Calcium/calmodulin-dependent Kinase II Inhibition Research Articles

CaMKII Inhibition is a Novel Therapeutic Strategy to Prevent Diabetic Cardiomyopathy

Increasing prevalence of diabetes mellitus worldwide has pushed the complex disease state to the foreground of biomedical research, especially concerning its multifaceted impacts on the cardiovascular system. Current therapies for diabetic cardiomyopathy have had a positive impact, but with diabetic patients still suffering from a significantly greater burden of cardiac pathology compared to the general population, the need for novel therapeutic approaches is great. A new therapeutic target, calcium/calmodulin-dependent kinase II (CaMKII), has emerged as a potential treatment option for preventing cardiac dysfunction in the setting of diabetes. Within the last 10 years, new evidence has emerged describing the pathophysiological consequences of CaMKII activation in the diabetic heart, the mechanisms that underlie persistent CaMKII activation, and the protective effects of CaMKII inhibition to prevent diabetic cardiomyopathy. This review will examine recent evidence tying cardiac dysfunction in diabetes to CaMKII activation. It will then discuss the current understanding of the mechanisms by which CaMKII activity is enhanced during diabetes. Finally, it will examine the benefits of CaMKII inhibition to treat diabetic cardiomyopathy, including contractile dysfunction, heart failure with preserved ejection fraction, and arrhythmogenesis. We intend this review to serve as a critical examination of CaMKII inhibition as a therapeutic strategy, including potential drawbacks of this approach.

P943The effects of ovariectomy on guinea pig cardiomyocyte intracellular calcium regulation and phosphorylation

Abstract Background Differences in cardiovascular disease risk between men and women have been partly attributed to the cardioprotective effects of oestrogen. Long-term oestrogen deficiency has been shown to alter cardiomyocyte intracellular calcium handling, but little is known about the mechanisms by which these changes occur. Oestrogen is thought to induce both genomic and non-genomic effects on cardiomyocytes, the latter including phosphorylation of calcium handling proteins. Purpose This study addresses the hypothesis that long-term oestrogen deficiency increases protein kinase A (PKA) and calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation in cardiomyocytes, resulting in altered intracellular calcium regulation. Methods Female guinea pigs underwent sham (n = 7) or ovariectomy (OVx) (n = 8) operations and 150 days later, left ventricular myocytes were enzymatically isolated and loaded with fluo-4AM to monitor intracellular calcium. Calcium transients (CaT) were recorded using confocal microscopy. PKA and CaMKII phosphorylation were inhibited by superfusing cells with specific inhibitors, PKI and AIP, respectively. Experiments were carried out both in the presence and absence of β-agonist, isoprenaline (ISO), and relative changes to CaT parameters compared between OVx and sham cells. Results CaT amplitude was greater (p < 0.05) in the OVx group (ΔF/Fo= 2.51 ± 0.57) compared with sham (ΔF/Fo = 2.16 ± 0.57). Inhibition of CaMKII phosphorylation increased CaT amplitude in the sham but not OVx group, both in the presence (by 22%, p < 0.01) and absence of ISO (by 19%, p < 0.01). Time to peak of the CaT increased to a greater extent following inhibition of PKA and CaMKII phosphorylation in the OVx group compared with sham, both in the presence (by 69%, p < 0.0001) and absence (by 162%, p < 0.0001) of ISO respectively. CaT decay time significantly increased (by 21%, p < 0.01) in the sham group following inhibition of PKA and CaMKII together, whilst decay times in the OVx group remained unchanged in the presence and absence of ISO. At higher pacing rates, time to peak of the CaT decreased significantly (by 48%, p < 0.01) in the OVx group but not sham with inhibition of phosphorylation. Conclusion Our findings suggest ovariectomy alters intracellular calcium regulation and some of these effects appear to be mediated by alterations in phosphorylation of calcium handling proteins and/or changes to sites of phosphorylation.

The Metastasis Suppressor Protein Nme1 Is a Concentration-Dependent Modulator of Ca2+/Calmodulin-Dependent Protein Kinase II.

