophic cardiomyocytes were observed under the conditions of low potassium (2 mmol/L), low magnesium (0.25 mmol/L) Tyrode's solution perfusion, and slow frequency (0.25-0.5 Hz) electrical stimulation. The incidence of EADs and I(ca, L) in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92 (KN-92 group) and KN-93 (KN-93 group). Eight weeks later, the model was successfully established. Under the conditions of low potassium, low magnesium Tyrode's solution perfusion, and slow frequency electrical stimulation, the incidence of EADs was 0/12, 11/12, 10/12, and 5/12 in sham group, LVH group, KN-92 group (0.5 μmol/L), and KN-93 group (0.5 μmol/L), respectively. When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group, the incidence of EADs was 10/12 and 2/12, respectively. At 0 mV, the current density was 6.7±1.0 and 6.3±0.7 PA·PF(-1) in LVH group and sham group, respectively (P>0.05, n=12). When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups, the peak I(Ca, L) at 0 mV was decreased by (9.4±2.8)% and (10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups, respectively (P>0.05, n=12). When the drug concentration was increased to 1 μmol/L, the peak I(Ca, L) values were lowered by (13.4±3.7)% and (40±4.9)%, respectively (P<0.01, n=12). KN-93, a specific inhibitor of CaMKII, can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I(Ca, L), which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.
Background:
The calcium-dependent signaling molecules calcineurin and calcium/calmodulin-dependent protein kinase II (CaMKII) both have been linked to decompensated hypertrophy and arrhythmias. CaMKII is also believed to be involved in acute modulation of ion channels.
Objective:
The purpose of this study was to determine the role of calcineurin and CaMKII in a dog model of compensated hypertrophy and a long QT phenotype.
Methods:
AV block was created in dogs to induce ventricular remodeling, including enhanced susceptibility to dofetilide-induced torsades de pointes arrhythmias. Dogs were treated with cyclosporin A for 3 weeks, which reduced calcineurin activity, as determined by mRNA expression levels of regulator of calcineurin 1 exon 4, but which was unable to prevent structural, contractile, or electrical remodeling and arrhythmias. Biopsies were taken before and at 2 or 9 weeks after AV block. Western blots were performed against phosphorylated and total CaMKII, phospholamban, Akt, and histone deacetylase 4 (HDAC4).
Results:
Chronic AV block showed an increase in Akt, CaMKII and phospholamban phosphorylation levels, but HDAC4 phosphorylation remained unaltered. Dofetilide induced torsades de pointes in vivo and early afterdepolarizations in cardiomyocytes, and increased [Ca(2+)](i) and CaMKII autophosphorylation. Both W-7 and KN-93 treatment counteracted this.
Conclusion:
The calcineurin pathway seems not to be involved in long-term cardiac remodeling of the chronic AV block dog. Although CaMKII is chronically activated, this does not translate to HDAC4 phosphorylation. However, acute CaMKII overactivation is able to initiate arrhythmias based on triggered activity.
The calmodulin kinase II (CaMKII) signaling pathway regulates divergent functions in heart including calcium cycling, pacemaking,
gene transcription, hypertrophy, and apoptosis. The unique structural and biophysical properties of CaMKII enable the kinase
to respond to critical intracellular second messengers important in pathophysiology including Ca2+ and reactive oxygen species. Precise localization of the kinase with a large number of intracellular substrates allows the
kinase to tightly regulate cell function. Importantly, studies in human patients and animal models clearly indicate a role
for CaMKII signaling in decreased cardiac function and arrhythmias in the diseased heart. Specifically, CaMKII dysfunction
occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII
inhibition prevents cardiac arrhythmia, improves heart function following myocardial infarction, and prevents the progression
of disease. Thus, the CaMKII signaling pathway represents an emerging candidate for targeted therapeutic intervention to preserve
myocardial function and prevent arrhythmias in heart disease.
Varieties of small animal models for arrhythmia study have been created using genetic and nongenetic techniques. However,
data obtained from small animals must be interpreted carefully compared to larger animals such as dogs or pigs, because some
electrophysiological properties of small animals such as constituents of action potentials are different from those of humans.
This chapter briefly describes various small animal nongenetic models such as the myocardial infarction model and chronic
atrioventricular block model as well as genetically engineered small animal models such as long QT syndrome and catecholaminergic
polymorphic ventricular tachycardia.
Membrane ion channels and transporters are key determinants of cardiac electrical function. Their expression is affected by cardiac region, hemodynamic properties, heart-rate changes, neurohormones and cardiac disease. One of the important determinants of ion-channel function is the level of ion-channel subunit mRNA expression, which governs the production of ion-channel proteins that traffic to the cell-membrane to form functional ion-channels. Ion-channel mRNA-expression profiling can be performed with cDNA microarrays or high-throughput reverse transcription/polymerase chain reaction (PCR) methods. Expression profiling has been applied to evaluate the dependence of ion-channel expression on cardiac region, revealing the molecular basis of regionally-controlled electrical properties as well as the molecular determinants of specialized electrical functions like pacemaking activity. Ion-channel remodeling occurs with cardiac diseases like heart failure, congenital repolarization abnormalities, and atrial fibrillation, and expression profiling has provided insights into the mechanisms by which these conditions affect cardiac electrical stability. Expression profiling has also shown how hormonal changes, antiarrhythmic drugs, cardiac development and altered heart rate affect ion-channel expression patterns to modify cardiac electrical function and sometimes to produce cardiac rhythm disturbances. This article reviews the information obtained to date with the application of cardiac ion-channel expression profiling. With increasing availability and efficiency of high-throughput PCR methods for ion-channel subunit mRNA-expression characterization, it is likely that the application of ion-channel expression profiling will increase and that it will provide important new insights into the determinants of cardiac electrical function in both physiological and pathological situations.
