Ca/calmodulin-dependent Protein Kinase II Research Articles

In vitro and in silico analysis of sodium-dependent regulation of mitochondrial calcium concentration and production of reactive oxygen species in rabbit ventricular myocytes

Increased intracellular Na concentration ([Na]i) is a hallmark of cardiomyocyte remodeling in heart failure (HF), a chronic condition characterized by impaired contractility, metabolic imbalance, and arrhythmia susceptibility. Although Na homeostasis is central to cardiomyocyte function, its contribution to the sequelae of disease is still largely unexplored. We previously demonstrated that Na overload plays a critical role in promoting Ca-dependent arrhythmias in failing ventricular myocytes by interacting with the Ca/calmodulin-dependent protein kinase II (CaMKII) signaling.

Abnormal P-waves found in patients with sleep-disordered breathing are associated with triggered pro-arrhythmic activity

Abstract Background Sleep-disordered breathing (SDB) is frequently associated with atrial arrhythmias, but diagnostic markers that predict the propensity for atrial arrhythmias are missing. Abnormal P-wave terminal force in lead V1 (PTFV1) has been associated with atrial structural/electrical remodeling and arrhythmias. Purpose Here we investigate the association of triggered pro-arrhythmic activity with abnormal PTFV1 in human right atrial appendage biopsies of patients without and with SDB. Methods 30 patients undergoing elective coronary artery bypass grafting were screened for SDB by polygraphy. An apnoea-hypopnea index (AHI) of ≥15/h defined SDB. PTFV1 was measured as product of negative P-wave amplitude and duration in lead V1 and was defined as abnormal if >4000 ms*μV. Ca/Calmodulin-dependent protein kinase II (CaMKII) activity was measured in human right atrial appendage biopsies by a specific HDAC4 pull-down assay. Premature atrial contractions (PACs) were triggered by exposure to 100 nM isoproterenol (at 3.5 mM [Ca]o) in human atrial trabeculae. PACs severity was classified by a score from 0 points (no arrhythmias) to 5 points (salve). In addition, atrial fibrosis was quantified by Masson's trichrome stain in cryo-sectioned atrial tissue. Multivariate linear regression analyses were performed accounting for age, sex, BMI, existing AF, heart failure, diabetes, and creatinine. Results Interestingly, the AHI was independently associated with the magnitude of PTFV1 (fig. A+B, B=57.47±21.03, R2=0.48, P=0.01). Importantly, patients with an abnormal PTFV1 had a significantly increased CaMKII activity (fig. C, P=0.04) and showed significantly more severe triggered PACs (fig. D, P=0.02). Moreover, the magnitude of PTFV1 correlated significantly and independently with PAC severity (fig. D, B=0.0005±0.0002, R2=0.50, P=0.03). Consistently, these arrhythmias could be significantly reduced by acute CaMKII inhibition (5 μM KN93, P<0.01), which abolished the correlation between PTFV1 and PAC severity (P=N.S.). Surprisingly, atrial fibrosis was significantly decreased in patients with an abnormal PTFV1 (fig. E, P=0.02), suggesting that abnormal PTFV1 is a marker of proarrhythmic atrial electrical remodeling of functional cardiomyocytes but not of fibrotic tissue. Conclusion The severity of sleep-disordered breathing (AHI) is independently associated with an abnormal PTFV1. Intriguingly, this abnormality is associated with an increased CaMKII activity and with CaMKII-dependent arrhythmias, suggesting PTFV1 to be a potential tool to evaluate the pro-arrhythmic risk of patients with SDB. Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Part of this study was funded by Philips Respironics.

