Autocamtide-2-related Inhibitory Peptide Research Articles

Empagliflozin inhibits increased Na influx in atrial cardiomyocytes of patients with HFpEF.

Heart failure with preserved ejection fraction (HFpEF) causes substantial morbidity and mortality. Importantly, atrial remodeling and atrial fibrillation is frequently observed in HFpEF. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have recently been shown to improve clinical outcomes in HFpEF, and post-hoc analyses suggest atrial antiarrhythmic effects. We tested if isolated human atrial cardiomyocytes from patients with HFpEF exhibit an increased Na influx, which is known to cause atrial arrhythmias, and if that is responsive to treatment with the SGTL2i empagliflozin. Cardiomyocytes were isolated from atrial biopsies of 124 patients (82 with HFpEF) undergoing elective cardiac surgery. Na influx was measured with the Na-dye Asante Natrium Green-2 AM (ANG-2). Compared to patients without heart failure (NF), Na influx was doubled in HFpEF patients (NF vs HFpEF: 0.21±0.02 vs 0.38±0.04 mmol/L/min (N=7 vs 18); p=0.0078). Moreover, late INa (measured via whole-cell patch clamp) was significantly increased in HFpEF compared to NF. Western blot and HDAC4 pulldown assay indicated a significant increase in CaMKII expression, CaMKII autophosphorylation, CaMKII activity, and CaMKII-dependent NaV1.5 phosphorylation in HFpEF compared to NF, whereas NaV1.5 protein and mRNA abundance remained unchanged. Consistently, increased Na influx was significantly reduced by treatment with the CaMKII inhibitor autocamtide-2 related inhibitory peptide (AIP), late INa inhibitor tetrodotoxin (TTX) but also with NHE1 inhibitor cariporide. Importantly, empagliflozin abolished both increased Na influx and late INa in HFpEF. Multivariate linear regression analysis, adjusting for important clinical confounders, revealed HFpEF to be an independent predictor for changes in Na handling in atrial cardiomyocytes. We show for the first time increased Na influx in human atrial cardiomyocytes from HFpEF patients, partly due to increased late INa and enhanced NHE1-mediated Na influx. Empagliflozin inhibits Na influx and late INa, which could contribute to antiarrhythmic effects in patients with HFpEF.

Hippocampal CaMKII inhibition induces reactivation-dependent amnesia for extinction memory and causes fear relapse

Hippocampal GluN2B subunit-containing NMDAR (GluN2B-NMDAR) activation during recall destabilizes fear extinction memory, which must undergo brain-derived neurotrophic factor (BDNF)-dependent reconsolidation to persist. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr protein kinase essential for hippocampus-dependent memory processing that acts downstream GluN2B-NMDAR and controls BDNF expression, but its participation in fear extinction memory reconsolidation has not yet been studied. Using a combination of pharmacological and behavioral tools, we found that in adult male Wistar rats, intra dorsal-CA1 administration of the CaMKII inhibitors autocamtide-2-related inhibitory peptide (AIP) and KN-93, but not of their inactive analogs scrambled AIP and KN-92, after fear extinction memory recall impaired extinction and caused GluN2B-NMDAR-dependent recovery of fear. Our results indicate that hippocampal CaMKII is necessary for fear extinction reconsolidation, and suggest that modulation of its activity around the time of recall controls the inhibition that extinction exerts on learned fear.

The CaMKII inhibitory peptide AIP alleviates renal fibrosis through the TGF-β/Smad and RAF/ERK pathways.

Renal fibrosis is characterized by the excessive deposition of extracellular matrix that destroys and replaces the functional renal parenchyma, ultimately leading to organ failure. It is a common pathway by which chronic kidney disease (CKD) can develop into end-stage renal disease (ESRD), which has high global morbidity and mortality, and there are currently no good therapeutic agents available. CaMKII has been indicated to be closely related to the occurrence of renal fibrosis, and its specific inhibitory peptide AIP, was shown to directly bind the active site of CaMKII. In this study, we examined the effect of AIP on the progression of renal fibrosis and its possible mechanism. The results showed that AIP could inhibit the expression of the fibrosis markers Fibronectin, Collagen I, MMP2 and α-SMA in vivo and in vitro. Further analysis revealed that AIP could inhibit the expression of various epithelial-to-mesenchymal transformation (EMT)-related markers, such as Vimentin and Snail 1, in vivo and in vitro. Mechanistically, AIP could significantly inhibit the activation of CaMKII, Smad 2, Raf and ERK in vitro and in vivo and reduce the expression of TGF-β in vivo. These results suggested that AIP could alleviate renal fibrosis by inhibiting CaMKII and blocking activation of the TGF-β/Smad2 and RAF/ERK pathways. Our study provides a possible drug candidate and demonstrates that CaMKII is a potential pharmacological target for the treatment of renal fibrosis. Significance Statement We have demonstrated that AIP significantly attenuated TGF-β1-induced fibrogenesis and ameliorated UUO-induced renal fibrosis through the CaMKII/TGF-β/Smad and CaMKII/RAF/ERK signaling pathways in vitro and in vivo. Our study provides a possible drug candidate and demonstrates that CaMKII can be a potential pharmacological target for the treatment of renal fibrosis.