Nucleoside diphosphate kinases (Nmes or NDPKs) have been implicated in a multitude of cellular processes, including an important role in metastasis suppression, and several enzymatic activities have been assigned to the Nme family. Nevertheless, for many of these processes, it has not been possible to establish a strong connection between Nme enzymatic activity and the relevant biological function. We hypothesized that, in addition to its known enzymatic functions, members of the Nme family might also regulate signaling cascades by acting on key signal transducers. Accordingly, here we show that Nme1 directly interacts with the calcium/calmodulin-dependent kinase II (CaMKII). Using purified proteins, we monitored the phosphorylation of a number of CaMKII substrates and determined that at nanomolar levels Nme1 enhances the phosphorylation of T-type substrates; this modulation shifts to inhibition at low micromolar concentrations. Specifically, the autophosphorylation of CaMKII at Thr286 is completely inhibited by 2 μM Nme1, a feature that distinguishes Nme1 from other known endogenous CaMKII inhibitors. Importantly, CaMKII inhibition does not require phosphotransfer activity by Nme1 because the kinase-dead Nme1 H118F mutant is as effective as the wild-type form of the enzyme. Our results provide a novel molecular mechanism whereby Nme1 could modulate diverse cellular processes in a manner that is independent of its known enzymatic activities.

CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGFβ and BMP through SMAD signaling

Upregulation of calcium/calmodulin-dependent kinase II (CaMKII) is implicated in the pathogenesis of osteoarthritis (OA) and reactivation of articular cartilage hypertrophy. However, direct inhibition of CaMKII unexpectedly augmented symptoms of OA in animal models. The role of CaMKII in OA remains unclear and requires further investigation. Analysis of CaMKII expression was performed in normal human and OA articular chondrocytes, and signaling mechanisms were assessed in articular, fetal and Pluripotent Stem Cell (PSC)-derived human chondrocytes using pharmacological (KN93), peptide (AC3-I) and small interfering RNA (siRNA) inhibitors of CaMKII. Expression levels of phospho-CaMKII (pCaMKII) were significantly and consistently increased in human OA specimens. BMP2/4 activated expression of pCaMKII as well as COLII and COLX in human adult articular chondrocytes, and also increased the levels and nuclear localization of SMADs1/5/8, while TGFβ1 showed minimal or no activation of the chondrogenic program in adult chondrocytes. Targeted blockade of CaMKII with specific siRNAs decreased levels of pSMADs, COLII, COLX and proteoglycans in normal and OA adult articular chondrocytes in the presence of both BMP4 and TGFβ1. Both human fetal and PSC-derived chondrocytes also demonstrated a decrease of chondrogenic differentiation in the presence of small molecule and peptide inhibitors of CaMKII. Furthermore, immunoprecipitation for SMADs1/5/8 or 2/3 followed by western blotting for pCaMKII showed direct interaction between SMADs and pCaMKII in primary chondrocytes. Current study demonstrates a direct role for CaMKII in TGF-β and BMP-mediated responses in primary and PSC-derived chondrocytes. These findings have direct implications for tissue engineering of cartilage tissue from stem cells and therapeutic management of OA.

Role of CaMKII in Ang-II-dependent small artery remodeling

Angiotensin-II (Ang-II) is a well-established mediator of vascular remodeling. The multifunctional calcium-calmodulin-dependent kinase II (CaMKII) is activated by Ang-II and regulates Erk1/2 and Akt-dependent signaling in cultured smooth muscle cells in vitro. Its role in Ang-II-dependent vascular remodeling in vivo is far less defined. Using a model of transgenic CaMKII inhibition selectively in smooth muscle cells, we found that CaMKII inhibition exaggerated remodeling after chronic Ang-II treatment and agonist-dependent vasoconstriction in second-order mesenteric arteries. These findings were associated with increased mRNA and protein expression of smooth muscle structural proteins. As a potential mechanism, CaMKII reduced serum response factor-dependent transcriptional activity. In summary, our findings identify CaMKII as an important regulator of smooth muscle function in Ang-II hypertension in vivo.

