Overexpression Of Calmodulin-dependent Protein Kinase II Research Articles

Abstract P3195: Mitochondrial CaMKII Drives Cardiometabolic Dysfunction In Heart Failure

Background: Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is an established negative regulator of cardiac injury. Both the expression and activity levels of CaMKII delta, the primary cardiac isoform of this kinase, are elevated in models of heart failure (HF) such as ischemia-reperfusion (I/R) injury and following myocardial infarction (MI). This upregulation is due in part to CaMKII’s role in the regulation of excitation-contraction coupling, apoptosis, activation of hypertrophic programming, arrhythmias and pro-inflammatory signaling. Recently, a pool of mitochondrial CaMKII (mtCaMKII) has been identified; we have demonstrated that there is an observed increase in mitochondrial CaMKII activation with left ventricular dilation acutely following injury in a mouse model of MI. This deleterious post-MI phenotype is rescued with genetic mitochondrial CaMKII inhibition; conversely, mice with myocardial mitochondrial CaMKII overexpression present with a severe dilated cardiomyopathy phenotype, alterations in electron transport chain (ETC) activity, and decreased ATP production. Objective: To date, the molecular mechanisms by which CaMKII regulates mitochondrial energetics are unknown and present a novel therapeutic target for HF which we are currently studying. Methods: As we have identified changes in the activity of enzymes, particularly complexes I and II, in the mitochondrial ETC as well as TCA cycle in response to increased CaMKII levels or activity, we are in the process of assessing changes in the mitochondrial CaMKII interactome between uninjured and failing hearts. Utilizing liquid chromatography-mass spectrometry (LCMS) and proteomics analysis of both scaffolding interactions alongside a novel ATP analogue labeling technique, we have mapped novel mitochondrial proteins which interact with mtCaMKII. Significance: The identification of previously unknown mitochondrial pathways involving CaMKII may uncover promising pharmacological targets for cardiovascular therapeutics, as this kinase is a critical player in the progression of HF.

Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Hepatitis B Virus Replication from cccDNA via AMPK Activation and AKT/mTOR Suppression.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.

Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) \u03b2-Dependent Phosphorylation of GABAB1 Triggers Lysosomal Degradation of GABAB Receptors via Mind Bomb-2 (MIB2)-Mediated Lys-63-Linked Ubiquitination

The G protein-coupled GABAB receptors, constituted from GABAB1 and GABAB2 subunits, are important regulators of neuronal excitability by mediating long-lasting inhibition. One factor that determines receptor availability and thereby the strength of inhibition is regulated protein degradation. GABAB receptors are constitutively internalized from the plasma membrane and are either recycled to the cell surface or degraded in lysosomes. Lys-63-linked ubiquitination mediated by the E3 ligase Mind bomb-2 (MIB2) is the signal that sorts GABAB receptors to lysosomes. However, it is unknown how Lys-63-linked ubiquitination and thereby lysosomal degradation of the receptors is regulated. Here, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) promotes MIB2-mediated Lys-63-linked ubiquitination of GABAB receptors. We found that inhibition of CaMKII in cultured rat cortical neurons increased cell surface GABAB receptors, whereas overexpression of CaMKIIβ, but not CaMKIIα, decreased receptor levels. This effect was conveyed by Lys-63-linked ubiquitination of GABAB1 at multiple sites mediated by the E3 ligase MIB2. Inactivation of the CaMKII phosphorylation site on GABAB1(Ser-867) strongly reduced Lys-63-linked ubiquitination of GABAB receptors and increased their cell surface expression, whereas the phosphomimetic mutant GABAB1(S867D) exhibited strongly increased Lys-63-linked ubiquitination and reduced cell surface expression. Finally, triggering lysosomal degradation of GABAB receptors by sustained activation of glutamate receptors, a condition occurring in brain ischemia, was accompanied with a massive increase of GABAB1(Ser-867) phosphorylation-dependent Lys-63-linked ubiquitination of GABAB receptors. These findings indicate that CaMKIIβ-dependent Lys-63-linked ubiquitination of GABAB1 at multiple sites controls sorting of GABAB receptors to lysosomes for degradation under physiological and pathological condition.

Ca2+/calmodulin-dependent protein kinase II promotes neurodegeneration caused by tau phosphorylated at Ser262/356 in a transgenic Drosophila model of tauopathy.