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Enhanced CaMKII-Dependent Late I Na Induces Atrial Proarrhythmic Activity in Patients With Sleep-Disordered Breathing

Sleep-disordered breathing (SDB) is frequently associated with atrial arrhythmias. Increased CaMKII (Ca/calmodulin-dependent protein kinase II) activity has been previously implicated in atrial arrhythmogenesis. We hypothesized that CaMKII-dependent dysregulation of Na current (INa) may contribute to atrial proarrhythmic activity in patients with SDB. We prospectively enrolled 113 patients undergoing elective coronary artery bypass grafting for cross-sectional study and collected right atrial appendage biopsies. The presence of SDB (defined as apnea-hypopnea index ≥15/h) was assessed with a portable SDB monitor the night before surgery. Compared with 56 patients without SDB, patients with SDB (57) showed a significantly increased level of activated CaMKII. Patch clamp was used to measure INa. There was a significantly enhanced late INa, but reduced peak INa due to enhanced steady-state inactivation in atrial myocytes of patients with SDB consistent with significantly increased CaMKII-dependent cardiac Na channel phosphorylation (NaV1.5, at serine 571, Western blotting). These gating changes could be fully reversed by acute CaMKII inhibition (AIP [autocamtide-2 related inhibitory peptide]). As a consequence, we observed significantly more cellular afterdepolarizations and more severe premature atrial contractions in atrial trabeculae of patients with SDB, which could be blocked by either AIP or KN93 (N-[2-[[[(E)-3-(4-chlorophenyl)prop-2-enyl]-methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide). In multivariable linear regression models incorporating age, sex, body mass index, existing atrial fibrillation, existing heart failure, diabetes mellitus, and creatinine levels, apnea-hypopnea index was independently associated with increased CaMKII activity, enhanced late INa and correlated with premature atrial contraction severity. In atrial myocardium of patients with SDB, increased CaMKII-dependent phosphorylation of NaV1.5 results in dysregulation of INa with proarrhythmic activity that was independent from preexisting comorbidities. Inhibition of CaMKII may be useful for prevention or treatment of arrhythmias in SDB. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02877745. Visual Overview: An online visual overview is available for this article.

P3508A novel mouse model of sleep-disordered breathing is associated with contractile dysfunction and CaMKII overexpression

Abstract Background Patients with sleep-disordered breathing (SDB) develop arrhythmias and contractile dysfunction, but the mechanisms are poorly understood, possibly due to the lack of mouse models that mimic airway obstruction in spontaneously sleeping mice. Interestingly, New Zealand obese mice have been shown to develop airway obstruction with inspiratory flow limitation resulting in apneas, but these mice also develop diabetes. Purpose We developed a novel mouse model of increased airway obstruction in spontaneously sleeping lean mice and investigated the impact on sleep-related apneas and contractile function. Methods and results Male C57BL6 mice at 8–12 weeks of age were subjected to polytetrafluoroethylene (PTFE) injection (100 μl) into the tongue. This resulted in an increased tongue volume and reduced pharyngeal luminal diameter. Conscious mice behave normal and there was no difference in body weight between PTFE injected mice and untreated littermates (control). Whole body plethysmography was used to monitor spontaneous breathing for 8h in a quiet environment. Interestingly, compared to control, mice with PTFE injection showed a significantly increased frequency of apneas (lasting >1s, fig. A, * indicated P<0.05, t-test). Echocardiographic analysis revealed that ejection fraction was significantly reduced in PTFE-treated mice 8 weeks after surgery (vs. control, fig. B). In accordance, the developed force of isolated papillary muscles from hearts of PTFE mice was significantly reduced compared to control (fig. C). Ca/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the development of heart failure. Intriguingly, compared to control, CaMKII expression was significantly increased in ventricular heart homogenates of PTFE-treated mice (fig. D). Moreover, the magnitude of CaMKII overexpression correlated significantly with the frequency of apneas (fig. E). Papillary muscle post-pause contractions can be used as measure of diastolic sarcoplasmic reticulum (SR) Ca leak, which is known to be enhanced by CaMKII. Compared to control, post-pause contraction amplitude was significantly smaller in PTFE-treated mice, indicating an increased SR Ca leak (fig. F). Figure 1 Conclusion PTFE injection in mice results in an increased frequency of spontaneous apneas. PTFE-treated mice develop mild contractile dysfunction, which is accompanied by CaMKII overexpression. This novel mouse model offers great opportunities for investigation of sleep-related breathing disorders. Acknowledgement/Funding This study was supported by the Medical Faculty of the University of Regensburg (ReForM programme).

