Jeffrey R. Erickson

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca2+ handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca2+ concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca2+ release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

Fluorescence Resonance Energy Transfer–Based Sensor Camui Provides New Insight Into Mechanisms of Calcium/Calmodulin-Dependent Protein Kinase II Activation in Intact Cardiomyocytes

Rationale: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited. Objective: We use Camui, a novel FRET-based biosensor in which full-length CaMKII is flanked by CFP and YFP, to measure CaMKII activation state in living rabbit myocytes. Methods and Results: We show that Camui and mutant variants that lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKII&dgr; activation state. Camui (wild-type or mutant) was expressed in isolated adult cardiac myocytes, and localization and CaMKII activation state were determined using confocal microscopy. Camui, like CaMKII&dgr;, is concentrated at the z-lines, with low baseline activation state. Camui activation increased directly with pacing frequency, but the maximal effect was blunted with the T286A, consistent with frequency-dependent phosphorylation of CaMKII at T286 mainly at high-frequency and high-amplitude Ca transients. Camui was also activated by 4 neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely through an oxidation-dependent mechanism, whereas isoproterenol- and phenylephrine-mediated mechanisms had a significant autophosphorylation-dependent component. Conclusions: Camui is a novel, nondestructive tool that allows spatiotemporally resolved measurement of CaMKII activation state in physiologically functioning myocytes. This represents a first step in using Camui to elucidate key mechanistic details of CaMKII signaling in live hearts and myocytes.Rationale Calcium/calmodulin dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited.

Mechanisms of CaMKII activation in the heart

Calcium/calmodulin (Ca2+/CaM) dependent protein kinase II (CaMKII) has emerged as a key nodal protein in the regulation of cardiac physiology and pathology. Due to the particularly elegant relationship between the structure and function of the kinase, CaMKII is able to translate a diverse set of signaling events into downstream physiological effects. While CaMKII is typically autoinhibited at basal conditions, prolonged rapid Ca2+ cycling can activate the kinase and allow post-translational modifications that depend critically on the biochemical environment of the heart. These modifications result in sustained, autonomous CaMKII activation and have been associated with pathological cardiac signaling. Indeed, improved understanding of CaMKII activation mechanisms could potentially lead to new clinical therapies for the treatment or prevention of cardiovascular disease. Here we review the known mechanisms of CaMKII activation and discuss some of the pathological signaling pathways in which they play a role.

CaMKIIδ mediates β-adrenergic effects on RyR2 phosphorylation and SR Ca2 + leak and the pathophysiological response to chronic β-adrenergic stimulation

Chronic activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the deleterious effects of β-adrenergic receptor (β-AR) signaling on the heart, in part, by enhancing RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) leak. We used CaMKIIδ knockout (CaMKIIδ-KO) mice and knock-in mice with an inactivated CaMKII site S2814 on the ryanodine receptor type 2 (S2814A) to investigate the involvement of these processes in β-AR signaling and cardiac remodeling. Langendorff-perfused hearts from CaMKIIδ-KO mice showed inotropic and chronotropic responses to isoproterenol (ISO) that were similar to those of wild type (WT) mice; however, in CaMKIIδ-KO mice, CaMKII phosphorylation of phospholamban and RyR2 was decreased and isolated myocytes from CaMKIIδ-KO mice had reduced SR Ca(2+) leak in response to isoproterenol (ISO). Chronic catecholamine stress with ISO induced comparable increases in relative heart weight and other measures of hypertrophy from day 9 through week 4 in WT and CaMKIIδ-KO mice, but the development of cardiac fibrosis was prevented in CaMKIIδ-KO animals. A 4-week challenge with ISO resulted in reduced cardiac function and pulmonary congestion in WT, but not in CaMKIIδ-KO or S2814A mice, implicating CaMKIIδ-dependent phosphorylation of RyR2-S2814 in the cardiomyopathy, independent of hypertrophy, induced by prolonged β-AR stimulation.

