CaMKIIδ Expression Research Articles

Abstract Mo137: Modulation of the Inflammatory and Fibrotic Effects of Angiotensin on Cardiomyocytes through YAP-mediated Transcription.

Introduction: Signaling through the transcriptional co-activator Yes-associated protein (YAP) has been shown to protect the myocardium against ischemic or mechanical stress. Research Question: In light of the critical role of inflammation on adverse cardiac responses we examined the possibility that YAP regulates inflammatory responses to Angiotensin II (AngII)-mediated stress. Approach: Cardiomyocyte specific YAP-KO mice were generated, implanted with AngII minipumps, infused with AngII (1.5 mg/kg/min) or vehicle for periods of 3 hrs to 3 days and hearts collected for analysis. Results: YAP deletion markedly enhanced AngII-induced mRNA expression of pro-inflammatory cytokines and chemokines (e.g. CCL2, CXCL2, IL-6) at 3 or 24 hrs in the cardiomyocyte, but not non-myocyte cell fraction. Expression of mRNA for the hypertrophic genes ANP and MHC-β, in contrast, was decreased in YAP KO mice. After 3 days of AngII infusion there was enhanced mRNA for pro-fibrotic genes (Col1a1, Col3a1 and Periostin) and increased staining for COL1A1 and F4/80, indicative of increased fibrosis and inflammatory cell recruitment in the absence of YAP signaling. Single cell RNA sequencing revealed specific subsets of genes that were upregulated in the cardiomyocyte subcluster from YAP KO mice. These included pro-inflammatory cytokines, cJun family genes, TLR4 and, surprisingly, CaMKIId. Elevated activation of CaMKIIδ in response to AngII in YAP KO hearts was confirmed by increased phosphorylation of phospholamban. Cardiomyocytes expressing siYAP also showed enhanced expression of TLR4 and CaMKIIδ. Conclusion: Upregulated expression of cJun, TLR4 and CaMKII occurs in the absence of YAP and this conspires to create a greatly enhanced inflammatory milieu which fuels adverse responses to external insults. YAP activation serves to suppress these responses and thus temper development of maladaptive inflammation.

Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

Nitric oxide (NO) has been identified as a signaling molecule generated during β-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-adrenergic receptor stimulation. We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (200 μM), and β-adrenergic agonist isoproterenol (100 nmol/L). Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent β-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain β-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Inflammatory Forms of Cardiomyocyte Cell Death in the Rat Model of Isoprenaline-Induced Takotsubo Syndrome.

Takotsubo syndrome (TTS) is associated with inflammatory response, therefore the aim of the study was to evaluate the presence and dynamics of inflammatory-associated forms of cell death, necroptosis, and pyroptosis in the female rat model of isoprenaline (ISO)-induced TTS. TTS was induced in female Sprague Dawley rats (n = 36) by ISO 150 mg/kg intraperitoneally. Animals were divided into four groups: TTSO (TTS+ovariectomy; n = 10), TTSP (TTS+sham operation; n = 10), CO (0.9% NaCl+ovariectomy; n = 8), CP (0.9% NaCl+sham operation; n = 8). Histopathological analysis, evaluation of plasma concentration, and myocardial expression of pyroptosis- and necroptosis-associated proteins were performed. TTSO and TTSP groups had higher plasma concentrations of interleukin-1β in comparison with the controls. Low myocardial protein expression of mixed lineage kinase domain-like pseudokinase (MLKL), caspase-1 (Casp-1), and calcium/calmodulin-dependent kinase type II isoform delta (CAMKIIδ) was visible 6 and/or 12 h post-ISO. Twenty-four hours post-ISO, high myocardial and vascular protein expression of CAMKIIδ was visible in TTSO but not TTSP rats, while high myocardial expression of MLKL and Casp-1 was visible both in TTSO and TTSP rats. The course of TTS is associated with activation of inflammatory-associated programmed cell death, necroptosis, and pyroptosis, therefore inflammation may be a primary response occurring simultaneously with cardiomyocyte death in TTS.

Cardiac-specific overexpression of insulin-like growth factor II receptor-α interferes with the regulation of calcium homeostasis in the heart under hyperglycemic conditions.