Misaligned Feeding May Aggravate Pain by Disruption of Sleep-Awake Rhythm.

Increasing evidence suggests that patients with eating disorders are more likely to develop chronic pain. A misaligned diet has been reported to disrupt the sleep-awake rhythms. Combined with our previous investigation on circadian pain, we aimed to investigate the role of misaligned diet in the pain sensitivity and the underlying mechanisms. Two-month-old C57BL/6J male mice were administered chronic constriction injury (CCI) surgery to establish neuropathic pain models. CCI mice were randomized to scheduled food access throughout the whole day (CCI-free), during the daytime (CCI-misaligned), and at night (CCI-aligned), respectively. The paw withdrawal mechanical threshold, indicating pain behavior, was measured by Von Frey. The gross motor activity pattern indicating the sleep-awake rhythm was monitored by Mini-Mitter. Melatonin (Mel) was administered to ameliorate the sleep-awake rhythm (CCI-free + Mel and CCI-misaligned + Mel). The expressions of circadian pain-related proteins were detected by quantitative polymerase chain reaction and western blot. The primary outcome is the pain threshold and the secondary outcome is the sleep-awake rhythm. Misaligned diet during the peri-CCI surgery period significantly decreased the paw withdrawal mechanical threshold compared with the CCI-free mice (day 14: 0.40 ± 0.09 vs 0.64 ± 0.15; P = .03;) and altered the sleep-awake rhythm. Mel pretreatment alleviated the increased pain (day 14, CCI-misaligned + Mel versus CCI-misaligned: day 14: 0.60 ± 0.13 vs 0.35 ± 0.12; P = .022) and the disrupted sleep-awake rhythm caused by misaligned feeding. The mRNA levels of N-methyl-D-aspartate receptor subtype 2B (NR2B), Ca/calmodulin-dependent protein kinase II (CaMKII), and cyclic adenosine monophosphate-response element binding protein (CREB) in the spinal dorsal horn increased in CCI-misaligned mice compared with the CCI-free mice. The phosphor-NR2B, phosphor-CaMKII, and phosphor-CREB also increased in CCI-misaligned mice compared with the CCI-free mice. However, the expressions of NR2B, CaMKII, and CREB were decreased in CCI-misaligned + Mel mice compared to CCI-misaligned mice at both transcriptional and translational levels. Misaligned diet might aggravate pain sensitivity through the disruption of the sleep-awake cycle, which could be recovered by Mel. NR2B-CaMKII-CREB may participate in the disruption of sleep-awake rhythm-mediated pain aggravation.

NMDA receptor subunit and CaMKII changes in rat hippocampus by congenital HCMV infection: a mechanism for learning and memory impairment.

The aim of this study was to investigate the effects of congenital human cytomegalovirus infection on the expression levels of N-methyl-D-aspartate receptors (NRs) and Ca/calmodulin-dependent protein kinase II (CaMKII) in the hippocampal neurons of neonatal Sprague-Dawley (SD) rats. Pregnant SD rats were divided into an experimental group and a control group (n=10 in each group). Spatial learning and memory of the offspring of SD rats were evaluated using the Morris water-maze test. Pathological studies of hippocampus sections were carried out. The concentration of [Ca] was measured using a dual-wavelength spectrophotometer method. The expression levels of NRs were detected by an immunohistochemical study. Western blot was performed to detect the expression level of CaMKII. In the Morris water-maze test, the rats in the experimental group showed significantly increased escape latency and distance traveled than the control group. Damaged and structural disorders of the dentate granule in the hippocampus were found in the experimental rats. Immunohistochemistry results showed that the expression levels of NR subunits in the hippocampus of the experimental group were significantly decreased. The concentration of [Ca] in the experimental group was significantly increased. In contrast, the level of CaMKII in the experimental group was significantly decreased. The expressions of the NR subunit and CaMKII were decreased in rat hippocampus by human cytomegalovirus congenital infection, which may be associated with the mechanism underlying the impairment of learning and memory function.