Calcium/calmodulin-dependent kinase II activity is required for maintaining learning-induced enhancement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic excitation.

Learning leads to changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic excitation. The mechanisms for maintaining such alterations needed for memory persistence remain to be clarified. Here, we report a novel molecular mechanism for maintaining learning-induced AMPAR-mediated enhancement of synaptic excitation. We show that training rats in a complex olfactory discrimination task, such that requires rule learning, leads to the enhancement of averaged amplitude of AMPAR-mediated miniature excitatory post-synaptic currents (mEPSCs) in piriform cortex pyramidal neurons for days after learning. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII) using KN93 or tatCN21days after learning, reduced the averaged mEPSC amplitude in neurons in piriform cortex of trained rats to the level where they are not significantly different from mEPSC of control animals. CaMKII inhibition leads to a decrease in single channel conductance and not to changes in the number of synaptic-activated channels. We conclude that the maintenance of learning-induced enhancement of AMPAR-mediated synaptic excitation requires the activity of calcium/calmodulin-dependent kinase II. We show that training rats in a complex olfactory discrimination task leads to the enhancement of averaged amplitude of AMPA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs) in piriform cortex pyramidal neurons for days after learning. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII) using KN93 or tatCN21days after learning, reduced the averaged mEPSC amplitude in neurons in piriform cortex of trained rats to the level where they are not significantly different from mEPSC of control animals. CaMKII inhibition leads to a decrease in AMPAR single channel conductance. We conclude that the maintenance of learning-induced enhancement of AMPAR-mediated synaptic excitation requires the activity of CaMKII.

Calcium/Calmodulin-Dependent Protein Kinase II and Its Endogenous Inhibitor α in Medullary Thyroid Cancer

Calcium/calmodulin-dependent kinase II (CaMKII) is involved in the regulation of cell proliferation. Its endogenous inhibitor (hCaKIINα) is expressed in some cell types. We determined the role of CaMKII in RET-stimulated proliferation and hCaMKIINα in medullary thyroid carcinoma (MTC). We analyzed the role of RET mutants on CaMKII activation in NIH3T3 and in MTC cell lines, and determined the effect of CaMKII inhibition on RET/ERK pathway and cell proliferation. Then the expression of hCaKIINα mRNA was determined by real-time PCR in primary MTC and it was correlated with some clinicopathologic parameters. RET(C634Y) and RET(M918T) mutants expressed in NIH3T3 cells induced CaMKII activation. CaMKII was activated in unstimulated MTC cells carrying the same RET mutants and it was inhibited by RET inhibition. Inhibition of CaMKII in these cells induced a reduction of Raf-1, MEK, and ERK phosphorylation, cyclin D expression, and cell proliferation. hCaKIINα mRNA expression in primary MTC was very variable and did not correlate with gender and age at diagnosis. Serum calcitonin, (R(2) = 0.032; P = 0.017), tumor volume (P = 0.0079), lymph node metastasis (P = 0.033), and staging (P = 0.0652) were negatively correlated with the hCaKIINα mRNA expression. CaMKII is activated by RET mutants and is activated at baseline in MTC cells where it mediates the oncogenic pathway leading to cell proliferation. The mRNA expression of its endogenous inhibitor hCaKIINα inversely correlates with the severity of MTC. CaMKII might represent a new target for MTC therapy and hCaKIINα is a marker of disease extension.