Calcium/calmodulin-dependent protein kinase II is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rat nucleus accumbens.

Synaptic plasticity contributes to nociceptive signal transmission and modulation, with calcium/calmodulin-dependent protein kinase II (CaMK II) playing a fundamental role in neural plasticity. This research was conducted to investigate the role of CaMK II in the transmission and regulation of nociceptive information within the nucleus accumbens (NAc) of naïve and morphine-tolerant rats. Randall Selitto and hot-plate tests were utilized to measure the hindpaw withdrawal latencies (HWLs) in response to noxious mechanical and thermal stimuli. To induce chronic morphine tolerance, rats received intraperitoneal morphine injection twice per day for seven days. CaMK II expression and activity were assessed using western blotting. Intra-NAc microinjection of autocamtide-2-related inhibitory peptide (AIP) induced an increase in HWLs in naïve rats in response to noxious thermal and mechanical stimuli. Moreover, the expression of the phosphorylated CaMK II (p-CaMK II) was significantly decreased as determined by western blotting. Chronic intraperitoneal injection of morphine resulted in significant morphine tolerance in rats on Day 7, and an increase of p-CaMK II expression in NAc in morphine-tolerant rats was observed. Furthermore, intra-NAc administration of AIP elicited significant antinociceptive responses in morphine-tolerant rats. In addition, compared with naïve rats, AIP induced stronger thermal antinociceptive effects of the same dose in rats exhibiting morphine tolerance. This study shows that CaMK II in the NAc is involved in the transmission and regulation of nociception in naïve and morphine-tolerant rats.

Empagliflozin inhibits increased Na influx in HFpEF cardiomyocytes and reduces arrhythmic activity in human atrial trabeculae

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is a major health problem associated with substantial morbidity and mortality. However, the underlying pathophysiological mechanisms are poorly understood, and effective treatment strategies are scarce. Importantly, SGLT2i, which have been suggested to improve cellular Na and Ca homeostasis in HFrEF, have recently been shown to also improve clinical outcomes in patients with HFpEF. Interestingly, post-hoc analyses of clinical data suggest an involvement of anti-arrhythmic effects of SGLT2i. Purpose We tested, if isolated human atrial cardiomyocytes from patients with HFpEF exhibit an increased Na influx that is responsive to treatment with the SGLT2i empagliflozin (Empa) and if Empa has anti-arrhythmic properties in human atrial trabeculae. Methods Atrial biopsies were obtained from 101 patients undergoing elective cardiac surgery. Na influx was measured as increase in [Na]i during Na/K-ATPase inhibition in isolated cardiomyocytes loaded with the Na-sensitive fluorescence dye Asante Natrium Green–2 AM (ANG-2). Western Blot and HDAC4 pulldown assay were used to investigate NaV1.5 expression/phosphorylation as well as CaMKII expression/autophosphorylation and activity. Anti-arrhythmic effects of Empa were evaluated as the reduction in premature atrial complexes (PACs), which were induced in electrically field-stimulated (1Hz) human atrial trabeculae by superfusion with isoproterenol (100 nM) and high Ca (3.5 mM). Results Compared to patients without heart failure (NF), Na influx was almost doubled in HFpEF patients (NF vs HFpEF: 0.21±0.02 vs 0.38±0.04 mmol/L/min (N=7 vs 18); p=0.005) (Fig. 1D, E). CaMKII expression, CaMKII autophosphorylation, CaMKII activity, and CaMKII-dependent NaV1.5 phosphorylation were significantly increased in atrial biopsies of HFpEF patients, whereas NaV1.5 protein abundance remained unchanged (Fig. 1A–C). Consistent with these results, the increased Na influx was significantly reduced by treatment with the specific CaMKII inhibitor autocamtide-2 related inhibitory peptide (AIP) and the late INa inhibitor tetrodotoxin (TTX) (Fig. 1D, E). Importantly, Empa also abolished the increased Na influx in HFpEF cardiomyocytes (Fig. 1D, E). Multivariate linear regression analysis, adjusting for clinical co-variates, revealed HFpEF to be an independent predictor of cardiomyocyte Na handling. In line with Empa-mediated inhibition of Na influx, the frequency of PACs in human atrial trabeculae was significantly reduced by Empa (Fig. 1F, G). Conclusion This is the first study to demonstrate increased Na influx in human cardiomyocytes from HFpEF patients potentially by an increased CaMKII-dependent NaV1.5 phosphorylation. Excitingly, treatment with Empa decreases this Na influx in HFpEF cardiomyocytes and reduces isoproterenol-induced arrhythmic activity in human atrial trabeculae, which could contribute to the cardioprotective effects of this drug in patients with HFpEF. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Else Kröner-Fresenius-Stiftung,Deutsche Forschungsgemeinschaft