CaMKII inhibition reduces isoproterenol-induced ischemia and arrhythmias in hypertrophic mice.

ObjectivesThe Ca/calmodulin-dependent protein kinase II (CaMKII), an arrhythmogenic molecule, is excessively activated in cardiac hypertrophy. Here, we investigated the effect of CaMKII inhibition in isoproterenol (ISO)-induced arrhythmias in hypertrophic mice.ResultsISO induced multiple types of arrhythmias in the hypertrophic mice but not in the normal mice. The QTc intervals were prolonged and the amplitudes of T waves were increased significantly by ISO prior to arrhythmia initiation. Inhibition of CaMKII prevented ISO-induced QTc prolongation and T wave elevation and abrogated arrhythmia induction.Materials and MethodsPressure-overload cardiac hypertrophy was induced in mice by thoracic aortic banding. Arrhythmias were recorded by electrocardiogram in conscious mice.ConclusionsCaMKII inhibition is effective in suppressing adrenergic activation-induced ventricular arrhythmias in cardiac hypertrophy, of which the ventricular ischemia-induced CaMKII activation plays an important role.

CaMKII inhibition promotes neuronal apoptosis by transcriptionally upregulating Bim expression.

The effects of Ca/calmodulin-dependent protein kinase II (CaMKII) on neuronal apoptosis are complex and contradictory, and the underlying mechanisms remain unclear. Bcl-2-interacting mediator of cell death (Bim) is an important proapoptotic protein under many physiological and pathophysiological conditions. However, there is no evidence that CaMKII and Bim are mechanistically linked in neuronal apoptosis. In this study, we showed that CaMKII inhibition by the inhibitors KN-62 and myristoylated autocamtide-2-related inhibitory peptide promoted apoptosis in cerebellar granule neurons in a dose-dependent manner. CaMKII inhibition increased Bim protein and messenger RNA levels. The expression of early growth response factor-1, a transcription factor of Bim, was also induced by CaMKII inhibitors. These data suggested that CaMKII repressed the transcriptional expression of Bim. Moreover, knockdown of Bim using small interfering RNAs attenuated the proapoptotic effects of CaMKII inhibition. Taken together, this is the first report to show that CaMKII inhibition transcriptionally upregulates Bim expression to promote neuronal apoptosis, providing new insights into the proapoptotic mechanism of CaMKII inhibition.

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance.

CaMKII and Heart Failure Promote a Pathological Ryanodine Receptor Conformation that Reduces Calmodulin Binding and Enhances SR Ca2+ Leak

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). RyR2 phosphorylation (at Ser2814) by Ca/calmodulin-dependent protein kinase II (CaMKII) is thought to be a critical promoter of leaky RyR2 in HF and arrhythmias. A conformational change in RyR2 (“domain unzipping”) observed in HF can increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and CaM binding affinity for RyR2 is reduced in HF. Here, we tested the hypothesis that CaMKII-dependent phosphorylation of RyR2 increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM-RyR2 binding affinity. Using non-phosphorylatable RyR2-S2814A and phosphomimetic RyR2-S2814D knock-in mice, fluorescence resonance energy transfer (FRET) was measured to directly detect binding of fluorescently labeled DPc10 (RyR2 unzipping peptide), FK506-binding protein12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In S2814D vs. S2814A: (1) Ca sparks and waves were increased; (2) CaM-RyR2 binding affinity was reduced 3-fold, but FRETmax was unaltered; and (3) F-DPc10 accessibility was 2-fold faster, reflecting unzipped-conformation. Dantrolene (1 μM), which stabilizes the zipped conformation: (1) restored normal CaM-RyR2 binding affinity; (2) reduced DPc10 access (zipped-conformation); and (3) reduced frequency of Ca sparks and waves in S2814D. Furthermore, in rabbit non-ischemic HF myocytes, in which RyR2 is more highly phosphorylated by CaMKII, vs. age-matched control: (1) The CaM-RyR2 binding affinity was reduced by 2.5-fold; and (2) F-DPc10 accessibility was 1.5-fold faster. Dantrolene also restored CaM-RyR2 binding affinity and kept zipped conformation in rabbit HF. We conclude that CaMKII-dependent phosphorylation of RyR2 promotes a pathological RyR2 conformation with reduced CaM binding affinity and elevated Ca leakiness. Dantrolene reverses this conformational change and restores normal CaM binding affinity in S2814D mouse and rabbit HF.