Diabetic cardiomyopathy is a progressive disease caused by inexplicit mechanisms, and a novel factor, insulin-like growth factor II receptor-α (IGF-IIRα), may contribute to aggravating its pathogenesis. We hypothesized that IGF-IIRα could intensify diabetic heart injury. To demonstrate the potential role of IGF-IIRα in the diabetic heart, we used (SD-TG [IGF-IIRα]) transgenic rat model with cardiac-specific overexpression of IGF-IIRα, along with H9c2 cells, to study the effects of IGF-IIRα in the heart under hyperglycemic conditions. IGF-IIRα was found to remodel calcium homeostasis and intracellular Ca2+ overload-induced autophagy disturbance in the heart during diabetes. IGF-IIRα overexpression induced intracellular Ca2+ alteration by downregulating phosphorylated phospholamban/sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a (PLB/SERCA2a), resulting in the suppression of Ca2+ uptake into the endoplasmic reticulum. Additionally, IGF-IIRα itself contributed to Ca2+ withdrawal from the endoplasmic reticulum by increasing the expression of CaMKIIδ in the active form. Furthermore, alterations in Ca2+ homeostasis significantly dysregulated autophagy in the heart during diabetes. Our study reveals the novel role of IGF-IIRα in regulating cardiac intracellular Ca2+ homeostasis and its related autophagy interference, which contribute to the development of diabetic cardiomyopathy. In future, the present study findings have implications in the development of appropriate therapy to reduce diabetic cardiomyopathy.

ISX-9 potentiates CaMKIIδ-mediated BMAL1 activation to enhance circadian amplitude

Circadian dysregulation associates with numerous diseases including metabolic dysfunction, sleep disorder, depression and aging. Given that declined circadian amplitude is a trait commonly found with compromised health, interventions that design in precluding circadian amplitude from dampening will aid to mitigate complex, circadian-related diseases. Here we identify a neurogenic small molecule ISX-9 that is able to support persistent and higher amplitude of circadian oscillations. ISX-9 improves diurnal metabolic rhythms in middle-aged mice. Moreover, the ISX-9-treated mice show better sleep homeostasis with increased delta power during the day time and higher locomotive activity in the dark period. ISX-9 augments CaMKIIδ expression and increases BMAL1 activity via eliciting CaMKIIδ-mediated phosphorylation on BMAL1 residues S513/S515/S516, accordingly composes a positive feedback effect on enhancing circadian amplitude. CaMKIIδ-targeting, and the use of ISX-9 may serve as decent choices for treating circadian-related disorders.

MiR-145-5p affects autophagy by targeting CaMKIIδ in atherosclerosis

BackgroundAtherosclerosis (AS) is a chronic progressive inflammatory disease involving many cells. miR-145-5p mediates the biological phenotypes of human aortic vascular smooth muscle cells (HAVSMCs) and influences the progression of AS, but the potential mechanism needs further study. MethodsTotal RNA was extracted from patient plasma and arteries to determine the expression of miR-145-5p. The CaMKIIδ pathway and genes were predicted as the target of miR-145-5p by bioinformatics approaches. The interaction between miR-145-5p and CaMKIIδ was confirmed by RT–qPCR and Dual Luciferase Reporter Assay System. Western blot analysis, immunofluorescence staining, transmission electron microscopy (TEM) and protein tracing on HAVSMCs transduced with mCherry-GFP-LC3 lentiviral vectors to determine the mechanism by which miR-145-5p affects the atherosclerotic disease process. ResultsThe expression of miR-145-5p was downregulated in blood and arteries specimens of patients with coronary stenosis. Correspondingly, CaMKIIδ was upregulated and miR-145-5p was downregulated in hypoxic HAVSMCs. CaMKIIδ was predicted and confirmed as a downstream target of miR-145-5p. In addition, CaMKIIδ induced the upregulation of autophagy-related proteins by activating the AMPK/mTOR/ULK1 signalling pathway. Moreover, we confirmed that miR-145-5p inhibits CaMKIIδ expression by binding to a specific sequence in the CaMKIIδ 3′ UTR and affects autophagy. Crucially, CaMKIIδ was promoted by the downregulation of miR-145-5p and then activating autophagy in HAVSMCs through the AMPK/mTOR/ULK1 signalling pathway to affect the AS progress. ConclusionsmiR-145-5p regulates CaMKIIδ, leading to altered autophagy in HAVSMCs. This alteration plays an important role in AS progression.