The search for alternative anti-arrhythmic strategies is fueled by an unmet medical need as well as by the opportunities arising from identification of novel targets and novel drugs. Na/Ca exchange is a potential target involved in several types of arrhythmias, such as those related to ischemia-reperfusion, heart failure and also some forms of genetic arrhythmias. Inhibition of Na/Ca exchange is theoretically not only anti-arrhythmic but also increases cellular Ca(2+) content. This could be an advantage in conditions of low inotropy, such as in heart failure, but may also worsen conditions such as the recovery from ischemia or relaxation abnormalities. With the available drugs such as KB-R7943 and SEA-0400 these theories have now been tested in a number of cellular and in vivo models. Experience is overall rather positive and seems less hampered by the potential drawbacks than expected. This may be because the currently available drugs are not highly selective, with additional benefit derived from concurrent effects. While this precludes a definite answer regarding the benefit of a pure NCX inhibitor, they indicate that Na/Ca exchange inhibition as part of a multi-target strategy is an avenue to be considered. Such studies will need further 'bench' work and testing in relevant preclinical models, including chronic disease.
The ionic basis of acquired QT prolongation and torsade de pointes (TdP) unrelated to drugs is not fully understood.
We created a rabbit model with chronic complete atrioventricular block (AVB) (n=34), which showed prominent QT prolongation (by 120%), high incidence of spontaneous TdP (71%), and cardiac hypertrophy. Patch-clamp experiments were performed in left ventricular myocytes from 9 rabbits (8 with TdP, 1 without TdP) at approximately 21 days of AVB and from 8 sham-operated controls with sinus rhythm. Action potential duration was prolonged in AVB myocytes compared with control (+61% at 0.5 Hz, +21% at 3 Hz). Both rapidly and slowly activating components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in AVB myocytes were significantly smaller than in control by 50% and 55%, respectively. There was no significant difference in Ca(2+)-independent transient outward current (I(to1)). L-type Ca(2+) current (I(Ca,L)) in control and AVB myocytes was similar in peak amplitude, but the half voltage for activation was shifted to the negative direction (5.9 mV) in AVB myocytes. Voltage dependence of I(Ca,L) inactivation was not different in control and AVB myocytes. The inward rectifier K(+) current (I(K1)) significantly increased in AVB myocytes compared with control.
In the rabbit, chronic AVB leads to prominent QT prolongation and high incidence of spontaneous TdP. Downregulation of both I(Kr) and I(Ks) in association with altered I(Ca,L) activation kinetics may underlie the arrhythmogenic ventricular remodeling.
beta(1)-adrenergic receptor (beta(1)AR) stimulation activates the classic cAMP/protein kinase A (PKA) pathway to regulate vital cellular processes from the change of gene expression to the control of metabolism, muscle contraction, and cell apoptosis. Here we show that sustained beta(1)AR stimulation promotes cardiac myocyte apoptosis by activation of Ca(2+)/calmodulin kinase II (CaMKII), independently of PKA signaling. beta(1)AR-induced apoptosis is resistant to inhibition of PKA by a specific peptide inhibitor, PKI14-22, or an inactive cAMP analogue, Rp-8-CPT-cAMPS. In contrast, the beta(1)AR proapoptotic effect is associated with non-PKA-dependent increases in intracellular Ca(2+) and CaMKII activity. Blocking the L-type Ca(2+) channel, buffering intracellular Ca(2+), or inhibiting CaMKII activity fully protects cardiac myocytes against beta(1)AR-induced apoptosis, and overexpressing a cardiac CaMKII isoform, CaMKII-deltaC, markedly exaggerates the beta(1)AR apoptotic effect. These findings indicate that CaMKII constitutes a novel PKA-independent linkage of beta(1)AR stimulation to cardiomyocyte apoptosis that has been implicated in the overall process of chronic heart failure.
Cardiac tissue in the pulmonary vein sleeves plays an important role in clinical atrial fibrillation. Mechanisms leading to pulmonary vein activity in atrial fibrillation remain unclear. Indirect experimental evidence points to pulmonary vein Ca(2+) handling as a potential culprit, but there are no direct studies of pulmonary vein cardiomyocyte Ca(2+) handling in the literature. We used the Ca(2+)-sensitive dye indo-1 AM to study Ca(2+) handling in isolated canine pulmonary vein and left atrial myocytes. Results were obtained at 35 degrees C and room temperature in cells from control dogs and in cardiomyocytes from dogs subjected to 7-day rapid atrial pacing. We found that basic Ca(2+)-transient properties (amplitude: 186 +/- 28 vs. 216 +/- 25 nM; stimulus to half-decay time: 192 +/- 9 vs. 192 +/- 9 ms; atria vs. pulmonary vein, respectively, at 1 Hz), beat-to-beat regularity, propensity to alternans, beta-adrenergic response (amplitude increase at 0.4 Hz: 96 +/- 52 vs. 129 +/- 61%), number of spontaneous Ca(2+)-transient events after Ca(2+) loading (in normal Tyrode: 0.9 +/- 0.2 vs. 1.3 +/- 0.2; with 1 microM isoproterenol: 7.6 +/- 0.3 vs. 5.1 +/- 1.8 events/min), and caffeine-induced Ca(2+)-transient amplitudes were not significantly different between atrial and pulmonary vein cardiomyocytes. In an arrhythmia-promoting model (dogs subjected to 7-day atrial tachypacing), Ca(2+)-transient amplitude and kinetics were the same in cells from both pulmonary veins and atrium. In conclusion, the similar Ca(2+)-handling properties of canine pulmonary vein and left atrial cardiomyocytes that we observed do not support the hypothesis that intrinsic Ca(2+)-handling differences account for the role of pulmonary veins in atrial fibrillation.
Torsade de pointes (TdP) is a life-threatening arrhythmia that develops as a consequence of a reduction in the repolarization reserve of cardiac cells leading to amplification of electrical heterogeneities in the ventricular myocardium as well as to the development of early after depolarization-induced triggered activity. Electrical heterogeneities within the ventricles are due to differences in the time course of repolarization of the three predominant cell types that make up the ventricular myocardium, giving rise to transmural voltage gradients and a dispersion of repolarization that contributes to the inscription of the electrocardiographic T wave. A number of non-antiarrhythmic drugs and antiarrhythmic agents with class III actions and/or the various mutations and cardiomyopathies associated with the long QT syndrome reduce net repolarizing current and amplify spatial dispersion of repolarization, thus creating the substrate for re-entry. This results in a prolongation of the QT interval, abnormal T waves, and development of TdP. Agents that prolong the QT interval but do not cause an increase in transmural dispersion of repolarization (TDR) do not induce TdP, suggesting that QT prolongation is not the sole or optimal determinant for arrhythmogenesis. This article reviews recent advances in our understanding of these mechanisms, particularly the role of TDR in the genesis of drug-induced TdP, and examines how these may guide us towards development of safer drugs.