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Transient Elevation of Glucose Increases Arrhythmia Susceptibility in Non-Diabetic Rat Trabeculae With Non-Uniform Contraction.

In non-diabetic patients with acute coronary syndrome, stress hyperglycemia occasionally occurs and is related to their mortality. Whether transient elevation of glucose affects arrhythmia susceptibility in non-diabetic hearts with non-uniform contraction was examined.Methods and Results:Force, intracellular Ca2+([Ca2+]i), and membrane potential were measured in trabeculae from rat hearts. Non-uniform contraction was produced by a jet of paralyzing solution. Ca2+waves and arrhythmias were induced by electrical stimulation (2.0 mmol/L [Ca2+]o). The activity of Ca2+/calmodulin-dependent protein kinaseII (CaMKII) was measured. An elevation of glucose from 150 to 400 mg/dL increased the velocity of Ca2+waves and the number of spontaneous action potentials triggered by electrical stimulation. Besides, the elevation of glucose increased the CaMKII activity. In the presence of 1 μmol/L KN-93, the elevation of glucose did not increase the velocity of Ca2+waves and the number of triggered action potentials. In addition, in the presence of 1 μmol/L autocamtide-2 related inhibitory peptide or 50 μmol/L diazo-5-oxonorleucine, the elevation of glucose did not increase the number of triggered action potentials. Furthermore, the elevation of glucose by adding L-glucose did not increase their number. In non-diabetic hearts with non-uniform contraction, transient elevation of glucose increases the velocity of Ca2+waves by activating CaMKII,probably through glycosylation with O-linked β-N-acetylglucosamine, thereby increasing arrhythmia susceptibility.

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.

The CaMKII phosphorylation site Thr1604 in the CaV1.2 channel is involved in pathological myocardial hypertrophy in rats

ABSTRACT Residue Thr1604 in the CaV1.2 channel is a Ca2+/calmodulin dependent protein kinase II (CaMKII) phosphorylation site, and its phosphorylation status maintains the basic activity of the channel. However, the role of CaV1.2 phosphorylation at Thr1604 in myocardial hypertrophy is incompletely understood. Isoproterenol (ISO) was used to induce cardiomyocyte hypertrophy, and autocamtide-2-related inhibitory peptide (AIP) was added as a treatment. Rats in a myocardial hypertrophy development model were subcutaneously injected with ISO for two or three weeks. The heart and left ventricle weights, each of which were normalized to the body weight and cross-sectional area of the myocardial cells, were used to describe the degree of hypertrophy. Protein expression levels were detected by western blotting. CaMKII-induced CaV1.2 (Thr1604) phosphorylation (p-CaV1.2) was assayed by coimmunoprecipitation. The results showed that CaMKII, HDAC, MEF2 C, and atrial natriuretic peptide (ANP) expression was increased in the ISO group and downregulated by AIP treatment in vitro. There was no difference in the expression of these proteins between the ISO 2-week group and the ISO 3-week group in vivo. CaV1.2 channel expression did not change, but p-CaV1.2 expression was increased after ISO stimulation and decreased by AIP. In the rat model, p-CaV1.2 levels and CaMKII activity were much higher in the ISO 3-week group than in the ISO 2-week group. CaMKII-induced CaV1.2 channel phosphorylation at residue Thr1604 may be one of the key features of myocardial hypertrophy and disease development.Abbreviations: CaMKII: Ca2+/calmodulin dependent protein kinase II; p-CaMKII: autophosphorylated Ca2+/calmodulin dependent protein kinase II; CaM: calmodulin; AIP: autocamtide-2-related inhibitory peptide; ECC: excitation-contraction coupling; ISO: isoproterenol; BW: body weight; HW: heart weight; LVW: left ventricle weight; HDAC: histone deacetylase; p-HDAC: phosphorylated histone deacetylase; MEF2C: myocyte-specific enhancer factor 2C; ANP: atrial natriuretic peptide; PKC: protein kinase C