Sulforaphane protects myocardium from ischemia-reperfusion injury by regulating CaMKIIN2 and CaMKIIδ

Myocardial ischemia/reperfusion (I/R) injury poses a significant threat to human health. High level of reactive oxygen species (ROS) and calcium overload are the foremost causes of myocardial damage in I/R. Sulforaphane (SFN) is known for its promising antioxidant effect. Whether or not SFN has myocardial protective effect against I/R is largely unknown. This study aimed to investigate if SFN can protect myocardium from I/R injury. We found that mice or cells pre-treated with SFN showed improved cardiac functions and cell survival. SFN treatment inhibited the production of inflammatory cytokines and the increase of intracellular calcium induced by hypoxia-reperfusion (H/R), while mitochondria membrane potential was effectively maintained. Transcriptome analysis showed that CaMKIIδ expression was down-regulated by SFN treatment in I/R myocardium, while CaMKIIN2, the inhibitor of CaMKII, was upregulated. Knockdown of CaMKIIN2 not only led to increased level of total CaMKIIδ and the phosphorylated CaMKIIδ but also blocked the pro-survival effect of SFN for H/R cells. Moreover, CaMKIIN2 overexpression was sufficient to suppress CaMKIIδ activation and improve cell survival under H/R. Taken together, this study demonstrated that SFN exerts cardioprotective effect toward I/R injury through upregulating CaMKIIN2 and down-regulating CaMKIIδ.

Abstract 9022: Chronic Beta3-Adrenergic Receptor Antagonist Therapy Rescues Diabetic Cardiomyopathy Through Ca 2+ /Calmodulin-Dependent Protein Kinase II Inhibition in a Type II Diabetes Murine Model

Background: Recent evidence showed that CaMKII inhibition with KN93 protects against diabetic cardiomyopathy (DCM). However, the off-target effects have limited its use. There are no CaMKII inhibitors available clinically. It may be best to use some complementary approaches for CaMKII inhibition. Previously we showed that diabetes produced upregulation of cardiac β 3 -AR-mediated inhibitory pathway promoting DCM progression. There is a significant crosstalk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β 3 -AR signaling in DCM. It is possible that the benefit of β 3 -AR antagonists (β 3 ANT) might involve modulation of CaMKII. We tested the hypothesis that chronic L-748-337, a selective β 3 ANT may inhibit CaMKII activity and limit DCM. Methods: LV myocyte CaMKIIδ expression and activity and myocyte function were determined in 3 groups (7/group) wild-type female mice over 14 weeks (W): Type 2 DM ( T2 ), 14 W fed high-fat diet (HFD), but after HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. 5 days); T2β 3 ANT , T2 mice at 10 W received L-748,337 (10 -7 M/kg/day, mini-pump) for 4 W; and Vehicle controls (C). Results: Versus C, T2 diabetes was induced in mice received HFD and low dose STZ with significantly elevated blood glucose. But only T2 myocytes had significantly increased protein levels of CaMKIIδ (T2: 29%, 0.53 vs C: 0.41) and CaMKIIδ phosphorylation (at Thr287) (PCaMKIIδ) (79%, 0.52 vs 0.29) followed by significantly reduced cell contraction (dL/dt max , 75 vs 134 μm/s), relaxation (dR/dt max , 60 vs 114 μ m/s) and [Ca 2+ ] iT (0.16 vs 0.21). The isoproterenol (ISO, 10 -8 M)-stimulated increases in dL/dt max (38% vs 58%), dR/dt max and [Ca 2+ ] iT (19% vs 30%) were also significantly reduced. By contrary, versus C, T2β 3 ANT myocytes had similar levels of CaMKIIδ (0.37) and PCaMKIIδ (0.31) with preserved cell contractility (131 μm/s), relaxation (109 μm/s) and [Ca 2+ ] iT (0.21). ISO caused increases in dL/dt max (57%), dR/dt max (52%) and [Ca 2+ ] iT (30%) were also closed to control values. Conclusions: Chronic β 3 ANT prevents Type II diabetes-caused upregulation of cardiac CaMKIIδ, thereby leading to the preservation of normal myocyte function, [Ca 2+ ] iT , and β-adrenergic reserve in a DCM murine model.