The recently described exchange protein directly activated by cAMP (Epac) has been implicated in distinct protein kinase A-independent cellular signalling pathways. We investigated the role of Epac activation in adrenergically mediated ventricular arrhythmogenesis. In contrast to observations in control conditions (n = 20), monophasic action potentials recorded in 2 of 10 intrinsically beating and 5 of 20 extrinsically paced Langendorff-perfused wild-type murine hearts perfused with the Epac activator 8-pCPT-2′-O-Me-cAMP (8-CPT, 1 μM) showed spontaneous triggered activity. Three of 20 such extrinsically paced hearts showed spontaneous ventricular tachycardia (VT). Programmed electrical stimulation provoked VT in 10 of 20 similarly treated hearts (P < 0.001; n = 20). However, there were no statistically significant accompanying changes (P > 0.05) in left ventricular epicardial (40.7 ± 1.2 versus 44.0 ± 1.7 ms; n = 10) or endocardial action potential durations (APD90; 51.8 ± 2.3 versus 51.9 ± 2.2 ms; n = 10), transmural (ΔAPD90) (11.1 ± 2.6 versus 7.9 ± 2.8 ms; n = 10) or apico-basal repolarisation gradients, ventricular effective refractory periods (29.1 ± 1.7 versus 31.2 ± 2.4 ms in control and 8-CPT-treated hearts, respectively; n = 10) and APD90 restitution characteristics. Nevertheless, fluorescence imaging of cytosolic Ca2+ levels demonstrated abnormal Ca2+ homeostasis in paced and resting isolated ventricular myocytes. Epac activation using isoproterenol in the presence of H-89 was also arrhythmogenic and similarly altered cellular Ca2+ homeostasis. Epac-dependent effects were reduced by Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition with 1 μM KN-93. These findings associate VT in an intact cardiac preparation with altered cellular Ca2+ homeostasis and Epac activation for the first time, in the absence of altered repolarisation gradients previously implicated in reentrant arrhythmias through a mechanism dependent on CaMKII activity.
Background— The ionic basis of acquired QT prolongation and torsade de pointes (TdP) unrelated to drugs is not fully understood.
Methods and Results— We created a rabbit model with chronic complete atrioventricular block (AVB) (n=34), which showed prominent QT prolongation (by 120%), high incidence of spontaneous TdP (71%), and cardiac hypertrophy. Patch-clamp experiments were performed in left ventricular myocytes from 9 rabbits (8 with TdP, 1 without TdP) at ≈21 days of AVB and from 8 sham-operated controls with sinus rhythm. Action potential duration was prolonged in AVB myocytes compared with control (+61% at 0.5 Hz, +21% at 3 Hz). Both rapidly and slowly activating components of the delayed rectifier K⁺ current (IKr and IKs) in AVB myocytes were significantly smaller than in control by 50% and 55%, respectively. There was no significant difference in Ca²⁺-independent transient outward current (Ito1). L-type Ca²⁺ current (ICa,L) in control and AVB myocytes was similar in peak amplitude, but the half voltage for activation was shifted to the negative direction (5.9 mV) in AVB myocytes. Voltage dependence of ICa,L inactivation was not different in control and AVB myocytes. The inward rectifier K⁺ current (IK1) significantly increased in AVB myocytes compared with control.
Conclusions— In the rabbit, chronic AVB leads to prominent QT prolongation and high incidence of spontaneous TdP. Downregulation of both IKr and IKs in association with altered ICa,L activation kinetics may underlie the arrhythmogenic ventricular remodeling.
Objective:
Persistent atrial fibrillation (AF) results in an impairment of atrial function. In order to elucidate the mechanism behind this phenomenon, we investigated the gene expression of proteins influencing calcium handling.
Methods:
Right atrial appendages were obtained from eight patients with paroxysmal AF, ten with persistent AF (> 8 months) and 18 matched controls in sinus rhythm. All controls underwent coronary artery bypass grafting, whereas most AF patients underwent Cox's MAZE surgery (n = 12). All patients had a normal left ventricular function. Total RNA was isolated and reversely transcribed into cDNA. In a semi-quantitative polymerase chain reaction the cDNA of interest and of glyceraldehyde-3-phosphate dehydrogenase were coamplified and separated by ethidium bromide-stained gel electrophoresis. Slot blot analysis was performed to study protein expression.
Results:
L-type calcium channel alpha 1 and sarcoplasmic reticulum Ca(2+)-ATPase mRNA (-57%, p = 0.01 and -28%, p = 0.04, respectively) and protein contents (-43%, p = 0.02 and -28%, p = 0.04, respectively) were reduced in patients with persistent AF compared to the controls. mRNA contents of phospholamban, ryanodine receptor type 2 and sodium/calcium exchanger were comparable. No changes were observed in patients with paroxysmal AF.
Conclusions:
Alterations in gene expression of proteins involved in the calcium homeostasis occur only in patients with long-term persistent AF. In the absence of underlying heart disease, the changes are rather secondary than primary to AF.