Human G109E-inhibitor-1 impairs cardiac function and promotes arrhythmias

A hallmark of human and experimental heart failure is deficient sarcoplasmic reticulum (SR) Ca-uptake reflecting impaired contractile function. This is at least partially attributed to dephosphorylation of phospholamban by increased protein phosphatase 1 (PP1) activity. Indeed inhibition of PP1 by transgenic overexpression or gene-transfer of constitutively active inhibitor-1 improved Ca-cycling, preserved function and decreased fibrosis in small and large animal models of heart failure, suggesting that inhibitor-1 may represent a potential therapeutic target. We recently identified a novel human polymorphism (G109E) in the inhibitor-1 gene with a frequency of 7% in either normal or heart failure patients. Transgenic mice, harboring cardiac-specific expression of G109E inhibitor-1, exhibited decreases in contractility, Ca-kinetics and SR Ca-load. These depressive effects were relieved by isoproterenol stimulation. Furthermore, stress conditions (2Hz +/− Iso) induced increases in Ca-sparks, Ca-waves (60% of G109E versus 20% in wild types) and after-contractions (76% of G109E versus 23% of wild types) in mutant cardiomyocytes. Similar findings were obtained by acute expression of the G109E variant in adult cardiomyocytes in the absence or presence of endogenous inhibitor-1. The underlying mechanisms included reduced binding of mutant inhibitor-1 to PP1, increased PP1 activity, and dephosphorylation of phospholamban at Ser16 and Thr17. However, phosphorylation of the ryanodine receptor at Ser2808 was not altered while phosphorylation at Ser2814 was increased, consistent with increased activation of Ca/calmodulin-dependent protein kinase II (CaMKII), promoting aberrant SR Ca-release. Parallel in vivo studies revealed that mutant mice developed ventricular ectopy and complex ventricular arrhythmias (including bigeminy, trigeminy and ventricular tachycardia), when challenged with isoproterenol. Inhibition of CaMKII activity by KN-93 prevented the increased propensity to arrhythmias. These findings suggest that the human G109E inhibitor-1 variant impairs SR Ca-cycling and promotes arrhythmogenesis under stress conditions, which may present an additional insult in the compromised function of heart failure carriers.

O‐GlcNAcylation of CaMKII in Cardiac Myocytes can be Pro‐Arrhythmic

We found that high extracellular [glucose] (Hi‐Gluc) increased the activity level of Ca‐Calmodulin dependent protein kinase II (CaMKII) in adult cardiac myocytes, using both traditional biochemical activity measurements as well as a FRET based activation state reporter, Camui. Hi‐Gluc can induce O‐GlcNAcylation (covalent O‐linked β‐D‐N‐acetylglucosamine, GlcNAc) at Ser/Thr residues, and is a ‐translational modification of proteins (often at or near kinase target sites). GlcNAcylation is dynamic and is catalyzed by O‐GlcNAc transferase (OGT) (which is inhibitable upstream by DON) and GlcNAc is removed by O‐GlcNAase (which can be blocked by Thiamet G). Hi‐Gluc‐induced CaMKII activation was blocked by DON, promoted by Thiamet G and isoproterenol (ISO), and also promoted autonomous CaMKII activation (similar to that induced by autophosphorylation and oxidation). Mutation of CaMKII (S279A), adjacent to the known oxidation site prevented Hi‐Gluc –dependent activation, but did not prevent autonomous activation by autophosphorylation or oxidation. Mass spec confirmed S279 as a site of GlcNAcylation, and CaMKII was more highly GlcNAcylated in diabetic hearts and brains (human and rat). Hi‐Gluc caused activation of cardiac myocyte Ca sparks, action potential alterations and cardiac arrhythmias. These effects were prevented by either CaMKII or GlcNAc inhibition (DON) and in genetic CaMKIIδ‐KO mice. This new pathway may be an important pathophysiological synergy between GlcNAcylation and CaMKII signaling relevant in diabetic hyperglycemia.