Abstract 9026: Endogenous Ca2+/Calmodulin-Dependent Protein Kinase II Overactivation Exacerbates Cardiac Dysfunction and Beta-Adrenergic Desensitization in a Mouse Model With Type 2 Diabetes

Background: Emergent evidence indicates that CaMKII overactivity in diabetes (DM) plays a crucial role in the pathophysiology of diabetic cardiomyopathy (DCM) by targeting a diverse array of proteins involved in cardiac electrical, structural and functional remodeling. CaMKII has been proposed to be a therapeutic target for DCM. However, although chronic inhibition of CaMKII has been associated with cardioprotection in several cardiovascular diseases, the direct cardiac effects of CaMKII activation in type 2 (T2) DM are unclear. Moreover, whether and how DCM alters cardiac responses to CaMKII are undefined. We tested the hypothesis that in DCM, CaMKII is upregulated, which alters cardiac contractile behavior. Endogenous CaMKII activation may produce direct depressions in LV myocyte basal function and β-adrenergic reserve. Acute inactivation of CaMKII with KN93 would have a beneficial impact in DCM. Methods: LV myocyte CaMKIIδ expression and activity and myocyte functional responses were compared in 2 groups (n=9/group) of wild-type female mice: Vehicle Control (C) , animals fed chow for 14 weeks (W); T2DM, i nduced with a high-fat diet (HFD) intake for 14 W, but after fed HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. for 5 days). In T2DM, we further assessed myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses with and without acute treatment of myocytes with KN93 (10 -6 M, 30 min). Results: Versus C, in T2DM myocytes, the CaMKIIδ protein levels (28%, 0.51 vs 0.40) and CaMKIIδ phosphorylation (79%, 0.52 vs 0.29) were significantly increased. These changes were followed by significantly reduced cell contraction (dL/dt max , 76.0 vs 137.3 μm/s), relaxation (dR/dt max , 61.7 vs 116.0 μm/s) and [Ca 2+ ] iT (0.16 vs 0.21). ISO-stimulated increases dL/dt max (39% vs 58%), dR/dt max (35% vs 54%) and [Ca 2+ ] iT (20% vs 30%) were also significantly reduced. Moreover, in T2DM myocytes, superfusion with KN93 significantly improved myocyte basal contraction (104.2 μm/s), relaxation (79.3 μm/s) and [Ca 2+ ] iT (0.21). Conclusions: In T2 diabetes-induced DCM, upregulation of CaMKIIδ has adverse functional effects. Acute endogenous CaMKII activation exacerbates LV myocyte dysfunction. The inhibition of CaMKII has a significant beneficial impact.

CaMKIIδ is upregulated by pro-inflammatory cytokine IL-6 in a JAK/STAT3-dependent manner to promote angiogenesis.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine threonine kinase with established roles in physiological and pathophysiological vascular remodeling. Based on our previous study demonstrating that CaMKIIδ promotes thrombin-induced endothelial permeability and recent reports that CaMKII may contribute to inflammatory remodeling in the heart, we investigated CaMKIIδ-dependent regulation of endothelial function downstream of an interleukin-6 (IL-6)/JAK/STAT3 signaling axis. Upon treatment with IL-6 and its soluble receptor (sIL-6r), CaMKIIδ expression is significantly induced in HUVEC. Using pharmacological inhibitors of JAK and siRNA targeting STAT3, we demonstrated that activation of STAT3 is sufficient to induce CaMKIIδ expression. Under these conditions, rather than promoting IL-6-induced permeability, we found that CaMKIIδ promotes endothelial cell migration as measured by live cell imaging of scratch wound closure and single-cell motility analysis. In a similar manner, endothelial cell proliferation was attenuated upon knockdown of CaMKIIδ as determined by growth curves, cell cycle analysis, and capacitance of cell-covered electrodes as measured by ECIS. Using inducible endothelial-specific STAT3 knockout mice, we demonstrate that STAT3 signaling promotes developmental angiogenesis in the neonatal mouse retina assessed at postnatal day 6. CaMKIIδ expression in retinal endothelium was attenuated in these animals as measured by qPCR. STAT3's effects on angiogenesis were phenocopied by the endothelial-specific knockout of CaMKIIδ, with significantly reduced vascular outgrowth and number of junctions in the developing P6 retina. For the first time, we demonstrate that transcriptional regulation of CaMKIIδ by STAT3 promotes endothelial motility, proliferation, and in vivo angiogenesis.