Fourteen patients with complete atrioventricular block with or without torsades de pointes (TdP) were included in this study. They were divided into 2 groups, 6 patients with TdP (TdP[+] group) and 8 patients without TdP (TdP[-] group). The patients were evaluated at 2 different periods, before (acute period) and after (chronic period) pacemaker implantation. In the acute period, the QRS and heart rate during the escape rhythm were not significantly different between the 2 groups; however, the QT and QTc intervals were significantly longer in the TdP(+) group than in the TdP(-) group: 753 +/- 57.5 vs 635 +/- 78.4 ms (p less than 0.01) and 585 +/- 44.8 vs 476 +/- 58.3 ms (p less than 0.01). In the chronic period (greater than 2 months after pacemaker implantation), we changed the pacemaker rate from 90 or 100 beats/min to 50 beats/min and examined the QT interval changes in relation to the heart rate. The QT interval in the TdP(+) group was significantly prolonged compared with the TdP(-) group when the pacing rate was decreased less than or equal to 60 beats/min: 551 +/- 40 vs 503 +/- 36 ms at 60 beats/min (p less than 0.05), and 700 +/- 46 vs 529 +/- 43 ms at 50 beats/min (p less than 0.001). Patients with complete atrioventricular block with TdP had a bradycardia-sensitive repolarization abnormality and this characteristic remained after pacemaker implantation. The critical heart rate that induced abnormal QT prolongation in the TdP(+) group was less than or equal to 60 beats/min.
Early afterdepolarizations (EADs) are one mechanism proposed to cause certain cardiac arrhythmias. We studied the effect of the Ca2+ channel agonist Bay K 8644 (1 x 10(-8) to 5 x 10(-5) M) on normally polarized sheep and canine cardiac Purkinje fiber short segments. EADs occurred with higher Bay K 8644 concentrations and had an average take-off potential of -34 mV. The initiation of EADs was preceded by lengthening of action potential duration and flattening of the plateau. Induction of EADs with Bay K 8644 was enhanced by low stimulation frequencies, lowering of [K]o, addition of tetraethylammonium chloride, or application of depolarizing constant current pulses during the plateau. EADs were abolished by increasing stimulation frequency, raising [K]o, the addition of tetrodotoxin, lidocaine, ethmozin, verapamil, and nitrendipine, or application of repolarizing constant current pulses. Using current pulses to modify the action potential plateau, a steep inverse relationship was found between the EAD peak voltage and its take-off potential, and EADs could be initiated over only a narrow range of take-off potentials. Thus, interventions that suppressed EADs shortened action potential duration or shifted the plateau away from the voltage range needed to initiate EADs. These observations suggest that mechanisms dependent on both time and voltage underlie EADs, and provide a unifying hypothesis for the induction of the EADs. We propose that induction of EADs requires 1) lengthening of action potential duration within a plateau voltage range where 2) recovery from inactivation and reactivation of an inward current possibly carried through Ca2+ channels can occur.
Early afterdepolarizations (EADs) are a type of triggered activity found in heart muscle. We used voltage-clamped sheep cardiac Purkinje fibers to examine the mechanism underlying EADs induced near action potential plateau voltages with the Ca2+ current agonist Bay K 8644 and the effect of several interventions known to suppress or enhance these EADs. Bay K 8644 produced an inward shift of the steady-state current-voltage relation near plateau voltages. Tetrodotoxin, lidocaine, verapamil, nitrendipine, and raising [K]o abolish EADs and shift the steady-state current-voltage relations outwardly. Using a two-pulse voltage-clamp protocol, an inward current transient was present at voltages where EADs were induced. The voltage-dependence of availability of the inward current transient and of EAD induction were similar. The time-dependence of recovery from inactivation of the inward current transient and of EAD amplitude were nearly identical. Without recovery of the inward current transient, EADs could not be elicited. The inward current transient was enhanced with Bay K 8644 and blocked by nitrendipine, but was not abolished by tetrodotoxin or replacement of [Na]o with an impermeant cation. These results support a hypothesis that the induction of EADs near action potential plateau voltages requires 1) a conditioning phase controlled by the sum of membrane currents present near the action potential plateau and characterized by lengthening and flattening of the plateau within a voltage range where, 2) recovery from inactivation and reactivation of L-type Ca2+ channels to carry the depolarizing charge can occur. Our results suggest an essential role for the L-type Ca2+ "window" current and provide a framework for understanding the role of several membrane currents in the induction and block of EADs.
The purpose of these experiments was to study the pharmacological response of quinidine induced early afterdepolarisations to gain insights into underlying ionic mechanisms. Quinidine (8.5 microM) induced stable early afterdepolarisations at low activation frequencies in 80% of canine cardiac Purkinje fibres superfused with a modified Tyrode's solution. Early afterdepolarisations arose from a secondary plateau in the voltage range of -30 to -60 mV. Calcium channel blockers (verapamil, 1 microM, in 3/6 preparations; verapamil, 10 microM in 6/6 preparations; nifedipine, 0.1 microM in 5/5 preparations) completely eliminated early afterdepolarisations, despite continued quinidine superfusion, without altering the underlying action potential. Isoprenaline (0.2-1 microM) restored them in 75% of these preparations during continued calcium blocker superfusion. Tetrodotoxin (5/5 preparations) eliminated early afterdepolarisations by abbreviating action potentials and reducing or eliminating the quinidine induced secondary plateau. While low concentrations of isoprenaline favoured the occurrence of early afterdepolarisations, larger concentrations eliminated them by enhancing spontaneous automaticity. These experiments suggest that voltage dependent and/or receptor regulated slow inward current plays an important role in quinidine induced early afterdepolarisations. Beta receptor stimulation can enhance or suppress early afterdepolarisations, depending on whether effects on slow inward current (tending to favour them) or on automaticity (suppressing them) predominate.
A model of the cardiac ventricular action potential that accounts for dynamic changes in ionic concentrations was used to study the mechanism, characteristics, and rate dependence of early after depolarizations (EADs). A simulation approach to the study of the effects of pharmacological agents on cellular processes was introduced. The simulation results are qualitatively consistent with experimental observations and help resolve contradictory conclusions in the literature regarding the mechanism of EADs. Our results demonstrate that: 1) the L-type calcium current, ICa, is necessary as a depolarizing charge carrier during an EAD; 2) recovery and reactivation of ICa is the mechanism of EAD formation, independent of the intervention used to induce the EADs (cesium, Bay K 8644, or isoproterenol were used in our simulations, following similar published experimental protocols); 3) high [Ca2+]i is not required for EADs to develop and calcium release by the sarcoplasmic reticulum does not occur during the EAD; 4) although the primary mechanism of EAD formation is recovery of ICa, other plateau currents can modulate EAD formation by affecting the balance of currents during a conditional phase before the EAD take-off; and 5) EADs are present at drive cycle lengths longer than 1000 ms. Because of the very long activation time constant of the delayed rectifier potassium current, IK, the activation gate of IK does not deactivate completely between consecutive stimuli at fast rates (drive cycle length < 1000 ms). As a result, IK plays a key role in determining the rate dependence of EADs.