Novel Epac fluorescent ligand reveals distinct Epac1 vs. Epac2 distribution and function in cardiomyocytes

Significance β-Adrenergic activation in heart has many important effects (physiological and pathological) that had been mainly attributed to cAMP and protein kinase A-dependent signaling. The discovery of exchange protein directly activated by cAMP (Epac1 and Epac2) has uncovered new parallel cAMP signaling pathways in β-adrenergic signaling, but Epac isoform localization and function were not well understood. Here we show, using a novel fluorescent Epac analog, that Epac1 and Epac2 are differentially concentrated at the nucleus vs. at Z lines in cardiomyocytes, and that this corresponds to functional roles in nucleus vs. calcium handling and arrhythmias, respectively. This finding may be essential to develop more targeted therapeutics for cardiomyopathy. Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in β-adrenergic–dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2′-O-methyladenosine-3′,5′-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3′-,5′-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT–induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.

S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ

Background: CaMKIIδ and NO can modulate cardiac signaling/pathology. Results: NO treatment after calcium/calmodulin binding prolongs CaMKIIδ activation, whereas NO pretreatment inhibits CaMKIIδ activation, effects mediated by Cys-290 and Cys-273, respectively. Conclusion: S-nitrosylation has a dual role in modulating CaMKIIδ in the heart. Significance: Dual regulation by NO is a new pathway by which CaMKII can modulate cardiac function. NO is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implied that NO can activate calcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) in neurons and the heart. Here we use our novel sensor of CaMKII activation, Camui, to monitor changes in the conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca2+/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the Cys-290 site. However, exposure of CaMKIIδ to GSNO prior to Ca2+/CaM exposure strongly suppresses kinase activation and conformational change by Ca2+/CaM. This NO-induced inhibition was ablated by mutation of the Cys-273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity.

Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes

Both the ATP‐sensitive potassium (KATP) channel and the gaseous messenger nitric oxide (NO) play fundamental roles in protecting the heart from injuries related to ischaemia. NO has previously been suggested to modulate cardiac KATP channels; however, the underlying mechanism remains largely unknown. In this study, by performing electrophysiological and biochemical assays, we demonstrate that NO potentiation of KATP channel activity in ventricular cardiomyocytes is prevented by pharmacological inhibition of soluble guanylyl cyclase (sGC), cGMP‐dependent protein kinase (PKG), Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and extracellular signal‐regulated protein kinase 1/2 (ERK1/2), by removal of reactive oxygen species and by genetic disruption of CaMKIIδ. These results suggest that NO modulates cardiac KATP channels via a novel cGMP–sGC–cGMP–PKG–ROS–ERK1/2–calmodulin–CaMKII (δ isoform in particular) signalling cascade. This novel intracellular signalling pathway may regulate the excitability of heart cells and provide protection against ischaemic or hypoxic injury, by opening the cardioprotective KATP channels.

Cardiac CaMKIIδ splice variants exhibit target signaling specificity and confer sex-selective arrhythmogenic actions in the ischemic-reperfused heart