Abstract 12731: Acute Calcium/Calmodulin-Dependent Protein Kinase II Inhibition Augments Cardiomyocyte Contractility and Improves β-Adrenergic Regulation in Experimental Hypertension of Transgenic Rats Expressing Human Angiotensinogen Gene

Background: Previously we showed that rats expressing the human angiotensinogen (AGT) gene (hAogen) exhibit sustained hypertension and cardiac dysfunction, associated with increased cardiac angiotensin II (Ang II). Recent evidence indicates that Ang II increases CaMKII activation which plays a crucial role in ANG II-related pathological processes associated with hypertension, hypertrophy and cardiac dysfunction. Although CaMKII inhibitors are being developed as safe and effective therapeutic agents, their mechanism of action remains to be fully understood. We tested the hypothesis that in hAogen rats, CaMKII is upregulated. Increased CaMKII activation may produce direct depression in LV myocyte basal function and β-adrenergic reserve. Acute inactivation of CaMKII with KN93 would have beneficial actions in hAogen. Methods: We compared LV myocyte CaMKIIδ expression and activity and myocyte functional performance in 10 Sprague Dawley (SD) control and 10 hAogen male rats. In hAogen, we further assessed myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to β-AR stimulation by isoproterenol (ISO, 10 -8 M) with and without pretreatment of myocytes with KN93 (10 -6 M, 30 min). Results: Versus SD, in hAogen myocytes, the CaMKIIδ protein levels (19%, 0.50 vs 0.42) and CaMKIIδ phosphorylation (at Thr 287 ) (35%, 0.27 vs 0.20) were significantly increased. These changes were followed by significantly reduced cell contraction (dL/dtmax, 104.9 vs 138.4 μm/s), relaxation (dR/dtmax, 93.0 vs 104.2 μm/s) and [Ca 2+ ] iT (0.15 vs 0.20). ISO-stimulated increases in dL/dtmax (43% vs 62%), dR/dtmax (32% vs 49%) and [Ca 2+ ] iT (19% vs 29%) were also significantly reduced. Moreover, in hAogen myocytes, pretreatment with KN93 markedly improved myocyte basal contraction (134.4 μm/s), relaxation (107.4 μm/s) and [Ca 2+ ] iT (0.20). ISO-induced increases in dL/dtmax (60%), dR/dtmax (50%), and [Ca 2+ ] iT (31%) were also significantly augmented. Conclusions: In rats harboring the human AGT gene, upregulation of CaMKIIδ has adverse functional effects. Acute endogenous CaMKII activation exacerbates LV myocyte dysfunction and β-AR desensitization. Inhibition of CaMKII has a significant beneficial impact in this established humanized model of hypertension.

Abstract 13451: Upregulation of CaMKII and Contrast Changes of Cardiac β 1 - and β 3 -Adrenergic Signaling Pathways in a Humanized Angiotensinogen Model of Hypertension