Ryanodine, a specific blocker of the Ca2+ release channel of the sarcoplasmic reticulum, and flunarizine, a [Ca2+]i overload blocker, possess antiarrhythmic effects against delayed afterdepolarizations (DADs) and DAD-dependent arrhythmias. In vitro controversy exists about their effect on early after-depolarizations (EADs): no effect was reported on cesium-induced EADs, while ryanodine did prevent EADs induced by isoproterenol. To study the possible role of intracellular Ca2+ overload in acquired EAD-dependent torsades de pointes (TdP) arrhythmias, we tested the effects of flunarizine and ryanodine in our animal model of TdP.
Anaesthetized dogs with chronic AV block received d-sotalol or almokalant followed by pacing. A subset of dogs with reproducible TdP (> or = 3 times) were selected to receive flunarizine (2 mg/kg per 2 min) or ryanodine (10 micrograms/kg per 10 min). After d-sotalol, TdP was induced at a mean cycle length of the idioventricular rhythm (CL-IVR) of 2070 +/- 635 msec and a QT(U) interval of 535 +/- 65 msec. Induction of TdP was prevented by flunarizine in all experiments (8/8): electrophysiologically this was associated with a decrease in CL-IVR, QT(U), and QTc interval (390 +/- 100 to 320 +/- 45, P < 0.05). Ryanodine prevented TdP induction in 4 of 5 experiments and decreased the CL-IVR, QT(U), and the QTc interval from 385 +/- 75 to 320 +/- 20 msec (P < 0.05). Both drugs also suppressed the almokalant-induced EADs and related ectopic activity. This antiarrhythmic action corresponded with the inability to reinduce TdP by pacing.
Blockade of the Ca2+ release channel of the sarcoplasmic reticulum by ryanodine or the reduction of [Ca2+]i overload by flunarizine prevents induction of EAD-dependent acquired TdP arrhythmias, suggesting a role for [Ca2+]i overload in acquired TdP.
It has been well established that antiarrhythmic drugs can also have proarrhythmic effects such as torsade de pointes (TdP) arrhythmias. It was the purpose of this study to create an animal model with a high incidence of reproducible TdP that occurs under clinically relevant circumstances. Experiments were performed in anesthetized dogs that had been in chronic atrioventricular block for 9 +/- 6 weeks. TdP inducibility was attempted using different pacing modes before and after the administration of 2 mg/kg d-sotalol. In some experiments, endocardial monophasic action potentials were recorded. d-Sotalol increased the cycle length of the idioventricular rhythm (1475 +/- 460 to 1730 +/- 570 ms, P < .01) and the QT time (390 +/- 65 to 480 +/- 85 ms, P < .01). In 10% of the experiments, spontaneous TdP occurred after d-sotalol. The incidence of pacing-dependent TdP was 52% (P < .01). In the inducible group, the cycle length of idioventricular rhythm and QT time were significantly longer despite equal percentage increases in these parameters after d-sotalol in both groups. The pacing modes consisting of more than one frequency change had a higher TdP induction rate (P < .05). Reproducibility of TdP induction (three times or more using the same pacing train) remained present for approximately 60 minutes after d-sotalol and was greater than 90% within the same animal over weeks. TdP induction was related to the presence of early afterdepolarizations on the monophasic action potential recordings: five of six in the inducible group versus two of six in the nonresponders. Inducibility could be further increased to 89% when a second bolus of d-sotalol was administered to noninducible dogs. On the other hand, decreasing QT time by faster basic pacing or administration of isoproterenol, or MgSO4 prevented TdP induction. This effect of MgSO4 coincided with the disappearance of early afterdepolarizations. Our animal model shows a high incidence of reproducible acquired TdP arrhythmias, allowing study of the mechanism and treatment of TdP. TdP induction was related to the combination of a slow ventricular rate, the prolongation of QT time, a sudden induced rate change that often required two or more cycle length changes, and the presence of early afterdepolarizations.
The ionic mechanisms for early afterdepolarizations (EADs) have not been fully clarified. It has been suggested that L-type Ca2+ current (ICaL) is the primary current generating EADs that occur near the plateau level (E-EADs) of the membrane potential (Vm) when ICaL is enhanced. The purpose of these studies was to determine accurately the range of Vm at which EADs occur in Purkinje fibers with K+ currents blocked by Cs+ and to investigate the importance of Na+:Ca2+ exchange current (INa:Ca) as opposed to ICaL and other currents in the generation of EADs occurring later during repolarization (L-EADs).
Shortened Purkinje strands from dogs and guinea pigs were superfused with physiologic solution containing Cs+ (3.6 mM) and a low [K+]o (3.0 or 2.0 mM) to induce EADs. The Vm of origin of EADs and their evolution were measured with the aid of phase plane plots of the rate of repolarization against Vm. L-EADs occurred over a wide range of Vm (-35 to -90 mV), generally more negative in guinea pig than in dog. Elevation of [Ca2+]o from 1.8 to 3.6 mM suppressed L-EADs within a few cycles, and they returned with continued exposure. After repeated exposures to high [Ca2+]o, L-EADs migrated toward less negative Vm when [Ca2+]o was reestablished to 1.8 mM in the presence of Cs+. Reduction of [Na+]o from 147.5 to 112.5 mM by substitution with Li+ or sucrose also rapidly depressed L-EADs.
The observation of Cs(+)-induced L-EADs over a wide range of Vm indicates that there is not a single inward gated current as a common ionic mechanism for L-EADs but does not exclude an important role for INa:Ca, which can operate over a wide range of Vm. The rapid suppression of L-EADs with elevated [Ca2+]o and reduced [Na+]o and the migration of EADs to more positive Vm after exposures to high [Ca2+]o are compatible with INa:Ca as the major charge carrier for L-EADs.