P3832Dantrolene reduces CaMKII-mediated arrhythmogenesis

Abstract Rationale In atrial and ventricular rhythm disorders, an increased diastolic sarcoplasmatic reticulum (SR) calcium leak can induce a depolarizing transient inward current, serving as a trigger for cellular arrhythmias. Dantrolene has been shown to also stabilize the cardiac ryanodine receptor. However, the detailed mechanism of the mode of action remains unknown. This study aims to investigate the effects of dantrolene on calcium/calmodulin-dependent kinase II (CaMKII) mediated arrhythmogenesis. Methods and results Right atrial cardiomyocytes (CM) were isolated from patients with atrial fibrillation. To investigate SR Ca2+ leak, measurements of diastolic SR Ca2+ sparks were performed by confocal microscopy using Fluo-4 AM. Dantrolene (10 μmol/l) potently reduced Ca2+-spark-frequency (CaSpF) by 90±26% (p<0.05, n=21 cells dantrolene vs. 19 cells control) leading to a reduction of the calculated diastolic SR-Ca2+-leak by 91±31% (p<0.05, n=21 vs. 19). Interestingly, CaMKII-inhibition using Autocamtide-2-Related Inhibitory Peptide (AIP) did not further reduced SR Ca2+ leak compared to dantrolene alone in human cardiomyocytes. This observation may suggest (secondary) inhibitory effects of dantrolene on CaMKII. To elucidate the role of CaMKII in dantrolene-mediated antiarrhythmic effects, we investigated atrial CM from mice overexpressing CaMKII (TG) and respective wildtype controls (WT). CaSpF and SR Ca2+ leak were reduced by dantrolene in both TG and WT mice (p<0.005, TG: dantrolene vs. vehicle n=132 vs 127 cells (9 mice); WT: dantrolene vs. vehicle n=61 vs 61 cells (5 mice)). However, proarrhythmogenic Ca2+ waves were only significantly reduced by dantrolene in TG mice (p<0.05, TG: dantrolene vs. vehicle 10.8% vs. 26.2%, n=154 vs 164 cells). Correspondingly, the incidence of delayed afterdepolarizations (DADs) in TG cells was significantly diminished by dantrolene (p<0.05, TG: dantrolene vs. vehicle 1/14 vs. 9/15 cells, n=5 mice). In contrast, DADs were not reduced by dantrolene in WT cells without increased CaMKII activity (p=n.s., WT: dantrolene vs vehicle 3/16 vs 2/13 cells, n=5 mice). In preliminary in vivo experiments, intraperitoneal injection of 40 mg/kg body weight dantrolene reduced the inducibility of arrhythmias by ventricular burst stimulation in CaMKII TG mice compared to vehicle (dantrolene 0/2 mice vs. vehicle 2/2 mice, p<0.05 Chi-Square). Conclusion Dantrolene beneficially altered Ca2+ homeostasis in human AF CM and murine CM. Dantrolene seems to exert its antiarrhythmic potential in a CaMKII-dependent manner. Thus, dantrolene as an already clinically approved compound might be a potential antiarrhythmic drug that merits clinical investigation. Acknowledgement/Funding Deutsche Forschungsgemeinschaft (MA 1981/5-1 and 7-1 to LSM). Marga und Walter Boll-Stiftung (SS).

Blockade of L-type Ca2+ channel attenuates doxorubicin-induced cardiomyopathy via suppression of CaMKII-NF-\u03baB pathway

