Calcium-Calmodulin Kinase II Mediates Digitalis-Induced Arrhythmias (original) (raw)
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AJP: Heart and Circulatory Physiology, 2008
In cardiac myocytes, the activity of the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is hypothesized to regulate Ca 2+ release from and Ca 2+ uptake into the sarcoplasmic reticulum via phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of [Ca 2+ ] i -transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wildtype or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37 ºC). Compared to mice lacking PLN only, combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca 2+ release rate by more than 50% at 10 Hz. While PLN ablation increased the rate of Ca 2+ uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca 2+ ] i -transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca 2+ ] itransient, indicating that PLN-mediated feedback on [Ca 2+ ] i removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes (1) combined chronic CaMKII inhibition and PLN ablation slowed Ca 2+ release at physiological frequencies, (2) frequency-dependent decay of the amplitude and shortening of the [Ca 2+ ] i -transient occurs independent of chronic CaMKII inhibition and PLN ablation, (3) PLN-mediated regulation of Ca 2+ uptake is diminished at higher stimulation frequencies within the physiological range.
Calcium, Calmodulin, and Calcium-Calmodulin Kinase II: Heartbeat to Heartbeat and Beyond
Journal of Molecular and Cellular Cardiology, 2002
34, 919À939. Calcium (Ca) is the key regulator of cardiac contraction during excitationÀcontraction (EÀC) coupling. However, differences exist between the amount of Ca being transported into the myocytes upon electrical stimulation as compared to Ca released from the sarcoplasmic reticulum (SR). Moreover, alterations in EÀC coupling occur in cardiac hypertrophy and heart failure. In addition to the direct effects of Ca on the myo®laments, Ca plays a pivotal role in activation of a number of Ca-dependent proteins or second messengers, which can modulate EÀC coupling. Of these proteins, calmodulin (CaM) and Ca-CaM-dependent kinase II (CaMKII) are of special interest in the heart because of their role of modulating Ca in¯ux, SR Ca release, and SR Ca uptake during EÀC coupling. Indeed, CaM and CaMKII may be associated with some ion channels and Ca transporters and both can modulate acute cellular Ca handling. In addition to the changes in Ca, CaM and CaMKII signals from beat-to-beat, changes may occur on a longer time scale. These may occur over seconds to minutes involving phosphorylation/dephosphorylation reactions, and even a longer time frame in altering gene transcription (excitationÀtranscription (EÀT) coupling) in hypertrophic signaling and heart failure. Here we review the classical role of Ca in EÀC coupling and extend this view to the role of the Ca-dependent proteins CaM and CaMKII in modulating EÀC coupling and their contribution to EÀT coupling.
Calmodulin Regulation of Excitation-Contraction Coupling in Cardiac Myocytes
Circulation Research, 2003
Calmodulin (CaM) as a ubiquitous Ca 2ϩ sensor interacts with multiple key molecules involved in excitationcontraction (EC) coupling. In the present study, we report that adenoviral expression of a mutant CaM lacking all of its four Ca 2ϩ-binding sites, CaM(1-4), at a level 6.5-fold over endogenous CaM markedly increases the amplitude and abbreviates the decay time of Ca 2ϩ transients and contraction in cultured rat ventricular myocytes. To determine the underlying mechanisms, we examined the properties of L-type Ca 2ϩ channels, Ca 2ϩ /CaM-dependent protein kinase II (CaMKII), and phospholamban (PLB) in the sarcoplasmic reticulum (SR). We found that CaM(1-4) expression markedly augmented L-type Ca 2ϩ current amplitude and slowed its inactivation. Surprisingly, overexpression of CaM(1-4) increased CaMKII activity and phosphorylation of PLB-Thr-17. Moreover, CaM(1-4) elevated diastolic Ca 2ϩ and caffeine-labile Ca 2ϩ content of the SR. Inhibition of CaMKII by KN-93 or a myristoylated autocamtide-2 related inhibitory peptide prevented the aforementioned PLB phosphorylation and reversed the positive inotropic and relaxant effects, indicating that CaMKII is essential to CaM(1-4) actions. These results demonstrate that CaM modulates Ca 2ϩ influx, SR Ca 2ϩ release, and Ca 2ϩ recycling during cardiac EC coupling. A novel finding of this study is that expression of a Ca 2ϩ-insensitive CaM mutant can lead to activation of CaMKII in cardiac myocytes. (Circ Res. 2003;92:659-667.) Key Words: calmodulin Ⅲ Ca 2ϩ /calmodulin-dependent protein kinase II Ⅲ L-type Ca 2ϩ channels Ⅲ phospholamban I ntracellular Ca 2ϩ acts as a universal and versatile second messenger that regulates vital biological processes, including cell contraction, synaptic transmission, hormone secretion, cell growth, and cell death. 1,2 The specificity and versatility of Ca 2ϩ signaling are in part endowed by the spatial and temporal patterning of both the amplitude and frequency of Ca 2ϩ signals. During eukaryotic evolution, nature has also evolved a sophisticated means to orchestrate the intricate Ca 2ϩ signaling, ie, the use of small Ca 2ϩ-binding proteins, such as calmodulin (CaM), as Ca 2ϩ transducer to sort the Ca 2ϩ signals toward different cellular effectors. The multifunctional CaM contains 148 highly conserved amino acid residues, with two pairs of Ca 2ϩ binding sites of affinities in its N-and C-terminal lobes, and an effector-binding motif connecting these two domains. 3 CaM binds to different effectors in either Ca 2ϩ-free (apoCaM) 4 or Ca 2ϩ-bound forms, 3 mediating the Ca 2ϩ-dependent regulation of diverse cellular processes. CaM is known to interact with multiple proteins involved in excitation-contraction (EC) coupling and Ca 2ϩ homeostasis. 