Enhanced late INa induces proarrhythmogenic SR Ca leak in a CaMKII-dependent manner

ObjectiveEnhanced late Na current (late INa) induces Na-dependent Ca overload as well as proarrhythmogenic events on the cellular level that include spatio-temporally uncoordinated diastolic Ca release from the sarcoplasmic reticulum (SR) and delayed afterdepolarizations (DADs). The Ca/calmodulin-dependent protein kinase II (CaMKII) gets activated upon increases in [Ca]i and mediates diastolic SR Ca leak as well as DADs. RationaleWe hypothesized that increased late INa (in disease-comparable ranges) exerts proarrhythmogenic events in isolated ventricular mouse myocytes in a manner depending on CaMKII-dependent SR Ca leak. We further tested whether inhibition of disease-related late INa may reduce proarrhythmogenic SR Ca leak in myocytes from failing human hearts. MethodsVentricular myocytes were isolated from healthy wildtype (WT), failing CaMKIIδC transgenic (TG) mouse, and failing human hearts. ATX-II (0.25–10nmol/L) was used to enhance late INa. Spontaneous Ca loss from the SR during diastole (Ca sparks), DADs, non-triggered diastolic Ca transients in myocytes and premature beats of isometrically twitching papillary muscles were used as readouts for proarrhythmogenic events. CaMKII autophosphorylation was assessed by immunoblots. Late INa was inhibited using ranolazine (Ran, 10μmol/L) or TTX (2μmol/L), and CaMKII by KN-93 (1μmol/L) or AIP (1μmol/L). ResultsIn WT myocytes, sub-nanomolar ATX-II exposure (0.5nmol/L) enhanced late INa by ~60%, which resulted in increased diastolic SR Ca loss despite unaltered SR Ca content. In parallel, DADs and non-triggered diastolic Ca transients arose. Inhibition of enhanced late INa by RAN or TTX significantly attenuated diastolic SR Ca loss and suppressed DADs as well as mechanical alternans in mouse and diastolic SR Ca loss in failing human myocytes. ATX-II caused Ca-dependent CaMKII-activation without changes in protein expression, which was reversible by Ran or AIP. Conversely, CaMKII-inhibition decreased diastolic SR Ca loss, DADs and non-triggered diastolic Ca transients despite ATX-II-exposure. Finally, failing mouse myocytes with increased CaMKII activity (TG CaMKIIδC) showed an even aggravated diastolic SR Ca loss that was associated with an increased frequency of non-triggered diastolic Ca transients upon enhanced late INa. ConclusionsIncreased late INa (in disease-comparable ranges) induces proarrhythmogenic events during diastole in healthy and failing mouse myocytes, which are mediated via CaMKII-dependent SR Ca loss. Inhibition of late INa not only attenuated these cellular arrhythmias in mouse myocytes but also in failing human myocytes indicating some antiarrhythmic potential for an inhibition of the elevated late INa/CaMKII signaling pathway in this setting.

A Novel Computational Model of Mouse Myocyte Electrophysiology to Assess the Synergy Between Na Loading and CaMKII