The multifunctional Ca++/calmodulin-dependent protein kinase II (CaM kinase) mediates Ca++-induced augmentation of L-type Ca++ current (ICa); therefore it may act as a proarrhythmic signaling molecule during early afterdepolarizations (EADs) due to ICa. To investigate the hypothesis that ICa-dependent EADs are favored by CaM kinase activation EADs were induced with clofilium in isolated rabbit hearts. All EADs were rapidly terminated with ICa antagonists. Hearts were pretreated with the CaM kinase inhibitor KN-93 or the inactive analog KN-92 (0.5 microM) for 10 min before clofilium exposure. EADs were significantly suppressed by KN-93 (EADs present in 4/10 hearts) compared to KN-92 (EADs present in 10/11 hearts) (P =.024). There were no significant differences in parameters favoring EADs such as monophasic action potential duration or heart rate in KN-93- or KN-92-treated hearts. CaM kinase activity in situ increased 37% in hearts with EADs compared to hearts without EADs (P =.015). This increase in CaM kinase activity was prevented by pretreatment with KN-93. In vitro, KN-93 potently inhibited rabbit myocardial CaM kinase activity (calculated Ki </= 2.58 microM), but the inactive analog KN-92 did not (Ki > 100 microM). The actions of KN-93 and KN-92 on ICa and other repolarizing K+ currents did not explain preferential EAD suppression by KN-93. These data show a novel association between CaM kinase activation and EADs and are consistent with the hypothesis that the ICa and CaM kinase activation both contribute to EADs in this model.
Although it is widely accepted that afterdepolarizations initiate arrhythmias when action potentials are prolonged, the underlying mechanisms are unclear. In this study, we tested the hypothesis that action potential prolongation would raise intracellular calcium and thereby activate the arrhythmogenic transient inward current (Iti). Furthermore, given that Iti can be activated by sarcoplasmic reticulum Ca2+ release, we tested the hypothesis that inhibition of calmodulin (CaM) kinase would prevent Iti. Isolated rabbit ventricular myocytes were studied with whole-cell-mode voltage clamp. Stimulation with a prolonged action potential clamp, under near-physiological conditions, increased [Ca2+]i. Iti was reproducibly induced in 60 of 60 cells, but Iti was not seen with the use of a shorter action potential waveform (n=12). Iti was associated with a secondary elevation in [Ca2+]i. When [Ca2+]i buffering was enhanced by dialysis with BAPTA (20 mmol/L, n=9), no Iti was present. The Na+/Ca2+ exchanger was likely responsible for Iti, because Iti was inhibited by the Na+/Ca2+ exchanger inhibitory peptide XIP (10 micromol/L, n=6), but not by an inactive scrambled peptide (10 micromol/L, n=5) or by the Cl- current antagonist niflumic acid (10 to 40 micromol/L, n=9). Activator Ca2+ from the sarcoplasmic reticulum was essential for development of Iti, because it was prevented by pretreatment with ryanodine (10 micromol/L, n=6) or thapsigargin (1 micromol/L, n=6). Two different CaM kinase inhibitory peptides (n=16) and a CaM inhibitory peptide (n=4) completely suppressed Iti. These results are consistent with the hypothesis that CaM kinase plays a role in arrhythmias related to increased [Ca2+]i.
Early afterdepolarizations (EAD) caused by L-type Ca2+ current (ICa, L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in ICa,L during repolarization. ICa,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. ICa,L augmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.
Acquired QT prolongation enhances the susceptibility to torsades de pointes (TdP). Clinical and experimental studies indicate ventricular action potential prolongation, increased regional dispersion of repolarization, and early afterdepolarizations as underlying factors. We examined whether K(+)-current alterations contribute to these proarrhythmic responses in an animal model of TdP: the dog with chronic complete atrioventricular block (AVB) and biventricular hypertrophy.
The whole-cell K(+) currents I(TO1), I(K1), I(Kr), and I(Ks) were recorded in left (LV) and right (RV) ventricular midmyocardial cells from dogs with 9+/-1 weeks of AVB and controls with sinus rhythm. I(TO1) density and kinetics and I(K1) outward current were not different between chronic AVB and control cells. I(Kr) had a similar voltage dependence of activation and time course of deactivation in chronic AVB and control. I(Kr) density was similar in LV myocytes but smaller in RV myocytes (-45%) of chronic AVB versus control. For I(Ks), voltage-dependence of activation and time course of deactivation were similar in chronic AVB and control. However, I(Ks) densities of LV (-50%) and RV (-55%) cells were significantly lower in chronic AVB than control.
Significant downregulation of delayed rectifier K(+) current occurs in both ventricles of the dog with chronic AVB. Acquired TdP in this animal model with biventricular hypertrophy is thus related to intrinsic repolarization defects.
The ventricular arrhythmia torsade de pointes (TdP) occurs after QT interval prolongation and is associated with sudden cardiac death. The afterdepolarizations that initiate TdP are facilitated by protein kinase A and the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase).
In this study, we evaluated the feasibility of suppression of TdP through systemic therapy with kinase inhibitory agents in an established animal model. Under control conditions, TdP was inducible in 6 of 8 rabbits. CaM kinase blockade with the calmodulin antagonist W-7 reduced TdP in a dose-dependent fashion (4 of 7 inducible at 25 micromol/kg and 1 of 7 inducible at 50 micromol/kg). Increased intracellular Ca(2+) has been implicated in the genesis of afterdepolarizations, but pretreatment with high-dose W-7 did not prevent TdP in response to the L-type Ca(2+) channel agonist BAY K 8644 (300 nmol/kg), suggesting that CaM kinase-independent activation of L-type Ca(2+) current was not affected by W-7. Compared with control animals, W-7 reduced TdP inducibility without shortening the QT interval, increasing heart rate, or reducing the blood pressure. The protein kinase A antagonist H-8 also caused a dose-dependent reduction in TdP inducibility (5 of 6 at 1 micromol/kg, 4 of 6 at 5 micromol/kg, and 0 of 6 at 10 micromol/kg), but unlike W-7, H-8 did so by shortening the QT interval.