Background: The transgenic rats expressing the human AGT gene [TGR (hAGT) L1623] (hAogen) is an established humanized model of hypertension. Recently we further found that this model is associated with declines in cardiac contractile function and β-adrenergic receptor (AR) reserve. However, the molecular basis is unclear. It has been suggested that pivotal restructuring of β-AR system is a critical determinant of the dysfunctional β-AR regulation in several cardiovascular diseases, including hypertension. But the alterations of subtypes of β-ARs have not been systemically evaluated in this model. Furthermore, there is significant crosstalk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in the abnormal contractile phenotype of cardiac hypertrophy. However, the changes in cardiac CaMKII in this model are unknown. We tested the hypothesis that in hAogen, CaMKII and β 3 -AR are upregulated in the myocardium, but β 1 -AR is down-regulated, which are important causes of cardiac dysfunction and β-AR desensitization. Methods: We compared LV myocyte CaMKIIδ expression and activity and β 1 - and β 3 -AR expression and determined myocyte functional performance and [Ca 2+ ] i transient ([Ca 2+ ] iT) response to β -AR stimulation in myocytes obtained from 6 Sprague Dawley (SD) control and 6 haogen male adult rats. Results: Compared with SD, in hAogen LV myocytes, β 3 -AR protein level significantly increased (26%, 0.29 vs 0.23), but β 1 -AR protein level (26%, 0.4 vs 0.54) significantly decreased. The CaMKIIδ protein levels (16%, 0.50 vs 0.43) and CaMKIIδ phosphorylation (at Thr287) (35%, 0.27 vs 0.20) were significantly increased. These changes were followed by significantly reduced cell contraction (dL/dt max , 102.1vs 138.8μm/s), relaxation (dR/dt max , 94.0 vs 104.7 μm/s) and [Ca 2+ ] iT (0.15 vs 0.21). ISO-stimulated increases in dL/dt max (42% vs 60%), dR/dt max (36% vs 51%) and [Ca 2+ ] iT (20% vs 31%) were also significantly reduced. Conclusions: The upregulation of LV myocyte CaMKIIδ and contrast changes in β 3 -AR and β 1 -AR may be the important cause of the abnormal contractile phenotype and β-AR desensitization in this humanized angiotensinogen model of hypertension of TGR (hAGT) L1623.

Spatiotemporal restriction of endothelial cell calcium signaling is required during leukocyte transmigration

Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.

CaMKIIδC Drives Early Adaptive Ca2+ Change and Late Eccentric Cardiac Hypertrophy.

CaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF. To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice. We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions. We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.

The Role Of Camk2δ-MEF2 Signaling Pathway In Aerobic Exercise-induced Improvement Of Cardiac Function In Hypertension

Objective: The purpose of this study was to investigate the effects of aerobic exercise on the Calcium/calmodulin-dependent protein kinase IIδ(CaMKIIδ)-Myocyte enhancer factor-2(MEF2) signaling pathway in SHR myocardial cells, and the role of the A-kinase anchoring protein 150(AKAP150). METHODS: 12-week-old male SHR and WKY rats were randomly assigned to sedentary groups (WKY-SED, SHR-SED) and exercise training groups (WKY-EX, SHR-EX). Exercise groups were performed a 12-week moderate intensity treadmill running. After 12 weeks, the myocardial cells were enzymatically isolated. The experimental methods include HE staining, the Langendorff technique of isolated heart perfusion, immunohistochemistry, immune cell fluorescence, Western blot. RESULTS: 1) After 12 weeks of exercise, SBP in both WKY-EX and SHR-EX were significantly lower than that of their sedentary counterparts. 2) Compared with the WKY-SED group, the SHR-SED group +dp/dtmax, -dp/dtmax significantly decreased, and the SHR-EX group was significantly higher than the SHR-SED group, +dp/dtmax significantly increased, -dp/dtmax decreased (P<0.01), LVSP increased (P<0.01). 3) The fluorescence intensity of CaM and AKAP150 in the SHR-SED group was higher than WKY-SED group (P<0.01), and the fluorescence intensity of the SHR-EX group AKAP150 was higher than SHR-SED group (P<0.01), and the expression of CaM was lower than the SHR-SED group (P<0.01). 4) The protein expression of p-CaMKIIδ, CaMKIIδ and AKAP150 in the SHR-SED group was higher than that in the WKY-SED group (P<0.01). The expression of p-CaMKIIδ/CaMKIIδ, p-HDAC4/HDAC4 and MEF2 of SHR-EX group are lower than the SHR-SED group (P<0.01). The expression of AKAP150 of SHR-EX was higher than SHR-SED group (P<0.01). CONCLUSION: Aerobic exercise reduced the activity of the CaMKIIδ-MEF2 signaling pathway and increased the expression of AKAP150 in the SHR myocardial, which is one of the molecular mechanisms to improve the function of the heart. Key words: cardiac function; CaMKIIδ-MEF2 signaling pathway; aerobic exercise; AKAP150