Ca/Calmodulin-dependent protein kinase II (CaMKII) hyperactivity in heart failure causes intracellular Na ([Na]i) loading (at least in part by enhancing the late Na current (INaL)). This [Na]i gain promotes intracellular Ca ([Ca]i) overload by altering the equilibrium of the Na/Ca exchanger to impair forward-mode (Ca extrusion), and favor reverse-mode (Ca influx) exchange. In turn, this Ca overload is expected to further activate CaMKII thereby forming a pathologic positive feedback loop of ever-increasing CaMKII activity, [Na]i, and [Ca]i. We developed a computational framework to interrogate this potentially arrhythmogenic positive feedback in the mouse ventricular myocyte in both control conditions and when CaMKIIδC is overexpressed as in transgenic mice. In control conditions, simulation of an increase in [Na]i causes the expected increases in [Ca]i, CaMKII activity, and target phosphorylation, which degenerate into unstable Ca handling and and electrophysiology at high [Na]i gain. Notably, clamping CaMKII to basal levels ameliorates but does not completely offset this outcome, suggesting that the increase in [Ca]i per se plays an important role. The effect of this CaMKII-Na-Ca-CaMKII feedback is more striking in CaMKIIδC overexpression, where high but not low [Na]i causes delayed afterdepolarizations, which can be prevented by clamping CaMKII phosphorylation of L-type Ca channels, ryanodine receptors and phospholamban to basal levels. In this setting, Na loading fuels a vicious loop whereby increased CaMKII activation perturbs Ca and membrane potential homeostasis. High [Na]i is also required to produce instability when CaMKII is further activated by increased Ca loading due to β-adrenergic activation. Our results support recent experimental findings of a synergistic interaction between perturbed Na fluxes and CaMKII, and suggest that pharmacological inhibition of intracellular Na loading can contribute to normalizing Ca and membrane potential dynamics in heart failure.

CaMKII regulation of cardiac K channels

Cardiac K channels are critical determinants of cardiac excitability. In hypertrophied and failing myocardium, alterations in the expression and activity of voltage-gated K channels are frequently observed and contribute to the increased propensity for life-threatening arrhythmias. Thus, understanding the mechanisms of disturbed K channel regulation in heart failure (HF) is of critical importance. Amongst others, Ca/calmodulin-dependent protein kinase II (CaMKII) has been identified as an important regulator of K channel activity. In human HF but also various animal models, increased CaMKII expression and activity has been linked to deteriorated contractile function and arrhythmias. This review will discuss the current knowledge about CaMKII regulation of several K channels, its influence on action potential properties, dispersion of repolarization, and arrhythmias with special focus on HF.

Ionizing radiation regulates cardiac Ca handling via increased ROS and activated CaMKII

Ionizing radiation (IR) is an integral part of modern multimodal anti-cancer therapies. IR involves the formation of reactive oxygen species (ROS) in targeted tissues. This is associated with subsequent cardiac dysfunction when applied during chest radiotherapy. We hypothesized that IR (i.e., ROS)-dependently impaired cardiac myocytes’ Ca handling might contribute to IR-dependent cardiocellular dysfunction. Isolated ventricular mouse myocytes and the mediastinal area of anaesthetized mice (that included the heart) were exposed to graded doses of irradiation (sham 4 and 20 Gy) and investigated acutely (after ~1 h) as well as chronically (after ~1 week). IR induced a dose-dependent effect on myocytes’ systolic function with acutely increased, but chronically decreased Ca transient amplitudes, which was associated with an acutely unaltered but chronically decreased sarcoplasmic reticulum (SR) Ca load. Likewise, in vivo echocardiography of anaesthetized mice revealed acutely enhanced left ventricular contractility (strain analysis) that declined after 1 week. Irradiated myocytes showed persistently increased diastolic SR Ca leakage, which was acutely compensated by an increase in SR Ca reuptake. This was reversed in the chronic setting in the face of slowed relaxation kinetics. As underlying cause, acutely increased ROS levels were identified to activate Ca/calmodulin-dependent protein kinase II (CaMKII). Accordingly, CaMKII-, but not PKA-dependent phosphorylation sites of the SR Ca release channels (RyR2, at Ser-2814) and phospholamban (at Thr-17) were found to be hyperphosphorylated following IR. Conversely, ROS-scavenging as well as CaMKII-inhibition significantly attenuated CaMKII-activation, disturbed Ca handling, and subsequent cellular dysfunction upon irradiation. Targeted cardiac irradiation induces a biphasic effect on cardiac myocytes Ca handling that is associated with chronic cardiocellular dysfunction. This appears to be mediated by increased oxidative stress and persistently activated CaMKII. Our findings suggest impaired cardiac myocytes Ca handling as a so far unknown mediator of IR-dependent cardiac damage that might be of relevance for radiation-induced cardiac dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-013-0385-6) contains supplementary material, which is available to authorized users.