Abnormal deposition of the microtubule-associated protein tau is a common pathological feature of multiple neurodegenerative diseases, including Alzheimer's disease (AD), and plays critical roles in their pathogenesis. Disruption of calcium homeostasis and the downstream kinase Ca2+/calmodulin-dependent protein kinase II (CaMKII) coincides with pathological phosphorylation of tau in AD brains. However, it remains unclear whether and how dysregulation of CaMKII affects tau toxicity. Using a Drosophila model, we found that CaMKII promotes neurodegeneration caused by tau phosphorylated at the AD-associated sites Ser262/356. Overexpression of CaMKII promoted, while RNA-mediated knockdown of CaMKII and inhibition of CaMKII activity by expression of an inhibitory peptide suppressed, tau-mediated neurodegeneration. Blocking tau phosphorylation at Ser262/356 by alanine substitutions suppressed promotion of tau toxicity by CaMKII, suggesting that tau phosphorylation at these sites is required for this phenomenon. However, neither knockdown nor overexpression of CaMKII affected tau phosphorylation levels at Ser262/356, suggesting that CaMKII is not directly involved in tau phosphorylation at Ser262/356 in this model. These results suggest that a pathological cascade of events, including elevated levels of tau phosphorylated at Ser262/356 and aberrant activation of CaMKII, work in concert to promote tau-mediated neurodegeneration.

Astragalus Granule Prevents Ca2+ Current Remodeling in Heart Failure by the Downregulation of CaMKII.

Background Astragalus was broadly used for treating heart failure (HF) and arrhythmias in East Asia for thousands of years. Astragalus granule (AG), extracted from Astragalus, shows beneficial effect on the treatment of HF in clinical research. We hypothesized that administration of AG prevents the remodeling of L-type Ca2+ current (ICa-L) in HF mice by the downregulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Methods HF mice were induced by thoracic aortic constriction (TAC). After 4 weeks of AG treatment, cardiac function and QT interval were evaluated. Single cardiac ventricular myocyte was then isolated and whole-cell patch clamp was used to record action potential (AP) and ICa-L. The expressions of L-type calcium channel alpha 1C subunit (Cav1.2), CaMKII, and phosphorylated protein kinase A (p-PKA) were examined by western blot. Results The failing heart manifested distinct electrical remodeling including prolonged repolarization time and altered ICa-L kinetics. AG treatment attenuated this electrical remodeling, supported by AG-related shortened repolarization time, decreased peak ICa-L, accelerated ICa-L inactivation, and positive frequency-dependent ICa-L facilitation. In addition, AG treatment suppressed the overexpression of CaMKII, but not p-PKA, in the failing heart. Conclusion AG treatment protected the failing heart against electrical remodeling and ICa-L remodeling by downregulating CaMKII.

Deletion of Interleukin-6 Attenuates Pressure Overload-Induced Left Ventricular Hypertrophy and Dysfunction.

The role of interleukin (IL)-6 in the pathogenesis of cardiac myocyte hypertrophy remains controversial. To conclusively determine whether IL-6 signaling is essential for the development of pressure overload-induced left ventricular (LV) hypertrophy and to elucidate the underlying molecular pathways. Wild-type and IL-6 knockout (IL-6(-/-)) mice underwent sham surgery or transverse aortic constriction (TAC) to induce pressure overload. Serial echocardiograms and terminal hemodynamic studies revealed attenuated LV hypertrophy and superior preservation of LV function in IL-6(-/-) mice after TAC. The extents of LV remodeling, fibrosis, and apoptosis were reduced in IL-6(-/-) hearts after TAC. Transcriptional and protein assays of myocardial tissue identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 3 (STAT3) activation as important underlying mechanisms during cardiac hypertrophy induced by TAC. The involvement of these pathways in myocyte hypertrophy was verified in isolated cardiac myocytes from wild-type and IL-6(-/-) mice exposed to prohypertrophy agents. Furthermore, overexpression of CaMKII in H9c2 cells increased STAT3 phosphorylation, and exposure of H9c2 cells to IL-6 resulted in STAT3 activation that was attenuated by CaMKII inhibition. Together, these results identify the importance of CaMKII-dependent activation of STAT3 during cardiac myocyte hypertrophy via IL-6 signaling. Genetic deletion of IL-6 attenuates TAC-induced LV hypertrophy and dysfunction, indicating a critical role played by IL-6 in the pathogenesis of LV hypertrophy in response to pressure overload. CaMKII plays an important role in IL-6-induced STAT3 activation and consequent cardiac myocyte hypertrophy. These findings may have significant therapeutic implications for LV hypertrophy and failure in patients with hypertension.