5,6 Studies in heterologous expression system have shown that CaM preassociates with the C-terminus of Ca 2ϩ channels located in the plasma membrane and bifurcately mediates Ca 2ϩ-dependent inactivation 7-9 and facilitation 7,9 of the channel. Cardiac sarcoplasmic reticulum (SR) vesicles bind approximately four CaM molecules per type 2 ryanodine receptor (RyR2) tetramer in the absence of Ca 2ϩ or 7.5 CaM molecules per tetramer in the presence of 100 mol/L Ca 2ϩ. 10 Similarly, skeletal muscle isoform of RyR (RyR1) can bind both Ca 2ϩ-free and Ca 2ϩ-bound CaM, at a stoichiometry of 4 CaM per RyR1 tetramer, 11 exerting bifunctional regulation of Ca 2ϩ-dependent channel activation and inactivation. 12 Furthermore, Ca 2ϩ-bound CaM can activate many types of protein kinases (eg, Ca 2ϩ /CaM-dependent kinase II, CaMKII) 13 or phosphatases (eg, calcineurin) 14 to regulate the phosphorylation status, thereby affecting the functioning of a wide spectrum of effector proteins, including L-type Ca 2ϩ channel (LCC), 15,16 Na ϩ channel, 17 and the SR Ca 2ϩ-ATPase regulator protein phospholamban (PLB). 18,19 To delineate potential roles of CaM in regulating cardiac EC coupling and Ca 2ϩ signaling, we used a molecular approach to alter the Ca 2ϩ-sensing ability of CaM in cultured rat ventricular myocytes, and characterized its functional consequences, including LCC Ca 2ϩ current (I Ca), intracellular
British Journal of Pharmacology, 2010
BACKGROUND AND PURPOSE Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms. EXPERIMENTAL APPROACH Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca 2+ ]i transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16. KEY RESULTS Urocortin 2 elicited time-and concentration-dependent positive inotropic and lusitropic effects (EC50: 19 nM) that were abolished by antisauvagine-30 (10 nM, n = 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF2 receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca 2+ ]i transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 mM) or KT5720 (1 mM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 mM), an inhibitor of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca 2+ ]i transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca 2+ ]i increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93.
Basic Research in Cardiology
Cardiomyocyte Na+ and Ca2+ mishandling, upregulated Ca2+/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibiti...
Circulation: Arrhythmia and Electrophysiology
See Editorial by Morrow and Marx BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca 2+ /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 Ca 2+ homeostasis examined by fluorescence (fluo-4) and patchclamp 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 Ca 2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca 2+ 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 Ca 2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca 2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.
Cardiac ryanodine receptors (RyR2s) play a critical role in excitation–contraction coupling by providing a pathway for the release of Ca 2+ from the sarcoplasmic reticulum into the cytosol. RyR2s exist as macromolecular complexes that are regulated via binding of Ca 2+ and protein phosphorylation/dephosphorylation. The present study examined the association of endogenous CaMKII (calcium/calmodulin-dependent protein kinase II) with the RyR2 complex and whether this enzyme could modulate RyR2 function in isolated rabbit ventricular myocardium. Endogenous phosphorylation of RyR2 was verified using phosphorylation site-specific antibodies. Co-immunoprecipitation studies established that RyR2 was physically associated with CaMKIIδ. Quantitative assessment of RyR2 protein was performed by [ 3 H]ryanodine binding to RyR2 immunoprecipitates. Parallel kinase assays allowed the endogenous CaMKII activity associated with these immunoprecipitates to be expressed relative to the amount of RyR2. The activity of RyR2 in isolated cardiac myocytes was measured in two ways: (i) RyR2-mediated Ca 2+ release (Ca 2+ sparks) using confocal microscopy and (ii) Ca 2+-sensitive [ 3 H]ryanodine binding. These studies were performed in the presence and absence of AIP (autocamtide-2-related inhibitory peptide), a highly specific inhibitor of CaMKII. At 1 µM AIP Ca 2+ spark duration, frequency and width were decreased significantly. Similarly, 1 µM AIP decreased [ 3 H]ryanodine binding. At 5 µM AIP, a more profound inhibition of Ca 2+ sparks and a decrease in [ 3 H]ryanodine binding was observed. Separate measurements showed that AIP (1–5 µM) did not affect sarcoplas-mic reticulum Ca 2+-ATPase-mediated Ca 2+ uptake. These results suggest the existence of an endogenous CaMKIIδ that associates directly with RyR2 and specifically modulates RyR2 activity.
American Journal of Physiology-heart and Circulatory Physiology, 2008
Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff perfused rat/mice hearts and rat isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. Removal of acidosis provoked ectopic beats that were blunted by 1 µM of the CaMKII inhibitor KN-93, 1 µM thapsigargin, to inhibit SR Ca 2+ uptake, and 30 nM ryanodine or 45 µM dantrolene, to inhibit SR Ca 2+ release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr 17 site of phospholamban (PT-PLN) and SR Ca 2+ load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca 2+ leak, when compared with control or with acidosis at the same SR Ca 2+ content. Ca 2+ leak occurred without changes in the phosphorylation of ryanodine receptors (RyR2) and was blunted by in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (DADs), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated to an increase in SR Ca 2+ load, which appears to be mainly due to the increase in PT-PLN.
Circulation, 2010
Background Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca 2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca 2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca 2+ /calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias. Methods and Results Mice in which the S2814 Ca 2+ /calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca 2+ release events, resulting in reduced sarcoplasmic reticulum Ca 2+ load on confocal microscopy. These Ca 2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts wi...