Role of late sodium current as a potential arrhythmogenic mechanism in the progression of pressure-induced heart disease

The aim of the study was to determine the characteristics of the late Na current (INaL) and its arrhythmogenic potential in the progression of pressure-induced heart disease. Transverse aortic constriction (TAC) was used to induce pressure overload in mice. After one week the hearts developed isolated hypertrophy with preserved systolic contractility. In patch-clamp experiments both, INaL and the action potential duration (APD90) were unchanged. In contrast, after five weeks animals developed heart failure with prolonged APDs and slowed INaL decay time which could be normalized by addition of the INaL inhibitor ranolazine (Ran) or by the Ca/calmodulin-dependent protein kinase II (CaMKII) inhibitor AIP. Accordingly the APD90 could be significantly abbreviated by Ran, tetrodotoxin and the CaMKII inhibitor AIP. Isoproterenol increased the number of delayed afterdepolarizations (DAD) in myocytes from failing but not sham hearts. Application of either Ran or AIP prevented the occurrence of DADs. Moreover, the incidence of triggered activity was significantly increased in TAC myocytes and was largely prevented by Ran and AIP. Western blot analyses indicate that increased CaMKII activity and a hyperphosphorylation of the Nav1.5 at the CaMKII phosphorylation site (Ser571) paralleled our functional observations five weeks after TAC surgery. In pressure overload-induced heart failure a CaMKII-dependent augmentation of INaL plays a crucial role in the AP prolongation and generation of cellular arrhythmogenic triggers, which cannot be found in early and still compensated hypertrophy. Inhibition of INaL and CaMKII exerts potent antiarrhythmic effects and might therefore be of potential therapeutic interest. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

Redox Regulation of Sodium and Calcium Handling

In heart failure (HF), contractile dysfunction and arrhythmias result from disturbed intracellular Ca handling. Activated stress kinases like cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), and Ca/calmodulin-dependent protein kinase II (CaMKII), which are known to influence many Ca-regulatory proteins, are mechanistically involved. Beside classical activation pathways, it is becoming increasingly evident that reactive oxygen species (ROS) can directly oxidize these kinases, leading to alternative activation. Since HF is associated with increased ROS generation, ROS-activated serine/threonine kinases may play a crucial role in the disturbance of cellular Ca homeostasis. Many of the previously described ROS effects on ion channels and transporters are possibly mediated by these stress kinases. For instance, ROS have been shown to oxidize and activate CaMKII, thereby increasing Na influx through voltage-gated Na channels, which can lead to intracellular Na accumulation and action potential prolongation. Consequently, Ca entry via activated NCX is favored, which together with ROS-induced dysfunction of the sarcoplasmic reticulum can lead to dramatic intracellular Ca accumulation, diminished contractility, and arrhythmias. While low amounts of ROS may regulate kinase activity, excessive uncontrolled ROS production may lead to direct redox modification of Ca handling proteins. Therefore, depending on the source and amount of ROS generated, ROS could have very different effects on Ca-handling proteins. The discrimination between fine-tuned ROS signaling and unspecific ROS damage may be crucial for the understanding of heart failure development and important for the investigation of targeted treatment strategies.

CaMKII-dependent SR Ca leak contributes to doxorubicin-induced impaired Ca handling in isolated cardiac myocytes

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR). We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca] i) handling. Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ −/−, KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 μmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS] i) and [Ca] i handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 μmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 μmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation. DOX increased [ROS] i and led to significant diastolic [Ca] i overload in rat myocytes. This was associated with reduced [Ca] i transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca] i overload after DOX superfusion and led to partially restored [Ca] i transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca] i overload and Ca spark frequency. DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca] i homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca] i. In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.