CaMKII inhibition in vascular smooth muscle improves angiotensin II–hypertension

The multifunctional calcium/calmodulin‐dependent kinase II (CaMKII) is expressed in vascular smooth muscle cells (VSMC) and activated by Angiotensin‐II (Ang‐II). To test the hypothesis that CaMKII in VSMC mediates Ang‐II‐hypertension, we developed transgenic mice that express the specific CaMKII peptide inhibitor CaMKIIN selectively in VSMC (Tg SM CaMKIIN). These mice have a 65 % reduced CaMKII activity in arteries (p<0.01) and identical baseline blood pressure compared to littermate controls. Following Ang‐II infusion at pressor dose, the increase in mean pressure was significantly attenuated (129±4 mmHg vs, 116±3 mmHg, p<0.05). Ang‐II increases Ca2+ influx through L‐type Ca2+ channels (LTCC). Cytosolic Ca2+ ([Ca2+]) rise leads to an increased vascular tone. Compared with littermates, freshly isolated mesenteric VSMC of Ang‐II treated Tg SM CaMKIIN mice had significantly reduced voltage gated L‐type Ca2+ current (ICa), ICa facilitation. Moreover, the [Ca2+] elevation in response to Ang‐II stimulation was reduced by 50 % in VSMC of TgSM CaMKIIN mice (0.045±0.002 vs, 0.025±0.003; ratio340/380; p<0.05) whereas, level of total releasable SR calcium was decreased by 35 %. In conclusion, inhibition of CaMKII in VSMC significantly attenuates Ang‐II induced hypertension through regulation of LTCC and by restoring the calcium homeostasis. CaMKII may be a novel target in treating hypertension.

Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) Inhibition Induces Neurotoxicity via Dysregulation of Glutamate/Calcium Signaling and Hyperexcitability

Aberrant glutamate and calcium signalings are neurotoxic to specific neuronal populations. Calcium/calmodulin-dependent kinase II (CaMKII), a multifunctional serine/threonine protein kinase in neurons, is believed to regulate neurotransmission and synaptic plasticity in response to calcium signaling produced by neuronal activity. Importantly, several CaMKII substrates control neuronal structure, excitability, and plasticity. Here, we demonstrate that CaMKII inhibition for >4 h using small molecule and peptide inhibitors induces apoptosis in cultured cortical neurons. The neuronal death produced by prolonged CaMKII inhibition is associated with an increase in TUNEL staining and caspase-3 cleavage and is blocked with the translation inhibitor cycloheximide. Thus, this neurotoxicity is consistent with apoptotic mechanisms, a conclusion that is further supported by dysregulated calcium signaling with CaMKII inhibition. CaMKII inhibitory peptides also enhance the number of action potentials generated by a ramp depolarization, suggesting increased neuronal excitability with a loss of CaMKII activity. Extracellular glutamate concentrations are augmented with prolonged inhibition of CaMKII. Enzymatic buffering of extracellular glutamate and antagonism of the NMDA subtype of glutamate receptors prevent the calcium dysregulation and neurotoxicity associated with prolonged CaMKII inhibition. However, in the absence of CaMKII inhibition, elevated glutamate levels do not induce neurotoxicity, suggesting that a combination of CaMKII inhibition and elevated extracellular glutamate levels results in neuronal death. In sum, the loss of CaMKII observed with multiple pathological states in the central nervous system, including epilepsy, brain trauma, and ischemia, likely exacerbates programmed cell death by sensitizing vulnerable neuronal populations to excitotoxic glutamate signaling and inducing an excitotoxic insult itself.

CaMKII Regulation of Calcium Homeostasis and Neuronal Activity

Calcium/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that regulates multiple ion channels and receptors that are essential for neuronal activity and plasticity. To explore the role of CaMKII in regulating neuronal calcium homeostasis, we applied pharmacological inhibitors of CaMKII (10 micromolar tat-CN21, tat-AIP, and myr-AIP) to cultured cortical and hippocampal neurons and measured changes in intracellular calcium levels using Fluo-4AM and Fura-2FF. CaMKII inhibition induced a rapid increase in intracellular calcium levels. Dysregulated calcium signaling was not limited to neurons, as CaMKII inhibitors also initiated calcium waves in cultured astrocytes that preceeded neuronal effects. Inhibitors that disrupt neuronal activity, VGSCs, and AMPA-Rs, block this calcium dysregulation in neurons. Although L-type calcium channel blockers had no effect, eliminating extracellular calcium, inhibiting NMDA-Rs or N-type calcium channels did prevent calcium dysregulation in neurons, implicating aberrant glutamate release and/or post-synaptic activity in this calcium dysregulation. Increased glutamate levels (2-4 micromolar) were observed in the media following CaMKII inhibition. Enzymatic buffering of glutamate in the media prevented the aberrant calcium influx. Finally, to directly test whether CaMKII inhibition increases neuronal activity, voltage-clamp electrophysiology was utilized to measure action potential generation to a depolarizing ramp current. Unlike inactive controls, CaMKII inhibitors induced a three-fold increase in the number of action potentials when applied in the patch pipette. In total, a loss of CaMKII activity results in dysregulation of intracellular calcium homeostasis and a subsequent dysregulation of glutamate signaling and neuronal excitability. Thus, CaMKII not only responds to neuronal activity via changes in intracellular calcium levels, it also appears to serve as a master regulator of neuronal excitability through regulation of calcium homeostasis.