BACKGROUND Ischemia-related arrhythmic incidence is generally lower in females (vs males), though risk is selectively increased in women with underlying cardiopathology. Ca(2+)/calmodulin dependent kinase II (CaMKII) has been implicated in ischemia/reperfusion arrhythmias, yet the role of CaMKII in the ischemic female heart has not been determined. The aim of this study was to define the role and molecular mechanism of CaMKII activation in reperfusion arrhythmias in male/female hearts. METHODS AND RESULTS Male and female rat hearts and cardiomyocytes were subjected to multiple arrhythmogenic challenges. An increased capacity to upregulate autophosphorylated CaMKII (P-CaMKII) in Ca(2+)-challenged female hearts was associated with an enhanced ability to maintain diastolic function. In ischemia/reperfusion, female hearts (vs male) exhibited less arrhythmias (59 ± 18 vs 548 ± 9, s, p<0.05), yet had augmented P-CaMKII (2.69 ± 0.30 vs 1.50 ± 0.14, rel. units, p<0.05) and downstream phosphorylation of phospholamban (1.71 ± 0.42 vs 0.90 ± 0.10, p<0.05). In contrast, hypertrophic female hearts had more reperfusion arrhythmias and lower phospholamban phosphorylation. Isolated myocyte experiments (fura-2) confirmed Ca(2+)-handling arrhythmogenic involvement. Molecular analysis showed target specificity of CaMKII was determined by post-translational modification, with CaMKIIδB and CaMKIIδC splice variants selectively co-localized with autophosphorylation and oxidative modifications of CaMKII respectively. CONCLUSIONS This study provides new mechanistic evidence that CaMKIIδ splice variants are selectively susceptible to autophosphorylation/oxidation, and that augmented generation of P-CaMKIIδB(Thr287) is associated with arrhythmia suppression in the female heart. Collectively these findings indicate that therapeutic approaches based on selective CaMKII splice form targeting may have potential benefit, and that sex-selective CaMKII intervention strategies may be valid.

The role of CaMKII in diabetic heart dysfunction

Diabetes mellitus (DM) is an increasing epidemic that places a significant burden on health services worldwide. The incidence of heart failure (HF) is significantly higher in diabetic patients compared to non-diabetic patients. One underlying mechanism proposed for the link between DM and HF is activation of calmodulin-dependent protein kinase (CaMKIIδ). CaMKIIδ mediates ion channel function and Ca2+ handling during excitation–contraction and excitation-transcription coupling in the myocardium. CaMKIIδ activity is up-regulated in the myocardium of diabetic patients and mouse models of diabetes, where it promotes pathological signaling that includes hypertrophy, fibrosis and apoptosis. Pharmacological inhibition and knockout models of CaMKIIδ have shown some promise of a potential therapeutic benefit of CaMKIIδ inhibition, with protection against cardiac hypertrophy and apoptosis reported. This review will highlight the pathological role of CaMKIIδ in diabetes and discuss CaMKIIδ as a therapeutic target in DM, and also the effects of exercise on CaMKIIδ.

FRET-based sensor Camui provides new insight into mechanisms of CaMKII activation in intact cardiomyocytes

Rationale: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited. Objective: We use Camui, a novel FRET-based biosensor in which full-length CaMKII is flanked by CFP and YFP, to measure CaMKII activation state in living rabbit myocytes. Methods and Results: We show that Camui and mutant variants that lack the sites of CaMKII autophosphorylation (T286A) and oxidative regulation (CM280/1VV) serve as useful biosensors for CaMKII&dgr; activation state. Camui (wild-type or mutant) was expressed in isolated adult cardiac myocytes, and localization and CaMKII activation state were determined using confocal microscopy. Camui, like CaMKII&dgr;, is concentrated at the z-lines, with low baseline activation state. Camui activation increased directly with pacing frequency, but the maximal effect was blunted with the T286A, consistent with frequency-dependent phosphorylation of CaMKII at T286 mainly at high-frequency and high-amplitude Ca transients. Camui was also activated by 4 neurohormonal agonists. Angiotensin II and endothelin-1 activated Camui, largely through an oxidation-dependent mechanism, whereas isoproterenol- and phenylephrine-mediated mechanisms had a significant autophosphorylation-dependent component. Conclusions: Camui is a novel, nondestructive tool that allows spatiotemporally resolved measurement of CaMKII activation state in physiologically functioning myocytes. This represents a first step in using Camui to elucidate key mechanistic details of CaMKII signaling in live hearts and myocytes.Rationale Calcium/calmodulin dependent protein kinase II (CaMKII) is a key mediator of intracellular signaling in the heart. However, the tools currently available for assessing dynamic changes in CaMKII localization and activation in living myocytes are limited.