These findings show that the acute systemic application of W-7 and H-8 is hemodynamically tolerated and indicate that kinase inhibition may be a viable antiarrhythmic strategy.
Background:
Ventricular arrhythmias are a major cause of sudden death in patients with heart failure and hypertrophy. The dog with chronic complete atrioventricular block (CAVB) has biventricular hypertrophy and ventricular arrhythmias and is a useful model to study underlying cellular mechanisms. We investigated whether changes in Ca(2+) homeostasis are part of the contractile adaptation to CAVB and might contribute to arrhythmogenesis.
Methods and results:
In enzymatically isolated myocytes, cell shortening, Ca(2+) release from the sarcoplasmic reticulum (SR), and SR Ca(2+) content were enhanced at low stimulation frequencies. Ca(2+) influx through L-type Ca(2+) channels was unchanged, but Ca(2+) influx via the Na/Ca exchanger was increased and contributed to Ca(2+) loading of the SR. Inward Na/Ca exchange currents were also larger. Changes in Ca(2+) fluxes were less pronounced in the right versus left ventricle.
Conclusions:
Enhanced Na/Ca exchange activity may improve contractile adaptation to CAVB but at the same time facilitate arrhythmias by (1) increasing the propensity to Ca(2+) overload, (2) providing more inward current leading to (nonhomogeneous) action potential prolongation, and (3) enhancing (arrhythmogenic) currents during spontaneous Ca(2+) release.
Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 micromol/L), or by KN-93 (0.1 to 3.0 micromol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 micromol/L AIP and 3 micromol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca(2+) current (I(Ca, L)), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 micromol/L), but not its inactive analog, KN-92, decreased I:(Ca, L) amplitude from 12+/-2 to 6+/-1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I(Ca, L) from inactivation. Similar results were observed using the fast Ca(2+) chelator BAPTA, whereas the slow Ca(2+) chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca(2+) transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I(Ca, L) inactivation and reactivation, and local Ca(2+) is critically involved in these processes.
Phospholamban (PLB) plays a primary role in regulating cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity. Dephosphorylated PLB suppresses the SR Ca(2+) pump activity, whereas phosphorylation of PLB leads to deinhibition. A widely accepted sequential model of dual site PLB phosphorylation states that PKA-dependent phosphorylation of Ser(16) is obligatory to phosphorylation of Thr(17) by Ca(2+)/calmodulin-dependent kinase II, and mainly accounts for beta-adrenergic receptor mediated cardiac relaxation. However, emerging evidence supports independent phosphorylation of Ser(16) and Thr(17) and their independent contributions to cardiac relaxation. Furthermore, concurrent activation of PKA and CaMKII signaling pathways exhibits a robust synergistic effect on phosphorylation of Thr(17), but not of Ser(16). Thus, the synergistic interaction may masquerade as a sequential phosphorylation of Ser(16) and Thr(17) under certain circumstances. Further studies are required to determine the exact process of dual site PLB phosphorylation and its functional roles in healthy and diseased hearts.
Recent studies have demonstrated that transgenic (TG) expression of either Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIdeltaB, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIdeltaB but also a cytoplasmic isoform, CaMKIIdeltaC. In the present study, we demonstrate that expression of the deltaC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic deltaC isoform of CaMKII can lead to phosphorylation of Ca2+ regulatory proteins and induce hypertrophy, we generated TG mice that expressed the deltaC isoform of CaMKII. Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2+ handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIdeltaC can mediate phosphorylation of Ca2+ regulatory proteins in vivo and provide evidence for the involvement of CaMKIIdeltaC activation in the pathogenesis of dilated cardiomyopathy and heart failure.
The purpose of this study was to investigate interactions between early afterdepolarizations (EADs) and reentry in long QT (LQT) syndromes.
EADs, a characteristic feature of congenital and acquired LQT syndromes, are classically bradycardia dependent. Mechanisms by which they promote tachyarrhythmias such as torsades de pointes and ventricular fibrillation are not fully understood. Recent evidence suggests that EADs also may occur at rapid heart rates as a sequela of spontaneous sarcoplasmic reticulum (SR) Ca(2+) release related to intracellular Ca(2+) overload. Here, we performed computer simulations to explore the arrhythmogenic consequences of this phenomenon.
We used a modified version of the Luo-Rudy dynamic model in one-dimensional and two-dimensional cardiac tissue with the time-dependent K(+) currents I(Kr) or I(Ks) reduced by 50% to simulate acquired and congenital LQT syndromes.
(1) Spontaneous SR Ca(2+) release prolonged action potential duration but did not induce overt EADs unless K(+) current density was reduced to simulate acquired and congenital LQT syndromes. (2) In simulated LQT syndromes, EADs were capable of both terminating and reinitiating one-dimensional reentry. (3) A similar phenomenon in simulated 2D tissue led to reinitiation of spiral wave reentry that otherwise would have self-terminated. (4) Reentry reinitiation occurred only when the L-type Ca(2+) current and SR Ca(i) cycling were potentiated to simulate moderate sympathetic stimulation, consistent with the known arrhythmogenic effects of sympathetic activation (and protection by beta-blockers) in LQT syndromes.
These computer simulations suggest that EADs related to spontaneous SR Ca(2+) release can enhance arrhythmogenesis in LQT syndromes by reinitiating reentry.
The QT interval is the electrocardiographic manifestation of ventricular repolarization, is variable under physiologic conditions, and is measurably prolonged by many drugs. Rarely, however, individuals with normal base-line intervals may display exaggerated QT interval prolongation, and the potentially fatal polymorphic ventricular tachycardia torsade de pointes, with drugs or other environmental stressors such as heart block or heart failure. This review summarizes the molecular and cellular mechanisms underlying this acquired or drug-induced form of long QT syndrome, describes approaches to the analysis of a role for DNA variants in the mediation of individual susceptibility, and proposes that these concepts may be generalizable to common acquired arrhythmias.
Sustained heart rate abnormalities produce electrical remodeling and susceptibility to arrhythmia. Uncontrolled tachycardia produces heart failure and ventricular tachyarrhythmia susceptibility, whereas bradycardia promotes spontaneous torsade de pointes (TdP). This study compared arrhythmic phenotypes and molecular electrophysiological remodeling produced by tachycardia versus bradycardia in rabbits.