Cardiac Calmodulin Kinase: A Potential Target for Drug Design

Therapeutic strategy for cardiac arrhythmias has undergone a remarkable change during the last decades. Currently implantable cardioverter defibrillator therapy is considered to be the most effective therapeutic method to treat malignant arrhythmias. Some even argue that there is no room for antiarrhythmic drug therapy in the age of implantable cardioverter defibrillators. However, in clinical practice, antiarrhythmic drug therapies are frequently needed, because implantable cardioverter defibrillators are not effective in certain types of arrhythmias (i.e. premature ventricular beats or atrial fibrillation). Furthermore, given the staggering cost of device therapy, it is economically imperative to develop alternative effective treatments. Cardiac ion channels are the target of a number of current treatment strategies, but therapies based on ion channel blockers only resulted in moderate success. Furthermore, these drugs are associated with an increased risk of proarrhythmia, systemic toxicity, and increased defibrillation threshold. In many cases, certain ion channel blockers were found to increase mortality. Other drug classes such as ßblockers, angiotensin-converting enzyme inhibitors, aldosterone antagonists, and statins appear to have proven efficacy for reducing cardiac mortality. These facts forced researchers to shift the focus of their research to molecular targets that act upstream of ion channels. One of these potential targets is calcium/calmodulin-dependent kinase II (CaMKII). Several lines of evidence converge to suggest that CaMKII inhibition may provide an effective treatment strategy for heart diseases. (1) Recent studies have elucidated that CaMKII plays a key role in modulating cardiac function and regulating hypertrophy development. (2) CaMKII activity has been found elevated in the failing hearts from human patients and animal models. (3) Inhibition of CaMKII activity has been shown to mitigate hypertrophy, prevent functional remodeling and reduce arrhythmogenic activity. In this review, we will discuss the structural and functional properties of CaMKII, the modes of its activation and the functional consequences of CaMKII activity on ion channels.

Calcium-calmodulin-dependent kinase II (CaMKII) mediates insulin-stimulated proliferation and glucose uptake

Cellular growth and glucose uptake are regulated by multiple signals generated by the insulin receptor. The mechanisms of individual modulation of these signals remain somewhat elusive. We investigated the role of CaMKII in insulin signalling in a rat skeletal muscle cell line, demonstrating that CaMKII modulates the insulin action on DNA synthesis and the negative feedback that down regulates glucose uptake. Insulin stimulation generated partly independent signals leading to the rapid activation of Akt, Erk-1/2 and CaMKII. Akt activation was followed by Glut-4 translocation to the plasma membrane and increase of glucose uptake. Then, IRS-1 was phosphorylated at S612, the IRS-1/p85PI3K complex was disrupted, Akt was no more phosphorylated and both Glut-4 translocation and glucose uptake were reduced. Inhibition of CaMKII abrogated the insulin-induced Erk-1/2 activation, DNA synthesis and phosphorylation of IRS-1 at S612. Inhibition of CaMKII also abrogated the down-regulation of insulin-stimulated Akt phosphorylation, Glut-4 membrane translocation and glucose uptake. These results demonstrate that: 1 — CaMKII modulates the insulin-induced Erk-1/2 activation and cell proliferation; 2 — after the initial stimulation of the IRS-1/Akt pathway, CaMKII mediates the down-regulation of stimulated glucose uptake. This represents a novel mechanism in the selective control of insulin signals, and a possible site for pharmacological intervention.