Songling Xuemaikang Capsule inhibits isoproterenol-induced cardiac hypertrophy via CaMKIIδ and ERK1/2 pathways

Ethnopharmacological relevanceCardiac hypertrophy is a key pathologic process in heart failure. Songling Xuemaikang Capsule (SXC), is a formulae of Chinese Medicine commonly used in China to treat hypertension and heart failure. However, its mechanism of effects on cardiac hypertrophy is still unclear. Aim of the studyThe aims of the present study were to investigate the cardio-protection roles and detailed mechanisms of SXC on cardiac hypertrophy in vivo and in vitro. Materials and methodsA rat model of cardiac hypertrophy was constructed by isoproterenol (ISO) intraperitoneal injection (i.p), 10 mg/kg/day for 3 days, and 4 groups were compared: CON (n = 8), ISO (n = 8), MET (metoprolol, positive drug treatment, n = 7), and SXC (SXC treatment, n = 6). Cardiac structure and function were evaluated with echocardiography in vivo. Dose-dependent curve was obtained with SXC different concentrations. In addition, H9C2 rat cardiomyocytes were cultured in vitro and the phosphorylation of ERK1/2, p38, JNK, AKT, and protein expression of CaN, CaMKIIδ, GATA4 were detected with Western blot test. ResultsThe results showed that SXC reduced diastolic thickness of left ventricular posterior wall, while did not change ejection fraction and fraction shortening significantly (P > 0.05). SXC inhibit ISO-induced cardiac hypertrophy dose-dependently with 50% inhibiting concentration (IC50) is 0.504 g/kg/day. Moreover, SXC inhibited the protein expression of CaMKIIδ, and the phosphorylation of ERK1/2, so inhibiting protein expression of GATA4 in nucleus, and brain natriuretic peptide in serum (P < 0.001). ConclusionThe mechanism of SXC in the treatment of heart diseases involves SXC dose-dependently inhibited the ISO-induced cardiac hypertrophy via inhibiting CaMKIIδ and ERK1/2/GATA4 signaling pathway.

Compromised cardiovascular function in aged rats corresponds with increased expression and activity of calcium/calmodulin dependent protein kinase IIδ in aortic endothelium

Ageing is the greatest risk factor for cardiovascular disease. Calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) plays a fundamental role in the pathology of heart disease yet a potential role for CaMKIIδ in cardiovascular pathology associated with ageing remains unclear. Taking a combined in vivo and in vitro approach, we have for the first time investigated whether CaMKIIδ expression and CaMKII activity may be altered following age-related cardiovascular deterioration. Both cardiac contractility and aortic blood flow are compromised in aged rats and we have shown that this occurs in parallel with increased inflammation and crucially, autonomous activation of CaMKII. Endothelial cells isolated from young and aged aortae exhibit differences in cell phenotype and physiology. In line with observations in aortic tissue, aged aortic endothelial cells also show increased basal levels of pro-inflammatory markers and oxidative stress with concurrent increased basal activation of CaMKII. These results are the first to demonstrate that elevated CaMKIIδ expression and CaMKII activation occur in parallel with the pathological progression associated with ageing of the heart and vasculature. Specifically, CaMKIIδ expression is significantly increased and activated in the endothelium of aged aorta. As such, CaMKIIδ could serve as an important marker of endothelial dysfunction that accompanies the ageing process and may be an appropriate candidate for investigating targeted therapeutic intervention.

Transcriptional Upregulation of CaMKIIδ through the JAK/STAT3 Pathway is Necessary for the IL‐6‐Dependent Increase in Endothelial Cell Migration