We evaluated mRNA and protein expression of subunits underlying rapid (IKr) and slow (IKs) delayed-rectifier and transient-outward K+ currents in ventricular tissues from sinus rhythm control rabbits and rabbits with AV block submitted to 3-week ventricular pacing either at 60 to 90 bpm (bradypaced) or at 350 to 370 bpm (tachypaced). QT intervals at matched ventricular pacing rates were longer in bradypaced than tachypaced rabbits (eg, by approximately 50% at 60 bpm; P<0.01). KvLQT1 and minK mRNA and protein levels were downregulated in both bradypaced and tachypaced rabbits, whereas ERG was significantly downregulated in bradypaced rabbits only. Kv4.3 and Kv1.4 were downregulated by tachypacing only. Patch-clamp experiments showed that IKs was reduced in both but IKr was decreased in bradypaced rabbits only. Continuous monitoring revealed spontaneous TdP in 75% of bradypaced but only isolated ventricular ectopy in tachypaced rabbits. Administration of dofetilide (0.02 mg/kg) to mimic IKr downregulation produced ultimately lethal TdP in all tachypaced rabbits.
Sustained tachycardia and bradycardia downregulate IKs subunits, but bradycardia also suppresses ERG/IKr, causing prominent repolarization delays and spontaneous TdP. Susceptibility of tachycardia/heart failure rabbits to malignant tachyarrhythmias is induced by exposure to IKr blockers. These results point to a crucial role for delayed-rectifier subunit remodeling in TdP susceptibility associated with rate-related cardiac remodeling.
Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.
Torsades de pointes (TdP) ventricular tachycardia typically occurs in the setting of early afterdepolarizations; it contributes to arrhythmias and sudden death in congenital and acquired heart disease. Window L-type Ca2+ current (ICaL) has a central role in the arrhythmogenesis and may be particularly important under beta-adrenergic stimulation. We studied the properties of ICaL in myocytes from the dog with chronic atrioventricular block (cAVB) that has cardiac hypertrophy and an increased susceptibility to TdP. Peak ICaL densities at baseline (K+ - and Na+ -free solutions, 10 mmol l(-1) [EGTA]pip) in cAVB were comparable to control, but inactivation was shifted to the right, resulting in a larger window current area in cAVB. Under beta-adrenergic stimulation, the window current area was increased and shifted to the left, but less so in cAVB (maximum at -27 mV, versus -32 mV in control). ICaL during a step to -35 mV showed a transient reduction immediately after the peak. Test steps to 0 mV, simultaneous recording of [Ca2+]i and manipulation of sarcoplasmic reticulum (SR) Ca2+ release showed that this resulted from inhibition and fast recovery of ICaL with SR Ca2+ release. The extent of this dynamic modulation was larger in cAVB than in control (23 +/- 2% of the initially available current, versus 13 +/- 3%; P<0.05). Early afterdepolarizations (EADs) in cAVB myocytes under beta-adrenergic stimulation typically occurred in the window current voltage range and after decline of [Ca2+]i. In conclusion, in cAVB, the larger window current, its rightward shift and enhanced dynamic modulation by SR Ca2+ release may contribute to an increased incidence of EADs in cAVB under beta-adrenergic stimulation.
The multifunctional Ca(2+) calmodulin-dependent protein kinase II (CaMKII) has emerged as a pro-arrhythmic signaling molecule. CaMKII can participate in arrhythmia signaling by effects on ion channel proteins, intracellular Ca(2+) uptake and release, regulation of cell death, and by activation of hypertrophic signaling pathways. The pleuripotent nature of CaMKII is reminiscent of another serine-threonine kinase, protein kinase A (PKA), which shares many of the same protein targets and is the downstream kinase most associated with beta-adrenergic receptor stimulation. The ability of CaMKII to localize and coordinate activity of multiple protein targets linked to Ca(2+) signaling set CaMKII apart from other "traditional" arrhythmia drug targets, such as ion channel proteins. This review will discuss some of the biology of CaMKII and focus on work that has been done on molecular, cellular, and whole animal models that together build a case for CaMKII as a pro-arrhythmic signal and as a potential therapeutic target for arrhythmias and structural heart disease.
This study was designed to analyze the relevance of ventricular activation patterns for ventricular electrical remodeling after atrioventricular (AV) block in dogs.
Bradycardia is thought to be the main contributor to ventricular electrical remodeling after complete AV block. However, an altered ventricular activation pattern or AV dyssynchrony may also contribute.
For 4 weeks, AV block dogs were either paced from the high-ventricular septum near the His bundle at lowest captured rate (n = 9, high-septal pacing [HSP]) or kept at idioventricular rate without controlled activation (n = 14, chronic AV block [CAVB]). Multiple electrocardiographic and electrophysiological parameters were measured under anesthesia at 0 and 4 weeks. Proarrhythmia was tested at 4 weeks by I(Kr) block (25 mug/kg dofetilide intravenous).
At 0 weeks, the 2 groups were comparable, whereas after 4 weeks of similar bradycardia, QT duration at unpaced conditions had increased from 300 +/- 5 to 395 +/- 18 ms in CAVB (+32 +/- 6%) and from 307 +/- 8 ms to 357 +/- 11 ms in HSP (+17 +/- 4%; p < 0.05). Frequency dependency of repolarization was less steep in HSP compared to CAVB dogs after 4 weeks remodeling. Beat-to-beat variability of repolarization, a proarrhythmic parameter, increased only in CAVB from 0 to 4 weeks. Torsades de pointes arrhythmias were induced at 4 weeks in 44% HSP versus 78% CAVB dogs (p = 0.17). Cumulative duration of arrhythmias per inducible dog was 87 +/- 36 s in CAVB and 30 +/- 21 s in HSP (p < 0.05).
High-septal pacing reduces the magnitude of ventricular electrical remodeling and proarrhythmia in AV block dogs, suggesting a larger role for altered ventricular activation pattern in the generation of ventricular electrical remodeling than previously assumed.
Calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA-/- mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.