Role of CaMKII and PKA in Early Afterdepolarization of Human Ventricular Myocardium Cell: A Computational Model Study.

Early afterdepolarization (EAD) plays an important role in arrhythmogenesis. Many experimental studies have reported that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and β-adrenergic signaling pathway are two important regulators. In this study, we developed a modified computational model of human ventricular myocyte to investigate the combined role of CaMKII and β-adrenergic signaling pathway on the occurrence of EADs. Our simulation results showed that (1) CaMKII overexpression facilitates EADs through the prolongation of late sodium current's (I NaL) deactivation progress; (2) the combined effect of CaMKII overexpression and activation of β-adrenergic signaling pathway further increases the risk of EADs, where EADs could occur at shorter cycle length (2000 ms versus 4000 ms) and lower rapid delayed rectifier K+ current (I Kr) blockage (77% versus 85%). In summary, this study computationally demonstrated the combined role of CaMKII and β-adrenergic signaling pathway on the occurrence of EADs, which could be useful for searching for therapy strategies to treat EADs related arrhythmogenesis.

Modeling CaMKII in cardiac physiology: from molecule to tissue

Post-translational modification of membrane proteins (e.g., ion channels, receptors) by protein kinases is an essential mechanism for control of excitable cell function. Importantly, loss of temporal and/or spatial control of ion channel post-translational modification is common in congenital and acquired forms of cardiac disease and arrhythmia. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates a number of diverse cellular functions in heart, including excitation-contraction coupling, gene transcription, and apoptosis. Dysregulation of CaMKII signaling has been implicated in human and animal models of disease. Understanding of CaMKII function has been advanced by mathematical modeling approaches well-suited to the study of complex biological systems. Early kinetic models of CaMKII function in the brain characterized this holoenzyme as a bistable molecular switch capable of storing information over a long period of time. Models of CaMKII activity have been incorporated into models of the cell and tissue (particularly in the heart) to predict the role of CaMKII in regulating organ function. Disease models that incorporate CaMKII overexpression clearly demonstrate a link between its excessive activity and arrhythmias associated with congenital and acquired heart disease. This review aims at discussing systems biology approaches that have been applied to analyze CaMKII signaling from the single molecule to intact cardiac tissue. In particular, efforts to use computational biology to provide new insight into cardiac disease mechanisms are emphasized.

Fluoxetine Epigenetically Alters the CaMKIIα Promoter in Nucleus Accumbens to Regulate ΔFosB Binding and Antidepressant Effects

Chronic social defeat stress in mice produces a susceptible phenotype characterized by several behavioral abnormalities consistent with human depression that are reversed by chronic but not acute exposure to antidepressant medications. Recent work in addiction models demonstrates that the transcription factor ΔFosB and protein kinase calmodulin-dependent protein kinase II (CaMKII) are co-regulated in nucleus accumbens (NAc), a brain reward region implicated in both addiction and depression models including social defeat. Previous work has also demonstrated that ΔFosB is induced in NAc after chronic social defeat stress or after chronic antidepressant treatment, wherein it mediates a pro-resilience or antidepressant-like phenotype. Here, using chromatin immunoprecipitation assays, we found that ΔFosB binds the CaMKIIα gene promoter in NAc and that this binding increases after mice are exposed to chronic social defeat stress. Paradoxically, chronic exposure to the antidepressant fluoxetine reduces binding of ΔFosB to the CaMKIIα promoter and reduces CaMKII expression in NAc, despite the fact that ΔFosB is induced under these conditions. These data suggest a novel epigenetic mechanism of antidepressant action, whereby fluoxetine induces some chromatin change at the CaMKIIα promoter, which blocks the ΔFosB binding. Indeed, chronic fluoxetine reduces acetylation and increases lysine-9 dimethylation of histone H3 at the CaMKIIα promoter in NAc, effects also seen in depressed humans exposed to antidepressants. Overexpression of CaMKII in NAc blocks fluoxetine's antidepressant effects in the chronic social defeat paradigm, whereas inhibition of CaMKII activity in NAc mimics fluoxetine exposure. These findings suggest that epigenetic suppression of CaMKIIα expression in NAc is behaviorally relevant and offer a novel pathway for possible therapeutic intervention in depression and related syndromes.