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and nuclear factor-kappa B (NF-κB) play crucial roles in pathogenesis of doxorubicin (DOX)-induced cardiomyopathy. Their activities are regulated by intracellular Ca2+. We hypothesized that blockade of L-type Ca2+ channel (LTCC) could attenuate DOX-induced cardiomyopathy by regulating CaMKII and NF-κB. DOX activated CaMKII and NF-κB through their phosphorylation and increased cleaved caspase 3 in cardiomyocytes. Pharmacological blockade or gene knockdown of LTCC by nifedipine or small interfering RNA, respectively, suppressed DOX-induced phosphorylation of CaMKII and NF-κB and apoptosis in cardiomyocytes, accompanied by decreasing intracellular Ca2+ concentration. Autocamtide 2-related inhibitory peptide (AIP), a selective CaMKII inhibitor, inhibited DOX-induced phosphorylation of NF-κB and cardiomyocyte apoptosis. Inhibition of NF-κB activity by ammonium pyrrolidinedithiocarbamate (PDTC) suppressed DOX-induced cardiomyocyte apoptosis. DOX-treatment (18 mg/kg via intravenous 3 injections over 1 week) increased phosphorylation of CaMKII and NF-κB in mouse hearts. Nifedipine (10 mg/kg/day) significantly suppressed DOX-induced phosphorylation of CaMKII and NF-κB and cardiomyocyte injury and apoptosis in mouse hearts. Moreover, it attenuated DOX-induced left ventricular dysfunction and dilatation. Our findings suggest that blockade of LTCC attenuates DOX-induced cardiomyocyte apoptosis via suppressing intracellular Ca2+ elevation and activation of CaMKII-NF-κB pathway. LTCC blockers might be potential therapeutic agents against DOX-induced cardiomyopathy.

Angiotensin II upregulates fibroblast-myofibroblast transition through Cx43-dependent CaMKII and TGF-β1 signaling in neonatal rat cardiac fibroblasts.

In cardiac fibroblasts, angiotensin II (Ang II) can increase connexin 43 (Cx43) expression and promote calmodulin-dependent protein kinase II (CaMKII) activation. Cx43 overexpression is crucial for the fibroblast-myofibroblast transition. The main purpose of the present study was to investigate the role of CaMKII in regulating Cx43 expression and to determine whether the CaMKII/Cx43 pathway is essential for controlling fibroblast activation and differentiation. In vivo, 4 weeks of Ang II infusion enhanced CaMKII activation but reduced Cx43 expression in hearts undergoing fibrosis remodeling, while in cultured neonatal rat fibroblasts, CaMKII activation upregulated Cx43 expression via transforming growth factor-beta1 (TGF-β1). CaMKII inhibition by Ang-(1-7) or autocamtide 2-related inhibitory peptide reversed the Ang II-induced changes in Cx43 expression and attenuated Ang II-induced upregulation of alpha smooth muscle actin and TGF-β1 in both Ang II-infused rats and cultured fibroblasts. Based on the in vivo and in vitro experimental results, CaMKII plays a pivotal role in the Ang II-mediated fibroblast-myofibroblast transition by modulating the expressions of TGF-β1 and Cx43. We conclude that Ang II mediates the fibroblast-myofibroblast transition partially via the Ang II/CaMKII/TGF-β1/Cx43 signaling pathway.

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca2+-Activated K+ Current in Patients with Chronic Atrial Fibrillation.

BackgroundIncreased small-conductance Ca2+-activated K+ current (SK), abnormal intracellular Ca2+ handling, and enhanced expression and activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been found in clinical and/or experimental models of atrial fibrillation (AF), but the cumulative effect of these phenomena and their mechanisms in AF are still unclear. This study aimed to test the hypothesis that CaMKII increases SK current in human chronic AF.Material/MethodsRight atrial appendage tissues from patients with either sinus rhythm (SR) or AF and neonatal rat atrial myocytes were used. Patch clamp, qRT-PCR, and Western blotting techniques were used to perform the study.ResultsCompared to SR, the apamin-sensitive SK current (IKAS) was significantly increased, but the mRNA and protein levels of SK1, SK2, and SK3 were significantly decreased. In AF, the steady-state Ca2+ response curve of IKAS was shifted leftward and the [Ca2+]i level was significantly increased. CaMKII inhibitors (KN-93 or autocamtide-2-related inhibitory peptide (AIP)) reduced the IKAS in both AF and SR. The inhibitory effect of KN-93 or AIP on IKAS was greater in AF than in SR. The expression levels of calmodulin, CaMKII, and autophosphorylated CaMKII at Thr287 (but not at Thr286) were significantly increased in AF. Furthermore, KN-93 inhibited the expression of (Thr287)p-CaMKII and SK2 in neonatal rat atrial myocytes.ConclusionsSK current is increased via the enhanced activation of CaMKII in patients with AF. This finding may explain the difference between SK current and channels expression in AF, and thus may provide a therapeutic target for AF.

CaMKII Regulates Synaptic NMDA Receptor Activity of Hypothalamic Presympathetic Neurons and Sympathetic Outflow in Hypertension.