CaMKII and Its Role in Cardiac Arrhythmia

Calcium/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine kinase widely distributed in a number of tissue types. Activation of CaMKII has been linked to important downstream physiological processes, including apoptosis, hypertrophy, and arrhythmia in the heart. Pharmacological or genetic inhibition of CaMKII has been shown to improve health outcomes in a number of animal models. In this review, we summarize the structural and functional properties of CaMKII and detail its role in cardiac arrhythmia, structural heart disease, and sudden death.

Calcium/calmodulin‐dependent kinase II regulates notch‐1 signaling in prostate cancer cells

Notch signaling is associated with prostate osteoblastic bone metastases and calcium/calmodulin-dependent kinase II (CaMKII) is associated with osteoblastogenesis of human mesenchymal stem cells. Here we show that prostate cancer cell lines C4-2B and PC3, both derived from bone metastases and express Notch-1, have all four isoforms of CaMKII (alpha, beta, gamma, delta). In contrast, prostate cancer cell lines LNcaP and DU145, which are not derived from bone metastases and lack the Notch-1 receptor, both lack the alpha isoform of CaMKII. In addition, DU145 cells also lack the beta-isoform. In C4-2B cells, inhibition of CaMKII by KN93 or gamma-secretase by L-685,458 inhibited the formation of the cleaved form of Notch-1 thus inhibiting Notch signaling. KN93 inhibited down stream Notch-1 signaling including Hes-1 gene expression, Hes-1 promoter activity, and c-Myc expression. In addition, both KN93 and L-685,458 inhibited proliferation and Matrigel invasion by C4-2B cells. The activity of gamma-secretase was unaffected by KN93 but markedly inhibited by L-685,458. Inhibition of the expression of alpha, beta, or gamma-isoform by siRNA did not affect Hes-1 gene expression, however when expression of one isoform was inhibited by siRNA, there were compensatory changes in the expression of the other isoforms. Over-expression of CaMKII-alpha increased Hes-1 expression, consistent with Notch-1 signaling being at least partially dependent upon CaMKII. This unique crosstalk between CaMKII and Notch-1 pathways provides new insight into Notch signaling and potentially provides new targets for pharmacotherapeutics.

Serotonin 5-HT1A Receptors Regulate NMDA Receptor Channels through a Microtubule-Dependent Mechanism

The serotonin system and NMDA receptors (NMDARs) in prefrontal cortex (PFC) are both critically involved in the regulation of cognition and emotion under normal and pathological conditions; however, the interactions between them are essentially unknown. Here we show that serotonin, by activating 5-HT(1A) receptors, inhibited NMDA receptor-mediated ionic and synaptic currents in PFC pyramidal neurons, and the NR2B subunit-containing NMDA receptor is the primary target of 5-HT(1A) receptors. This effect of 5-HT(1A) receptors was blocked by agents that interfere with microtubule assembly, as well as by cellular knock-down of the kinesin motor protein KIF17 (kinesin superfamily member 17), which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Inhibition of either CaMKII (calcium/calmodulin-dependent kinase II) or MEK/ERK (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) abolished the 5-HT(1A) modulation of NMDAR currents. Biochemical evidence also indicates that 5-HT(1A) activation reduced microtubule stability, which was abolished by CaMKII or MEK inhibitors. Moreover, immunocytochemical studies show that 5-HT(1A) activation decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Together, these results suggest that serotonin suppresses NMDAR function through a mechanism dependent on microtubule/kinesin-based dendritic transport of NMDA receptors that is regulated by CaMKII and ERK signaling pathways. The 5-HT(1A)-NMDAR interaction provides a potential mechanism underlying the role of serotonin in controlling emotional and cognitive processes subserved by PFC.