Berbamine Inhibits the Growth of Liver Cancer Cells and Cancer-Initiating Cells by Targeting Ca2+/Calmodulin-Dependent Protein Kinase II

Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here, we report that berbamine and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca(2+)/calmodulin-dependent protein kinase II (CAMKII). Furthermore, berbamine inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice and downregulated the self-renewal abilities of liver cancer-initiating cells. Chemical inhibition or short hairpin RNA-mediated knockdown of CAMKII recapitulated the effects of berbamine, whereas overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to berbamine treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peritumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of berbamine for liver cancer therapies. Our data suggest that berbamine and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII. Mol Cancer Ther; 12(10); 2067-77. ©2013 AACR.

Theoretical investigation of the mechanism of heart failure using a canine ventricular cell model: Especially the role of up-regulated CaMKII and SR Ca2 + leak

Heart failure (HF) is associated with susceptibility to sudden cardiac death. However, the underlying mechanism of electrical instability and mechanical dysfunction associated with HF remains poorly understood. In this study, a new canine ventricular cell model based on the Hund–Rudy dynamic (HRd) model and recently published experimental data was developed to investigate the electrical changes and calcium handling dysfunction in HF. Simulation results suggest that: 1) acute Ca2+/calmodulin-dependent protein kinase II (CaMKII) over-expression (CaMKII-OE) affects the action potential (AP) profile, while AP prolongation is mainly caused by the down-regulation of K+ currents; 2) enhanced late Na+ current (INaL) alone cannot adequately lead to [Na+] elevation in HF; 3) enhanced sarcoplasmic reticulum (SR) leak current (Ileak) causes disturbed Ca2+ handling and there is little contribution from Na+/Ca2+ exchanger (NCX); 4) at high SR Ca2+ load, a steeper fractional SR Ca2+ release is observed in HF than that in control, causing alternans to occur more easily; and 5) Ileak block restores the contraction and relaxation function, but cannot eliminate alternans. By inhibiting CaMKII, alternans is eliminated, but contractility is not improved. Partial CaMKII inhibition in combination with Ileak block could augment mechanical function and depress alternans, suggesting a new possible therapeutic target for HF treatment.

Ca2+-calmodulin-dependent protein kinase II regulation of cardiac excitation-transcription coupling

The multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) has emerged as a nodal point in cardiac muscle function and adaptation. This stems from CaMKII’s demonstrated role in phosphorylation and regulation of many key molecules known to be critical in excitation-contraction coupling and arrhythmias, and because it increases in expression and activation during hypertrophy and heart failure (HF) and CaMKII overexpression and suppression induce and suppress HF respectively. For many years, there were indications that intracellular Ca could activate altered gene expression, but only in the past ten years has it been recognized that CaMKII and a Ca-calmodulin-dependent phosphatase calcineurin are major Ca-dependent mediators of transcriptional regulation. Moreover, these pathways contribute to changes in gene expression of proteins involved in the HF phenotype, including some of the ion channels and Ca transporters that are acutely involved in systolic dysfunction and arrhythmias.

Synergy between CaMKII Substrates and β-Adrenergic Signaling in Regulation of Cardiac Myocyte Ca 2+ Handling

Cardiac excitation-contraction coupling is a highly coordinated process that is controlled by protein kinase signaling pathways, including Ca 2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA). Increased CaMKII expression and activity (as occurs during heart failure) destabilizes EC coupling and may lead to sudden cardiac death. To better understand mechanisms of cardiac CaMKII function, we integrated dynamic CaMKII-dependent regulation of key Ca 2+ handling targets with previously validated models of cardiac EC coupling, Ca 2+/calmodulin-dependent activation of CaMKII, and β-adrenergic activation of PKA. Model predictions are validated against CaMKII-overexpression data from rabbit ventricular myocytes. The model demonstrates how overall changes to Ca 2+ handling during CaMKII overexpression are explained by interactions between individual CaMKII substrates. CaMKII and PKA activities during β-adrenergic stimulation may synergistically facilitate inotropic responses and contribute to a CaMKII-Ca 2+-CaMKII feedback loop. CaMKII regulated early frequency-dependent acceleration of relaxation and EC coupling gain (which was highly sarcoplasmic reticulum Ca 2+ load-dependent). Additionally, the model identifies CaMKII-dependent ryanodine receptor hyperphosphorylation as a proarrhythmogenic trigger. In summary, we developed a detailed computational model of CaMKII and PKA signaling in cardiac myocytes that provides unique insights into their regulation of normal and pathological Ca 2+ handling.