NMDAR activity in the hypothalamic paraventricular nucleus (PVN) is increased and critically involved in heightened sympathetic vasomotor tone in hypertension. Calcium/calmodulin-dependent protein kinase II (CaMKII) binds to and modulates NMDAR activity. In this study, we determined the role of CaMKII in regulating NMDAR activity of PVN presympathetic neurons in male spontaneously hypertensive rats (SHRs). NMDAR-mediated EPSCs and puff NMDA-elicited currents were recorded in spinally projecting PVN neurons in SHRs and male Wistar-Kyoto (WKY) rats. The basal amplitude of evoked NMDAR-EPSCs and puff NMDA currents in retrogradely labeled PVN neurons were significantly higher in SHRs than in WKY rats. The CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP) normalized the increased amplitude of NMDAR-EPSCs and puff NMDA currents in labeled PVN neurons in SHRs but had no effect in WKY rats. Treatment with AIP also normalized the higher frequency of NMDAR-mediated miniature EPSCs of PVN neurons in SHRs. CaMKII-mediated phosphorylation level of GluN2B serine 1303 (S1303) in the PVN, but not in the hippocampus and frontal cortex, was significantly higher in SHRs than in WKY rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter the increased level of phosphorylated GluN2B S1303 in the PVN. In addition, microinjection of AIP into the PVN significantly reduced arterial blood pressure and lumbar sympathetic nerve discharges in SHRs. Our findings suggest that CaMKII activity is increased in the PVN and contributes to potentiated presynaptic and postsynaptic NMDAR activity to elevate sympathetic vasomotor tone in hypertension.SIGNIFICANCE STATEMENT Heightened sympathetic vasomotor tone is a major contributor to the development of hypertension. Although glutamate NMDA receptor (NMDAR)-mediated excitatory drive in the hypothalamus plays a critical role in increased sympathetic output in hypertension, the molecular mechanism involved in potentiated NMDAR activity of hypothalamic presympathetic neurons remains unclear. Here we show that the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) is increased and plays a key role in the potentiated presynaptic and postsynaptic NMDAR activity of hypothalamic presympathetic neurons in hypertension. Also, the inhibition of CaMKII in the hypothalamus reduces elevated blood pressure and sympathetic nerve discharges in hypertension. This new knowledge extends our understanding of the mechanism of synaptic plasticity in the hypothalamus and suggests new strategies to treat neurogenic hypertension.

O22-4 - Nifedipine Attenuates Doxorubicin-induced Cardiomyopathy by Suppressing CaMKII-NF-kB Pathway

Background: Activation of CaMKII induces cardiomyocyte death in doxorubicin (DOX)-induced cardiomyopathy. Nifedipine, L-type calcium channel blocker, has been shown to inhibit CaMKII activity in hypertrophic heart. The purpose of this study was to determine whether nifedipine could attenuate DOX-induced cardiomyopathy by inhibiting CaMKII activity and to elucidate its downstream signaling. Methods and Results: Mice were treated with DOX (18 mg/kg via 3 intravenous injections over 1 week) with or without subpressor dose of nifedipine (10 mg/kg/day). Two weeks after DOX treatment, nifedipine prevented DOX-induced decrease in left ventricular fractional shortening (25.7 ± 1.5% vs. 32.9 ± 0.3%, P < .05). Nifedipine decreased Dox-induced cardiomyocyte death as assessed by Billingham score (2.88 ± 0.25 vs. 1.88 ± 0.48 P < .05). It also inhibited DOX-induced phosphorylation of CaMKII and NF-kB, their active forms, by 42% and 30%, respectively (P < .05), in the heart. In cultured neonatal rat ventricular myocytes (NRVMs), DOX (10 µM, 24h) increased phosphorylated CaMKII and NF-kB and cleaved caspase-3, and LDH release, a marker of cell death. Treatment with nifedipine (10 µM, 24h) or autocamtide 2-related inhibitory peptide (AIP, 10 µM, 24h), a selective inhibitor of CaMKII, significantly suppressed DOX-induced increases in phosphorylated CaMKII, cleaved caspase-3 and LDH release in NRVMs, which was accompanied by a decrease in phosphorylated NF-kB. Conclusions: Nifedipine attenuated DOX-induced cardiomyopathy by suppressing CaMKII-NF-kB pathway. Therapeutic strategy designed to interfere with this pathway might be beneficial in its prevention.