Multifunctional Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a well‐characterized family of proteins comprised of four separate isoforms: α, β, δ, and γ. These isoforms are differentially expressed in a tissue‐specific manner and contribute to diverse physiological and pathophysiological conditions. However, little is known regarding CaMKII isoform expression and function in the endothelium. Previous data from our lab has shown that CaMKIIδ6 is the primary isoform expressed in human umbilical vein endothelial cells (HUVECs) and promote thrombin‐induced loss of barrier function. Interleukin 6 (IL‐6) is a proinflammatory cytokine that induces loss of barrier function in HUVECs through a JAK/STAT3 signaling cascade. The purpose of this study was to test if CaMKIIδ mediates IL‐6‐induced increase in endothelial barrier permeability. Using qPCR, we determined that CaMKIIδ is the most abundant isoform of the enzyme present in HUVECs. Upon treatment with IL‐6, significant increases in both CaMKIIδ transcription and protein expression were observed. Treatment of HUVECs with JAK inhibitor ruxolitinib and siRNA for STAT3 decreased CaMKIIδ expression. However, adenoviral overexpression of various mutant CaMKII isoforms or siRNA knock down of CaMKIIδ did not affect IL‐6‐dependent loss of barrier function as measured by electric cell‐substrate impedence sensing (ECIS). It has been reported that IL‐6 signaling not only induces a loss of endothelial barrier function, but also increases endothelial cell migration. Indeed, siRNA knock down of CaMKIIδ attenuated IL‐6‐induced endothelial cell migration in a scratch‐wound assay. For the first time, our findings thus far have identified transcriptional induction of CaMKIIδ by IL‐6 that is necessary for the subsequent increase in endothelial cell migration.Support or Funding InformationRO1HL49426‐21 to Harold A. SingerThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two Polymorphic Gene Loci Associated with Treprostinil Dose in Pulmonary Arterial Hypertension

Purpose Prostacyclin infusion is an effective therapy for pulmonary arterial hypertension (PAH) but has variable clinical response and dosing requirements. We performed a genome-wide association study (GWAS) to identify biologically-based predictors of prostacyclin response heterogeneity and gene variants associated with PAH pathogenesis . Methods Ninety-eight patients with hemodynamically-defined PAH treated at two academic centers were included. Stably dosed treprostinil therapy was the quantitative phenotype for the GWAS. Candidate genes with the largest effect sizes and strongest statistical associations were further characterized in silico and in vitro to confirm mechanistic hypotheses, and at cardiac catheterization to assess for mechanistically consistent physiological effects. Results Three loci associated with treprostinil dose with P encoded sequence changes with predictable consequences. Production of the prostacyclin-induced pulmonary vasodilator cyclic adenosine monophosphate was reduced in human cell lines by the missense variant rs11078738. The allele of rs10023113 in the promoter of CAMK2D associated with higher treprostinil dose, conferred a reduced affinity for the FOXP1 transcriptional repressor , was associated with increased expression of CAMKIIδ in ventricular tissue, and was associated with elevated right mean atrial and ventricular diastolic pressures in a separate cohort of patients at cardiac catheterization. Conclusion The conduct of effective GWAS is challenging with small numbers of patients. However, by employing the quantitative phenotype of stable treprostinil dose we identified 2 novel loci for treatment response in PAH, gene variants associated with more severe right ventricular dysfunction and higher ultimate dose of treprostinil. Work is ongoing to further characterize these variants.

Targeting the Ca2+/Calmodulin-dependent protein kinase II by Tetrandrine in human liver cancer cells

Hepatocellular carcinoma (HCC) is the most prevalent malignancy in liver and a leading cause of cancer-related deaths. Despite the pressing need for treatment options, patients with HCC develop significant resistance and adverse side effects to current approved drugs that becomes a major barrier to effective treatment. A natural product Tetrandrine (TET) is a potential alternative treatment option for HCC, with demonstrated effectiveness and low toxicity. However, the mechanisms by which Tetrandrine inhibits HCC are unclear. In the current study, we identify Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) as a potential TET drug target through structural modeling. Screening of a panel of HCC cell lines reveal differential sensitivities toward TET treatment. Interestingly, IC50 of TET inhibition of HCC cell proliferation is positively correlated with CaMKIIδ expression level in these distinct HCC cells. Furthermore, TET treatment resulted in a marked reduction of CaMKIIδ phosphorylation level, and knockdown of CaMKIIδ reduced the sensitivity of HCC cells to TET. Most importantly, CaMKIIδ protein levels in high-grade human HCC samples were significantly elevated as compared to normal liver tissues. Taken together, our studies demonstrate that the natural compound TET targets CaMKIIδ in HCC cells, and that CaMKIIδ level is a potential biomarker to identify HCC patient populations sensitive to Tetrandrine treatment.