β-Amyloid Impairs AMPA Receptor Trafficking and Function by Reducing Ca2+/Calmodulin-dependent Protein Kinase II Synaptic Distribution

A fundamental feature of Alzheimer disease (AD) is the accumulation of beta-amyloid (Abeta), a peptide generated from the amyloid precursor protein (APP). Emerging evidence suggests that soluble Abeta oligomers adversely affect synaptic function, which leads to cognitive failure associated with AD. The Abeta-induced synaptic dysfunction has been attributed to the synaptic removal of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs); however, it is unclear how Abeta induces the loss of AMPARs at the synapses. In this study we have examined the potential involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), a signaling molecule critical for AMPAR trafficking and function. We found that the synaptic pool of CaMKII was significantly decreased in cortical neurons from APP transgenic mice, and the density of CaMKII clusters at synapses was significantly reduced by Abeta oligomer treatment. In parallel, the surface expression of GluR1 subunit as well as AMPAR-mediated synaptic response and ionic current was selectively decreased in APP transgenic mice and Abeta-treated cultures. Moreover, the reducing effect of Abeta on AMPAR current density was mimicked and occluded by knockdown of CaMKII and blocked by overexpression of CaMKII. These results suggest that the Abeta-induced change in CaMKII subcellular distribution may underlie the removal of AMPARs from synaptic membrane by Abeta.

Role of CaMKII for signaling and regulation in the heart

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the CaMK isoform predominantly found in the heart. Cardiac myocytes signaling during excitation-contraction coupling (ECC) is described by the increase in intracellular Ca2+ concentration. In consequence, CaMKII is activated thereby phosphorylating several important Ca2+ handling proteins with multiple functional consequences for cardiac myocytes. Specific CaMKII overexpression in the heart and in isolated myocytes of animals can exert distinct and novel effects on ECC. CaMKII activity and expression are reported to be increased in cardiac hypertrophy, in human heart failure, as well as in animal models thereby contributing to cardiac disease through a regulation process termed excitation-transcription coupling (ETC). In the present review important aspects of the role of CaMKII in ECC and ETC are summarized with an emphasis on recent novel findings.

The role of CaMKII as an F-actin-bundling protein crucial for maintenance of dendritic spine structure

Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase critically involved in synaptic plasticity in the brain. It is highly concentrated in the postsynaptic density fraction, exceeding the amount of any other signal transduction molecules. Because kinase signaling can be amplified by catalytic reaction, why CaMKII exists in such a large quantity has been a mystery. Here, we provide biochemical evidence that CaMKII is capable of bundling F-actin through a stoichiometric interaction. Consistent with this evidence, in hippocampal neurons, RNAi-mediated down-regulation of CaMKII leads to a reduction in the volume of dendritic spine head that is mediated by F-actin dynamics. An overexpression of CaMKII slowed down the actin turnover in the spine head. This activity was associated with beta subunit of CaMKII in a manner requiring its actin-binding and association domains but not the kinase domain. This finding indicates that CaMKII serves as a central signaling molecule in both functional and structural changes during synaptic plasticity.

CaMKII inhibition protects against necrosis and apoptosis in irreversible ischemia–reperfusion injury