Inhibitions of late INa and CaMKII act synergistically to prevent ATX-II-induced atrial fibrillation in isolated rat right atria

AimsIncreases in late Na+ current (late INa) and activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) are associated with atrial arrhythmias. CaMKII also phosphorylates Nav1.5, further increasing late INa. The combination of a CaMKII inhibitor with a late INa inhibitor may be superior to each compound alone to suppress atrial arrhythmias. Therefore, we investigated the effect of a CaMKII inhibitor in combination with a late INa inhibitor on anemone toxin II (ATX-II, a late INa enhancer)-induced atrial arrhythmias. Methods and resultsRat right atrial tissue was isolated and preincubated with either the CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP), the late INa inhibitor GS458967, or both, and then exposed to ATX-II. ATX-II increased diastolic tension and caused fibrillation of isolated right atrial tissue. AIP (0.3μmol/L) and 0.1μmol/L GS458967 alone inhibited ATX-II-induced arrhythmias by 20±3% (mean±SEM, n=14) and 34±5% (n=13), respectively, whereas the two compounds in combination inhibited arrhythmias by 81±4% (n=10, p<0.05, vs either AIP or GS458967 alone or the calculated sum of individual effects of both compounds). AIP and GS458967 also attenuated the ATX-induced increase of diastolic tension. Consistent with the mechanical and electrical data, 0.3μmol/L AIP and 0.1μmol/L GS458967 each inhibited ATX-II-induced CaMKII phosphorylation by 23±3% and 32±4%, whereas the combination of both compounds inhibited CaMKII phosphorylation completely. ConclusionThe effects of an enhanced late INa to induce arrhythmic activity and activation of CaMKII in atria are attenuated synergistically by inhibitors of late INa and CaMKII.

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

The ATP-sensitive potassium (KATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of KATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal KATP (sarcKATP) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK) 293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e., the principal ventricular-type KATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+ / calmodulin-dependent protein kinase II (CaMKII) and U0126 [an extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-α,β-d-glucopyranose-2-)-N2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcKATP channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcKATP channel activity in intact cardiomyocytes, but the H2O2-induced KATP channel stimulation was obliterated when ERK1/2 or CaMKII activity was suppressed, implying that H2O2 is positioned upstream of ERK1/2 and CaMKII for K(ATP) channel modulation. Furthermore, genetic ablation (i.e., knockout) of CaMKIIδ, the predominant cardiac CaMKII isoform, diminished ventricular sarcK(ATP) channel stimulation elicited by activation of PKG, unveiling CaMKIIδ as a crucial player. Additionally, evidence from kinase activity and Western blot analyses revealed that activation of NO-PKG signalling augmented CaMKII activity in rabbit ventricular myocytes and, importantly, CaMKII activation by PKG occurred in an ERK1/2-dependent manner, placing ERK1/2 upstream of CaMKII. Taken together, these findings suggest that NO modulates ventricular sarcK(ATP) channels via a novel sGC-cGMP-PKG-ROS(H2O2)-ERK1/2-calmodulin-CaMKII (δ isoform in particular) signalling cascade, which heightens K(ATP) channel activity by destabilizing the long closed states while facilitating closed-to-open state transitions. This pathway may contribute to regulation of cardiac excitability and cytoprotection against ischaemia-reperfusion injury, in part, by opening myocardial sarcK(ATP) channels.

Increased CAMKII Activity Impairs Contractile Function in Human HCM Myocardium

Background and Rationale: we have previously shown that the myocardium of patients with Hypertrophic Cardiomyopathy (HCM) shows increased CaMKII activity with respect to non-HCM non-failing surgical patients (controls). We found higher phosphorylation levels of several tested CaMKII targets, such as L-Type Ca2+ channels, Na+ channels and phospholamban (Coppini et al., Circulation 2013). Such changes are likely to determine significant deleterious effects on the electrical and mechanical function of HCM myocardium.Methods: we assessed the effects of a selective inhibitor of CaMKII, the cell-permeant Autocamtide-related Inhibitory Peptide II (AIP-II), on force and intracellular Ca2+ in intact trabeculae and cardiomyocytes isolated from surgical septal samples of HCM patients who underwent myectomy.Results: HCM trabeculae display an increased diastolic tension and a slower kinetics of isometric force generation when compared to control trabeculae. AIP-II determines a reduction of diastolic tension, with larger effects at fast pacing rates (−13±3 % at 2Hz). Additionally, AIP-II accelerated the kinetics of force generation in HCM muscles (time to peak contraction at 1Hz: 255±17 ms at baseline vs. 232±10 ms with AIP-II, p<0.05). Accordingly, in HCM cardiomyocytes AIP-II lowered intracellular diastolic Ca2+ levels (from 278 ± 40 nM to 159 ± 28 nM, p<0.01) and reduced the frequency-dependent increase of diastolic [Ca2+]. The kinetics of Ca2+ transients, however, were not significantly affected by AIP-II. This suggests that reduced CaMKII phosphorylation of myofilament proteins may underlie the accelerated contractile kinetics observed in HCM trabeculae following AIP-II exposure.Discussion: The results indicate that increased activation of CaMKII plays a significant role in determining the abnormalities of myocardial contraction in HCM. Pharmacological inhibition of CaMKII may represent a viable option to reduce diastolic dysfunction in HCM patients.

Regulation of ASIC1 by Ca2+/calmodulin-dependent protein kinase II in human glioblastoma multiforme

Recent studies have implicated the acid-sensing ion channel 1 (ASIC1), a proton-gated cation channel that belongs to the epithelial sodium channel (ENaC)/Degenerin family, plays an important role in glioma cell migration. Among the ASIC subunits, only ASIC1a has been found be calcium permeable. However, it has not been determined whether Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates ASIC1 in glioblastoma multiforme (GBM). Herein, we report that ASIC1 and CaMKII assemble to form a functional complex at the plasma membrane of GBM cells. We found that migration ability was significantly attenuated in GBM cells that were pre-treated with autocamtide-2-related inhibitory peptide (AIP), a CaMKII-specific inhibitor, or psalmotoxin 1 (PcTX-1), a selective ASIC1 blocker. Furthermore, the inhibitory effect of AIP or PcTX-1 on migration was diminished when ASIC1 was knocked down in GBM cells; when ASIC1 knockdown GBM cells were concurrently treated with these two inhibitors, cell migration was slightly but significantly decreased. Using whole-cell patch-clamp recordings, we detected an amiloride-sensitive current in GBM cells, and this current was significantly inhibited by both PcTX-1 and AIP. Moreover, the magnitude of this current was dramatically decreased when ASIC1 was knocked down in GBM cells. The addition of AIP failed to further decrease the amplitude of this current. Taken together, these data suggest that ASIC1 and CaMKII form a functional complex in GBM cells. Furthermore, it can be concluded that CaMKII regulates the activity of ASIC1, which is associated with the ability of GBM cells to migrate.

Selective Modulation of Coupled Ryanodine Receptors During Microdomain Activation of Calcium/Calmodulin-Dependent Kinase II in the Dyadic Cleft

In ventricular myocytes of large mammals with low T-tubule density, a significant number of ryanodine receptors (RyRs) are not coupled to the sarcolemma; cardiac remodeling increases noncoupled RyRs. Our aim was to test the hypothesis that coupled and noncoupled RyRs have distinct microdomain-dependent modulation. We studied single myocytes from pig left ventricle. The T-tubule network was analyzed in 3-dimension (3D) to measure distance to membrane of release sites. The rising phase of the Ca(2+) transient was correlated with proximity to the membrane (confocal imaging, whole-cell voltage-clamp, K5fluo-4 as Ca(2+) indicator). Ca(2+) sparks after stimulation were thus identified as resulting from coupled or noncoupled RyRs. We used high-frequency stimulation as a known activator of Ca(2+)/calmodulin-dependent kinase II. Spark frequency increased significantly more in coupled than in noncoupled RyRs. This specific modulation of coupled RyRs was abolished by the Ca(2+)/calmodulin-dependent kinase II blockers autocamtide-2-related inhibitory peptide and KN-93, but not by KN-92. Colocalization of Ca(2+)/calmodulin-dependent kinase II and RyR was not detectably different for coupled and noncoupled sites, but the F-actin disruptor cytochalasin D prevented the specific modulation of coupled RyRs. NADPH oxidase 2 inhibition by diphenyleneiodonium or apocynin, or global reactive oxygen species scavenging, also prevented coupled RyR modulation. During stimulated Ca(2+) transients, frequency-dependent increase of the rate of Ca(2+) rise was seen in coupled RyR regions only and abolished by autocamtide-2-related inhibitory peptide. After myocardial infarction, selective modulation of coupled RyR was lost. Coupled RyRs have a distinct modulation by Ca(2+)/calmodulin-dependent kinase II and reactive oxygen species, dependent on an intact cytoskeleton and consistent with a local Ca(2+)/reactive oxygen species microdomain, and subject to modification with disease.