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of cardiac excitation-contraction coupling (ECC) as well as in apoptotic signaling and adverse remodeling. The goal of the present study is to investigate the role of CaMKII in irreversible ischemia and reperfusion (I/R) injury. Isovolumic Langendorff perfused rat hearts were subjected to global no-flow I/R (45 min/120 min), and isolated myocytes were subjected to a protocol of simulated I/R (45 min simulated ischemia/60 min reoxygenation) either in the absence or presence of CaMKII inhibition [KN-93 (KN) or the CaMKII inhibitory peptide (AIP)]. In I/R hearts, an increase in CaMKII activity at the beginning of reperfusion was confirmed by the significantly increased phosphorylation of the Thr(17) site of phospholamban. In the presence of KN, contractile recovery at the end of reperfusion was almost double that of I/R hearts. This recovery was associated with a significant decrease in the extent of infarction, lactate dehydrogenase release (necrosis), TUNEL-positive cells, caspase-3 activity, and an increase in the Bcl-2/Bax ratio (apoptosis). In isolated myocytes, both KN and AIP prevented simulated I/R-induced spontaneous contractile activity and cell mortality. Similar results were obtained when inhibiting the reverse mode Na(+)/Ca(2+) exchanger (NCX) with KB-R7943, sarcoplasmic reticulum (SR) function with ryanodine and thapsigargin, or SR Ca(2+) release with tetracaine. In contrast, overexpression of CaMKII decreased cell viability from 52+/-3% to 26+/-2%. Taken together, the present findings are the first to establish CaMKII as a fundamental component of a cascade of events integrating the NCX, the SR, and mitochondria that promote cellular apoptosis and necrosis in irreversible I/R injury.

Phosphorylation of dystrophin Dp71d by Ca2+/calmodulin‐dependent protein kinase II modulates the Dp71d nuclear localization in PC12 cells

We have shown that the splicing isoform of Dp71 (Dp71d) localizes to the nucleus of PC12 cells, an established cell line derived from a rat pheochromocytoma; however, the mechanisms governing its nuclear localization are unknown. As protein phosphorylation modulates the nuclear import of proteins, and as Dp71d presents several potential sites for phosphorylation, we analyzed whether Dp71d is phosphorylated in PC12 cells and the role of phosphorylation on its nuclear localization. We demonstrated that Dp71d is phosphorylated under basal conditions at serine and threonine residues by endogenous protein kinases. Dp71d phosphorylation was activated by 2-O-tetradecanoyl phorbol 13-acetate (TPA), but this effect was blocked by EGTA. Supporting the role of intracellular calcium on Dp71d phosphorylation, we observed that the stimulation of calcium influx by cell depolarization increased Dp71d phosphorylation, and that the calcium-calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7) blocked such induction. The blocking action of bisindolylmaleimide I (Bis I), a specific inhibitor for Ca2+/diacylglicerol-dependent protein kinase (PKC), on Dp71d phosphorylation suggested the participation of PKC in this event. In addition, transfection experiments with Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression vectors as well as the use of KN-62, a CaMKII-specific inhibitor, demonstrated that CaMKII is also involved in Dp71d phosphorylation. Stimulation of Dp71d phosphorylation by cell depolarization and/or the overexpression of CaMKII favored the Dp71d nuclear accumulation. Overall, our results indicate that CAMKII-mediated Dp71d phosphorylation modulates its nuclear localization.

The Fire From Within

See related article, pages 1314–1322 The multifunctional Ca2+ and calmodulin (CaM)-dependent protein kinase II (CaMKII) is a serine threonine kinase that is abundant in heart where it phosphorylates Ca2+i homeostatic proteins. It seems likely that CaMKII plays an important role in cardiac physiology because these target proteins significantly overlap with the more extensively studied serine threonine kinase, protein kinase A (PKA), which is a key arbiter of catecholamine responses in heart. However, the physiological functions of CaMKII remain poorly understood, whereas the potential role of CaMKII in signaling myocardial dysfunction and arrhythmias has become an area of intense focus. CaMKII activity and expression are upregulated in failing human hearts and in many animal models of structural heart disease.1 CaMKII inhibitory drugs can prevent cardiac arrhythmias2,3 and suppress afterdepolarizations4 that are a probable proximate focal cause of arrhythmias in heart failure. CaMKII inhibition in mice reduces left ventricular dilation and prevents disordered intracellular Ca2+ (Ca2+i) homeostasis after myocardial infarction.5 CaMKII overexpression in mouse heart causes severe cardiac hypertrophy, dysfunction, and sudden death that is heralded by increased SR Ca2+ leak6; these findings go a long way to making a case for CaMKII as a causative signal in heart disease and arrhythmias but do not identify critical molecular targets or test the potential role of CaMKII in a large non-rodent animal model. The work by Ai et al in this issue of Circulation Research makes an important contribution by demonstrating CaMKII upregulation causes increased Ca2+ leak from ryanodine receptor (RyR) Ca2+ release channels in a clinically-relevant model of structural heart disease.7 Ca2+i release controls cardiac contraction, and most of the Ca2+i for contraction is released from the intracellular sarcoplasmic reticulum (SR) …