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Activation of Ca(2+)/calmodulin kinase II (CaMKII) and the N-Methyl D-aspartate receptor (NMDAR), particularly its GluN2B subunit, contribute to the central sensitization of nociceptive pathways and persistent pain. Using mutant mice wherein the activity-driven binding of CaMKII to S1303 in GluN2B is abrogated (GluN2BKI), this study investigated the importance of this interaction for acute and persistent inflammatory nociception. GluN2BKI, wild type and heterozygote mice did not differ in responses to acute noxious heat stimuli as measured with tail flick, paw flick, or hot plate assays, nor did they differ in their responses to mechanical stimulation with von Frey filaments. Surprisingly, the three genotypes exhibited similar spontaneous pain behaviors and hypersensitivity to heat or mechanical stimuli induced by intraplantar injection of capsaicin; however, GluN2BKI mice did not immediately attend to the paw. WT and GluN2BKI mice also did not differ in the nociceptive behaviors elicited by intraplantar injection of formalin, even though MK801 greatly reduced these behaviors in both genotypes concordant with NMDAR dependence. CaMKII binding to GluN2B at S1303 therefore does not appear to be critical for the development of inflammatory nociception. Finally, intrathecal KN93 reduced formalin-induced nociceptive behaviors in GluN2BKI mice. KN93 does not inhibit CaKMII, but rather binds Ca(2+)/calmodulin. It has multiple other targets including Ca(2+)-, Na(+)- and K(+)-channels, as well as various kinases. Therefore, the use of GluN2BKI mice provided genetic specificity in assessing the role of CaMKII in inflammatory pain signaling cascades. These results challenge current thinking on the involvement of the CaMKII-NMDAR interaction in inflammatory pain.

The multifunctional Ca 2+ /calmodulin-dependent kinase II (CaMKII) is activated by vasoconstrictors in vascular smooth muscle cells (VSMC), but its impact on vasoconstriction remains unknown. We hypothesized that CaMKII inhibition in VSMC decreases vasoconstriction. Using novel transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), we investigated the effect of CaMKII inhibition on L-type Ca 2+ channel current ( I Ca ), cytoplasmic and sarcoplasmic reticulum Ca 2+ , and vasoconstriction in mesenteric arteries. In mesenteric VSMC, CaMKII inhibition significantly reduced action potential duration and the residual I Ca 50 ms after peak amplitude, indicative of loss of L-type Ca 2+ channel–dependent I Ca facilitation. Treatment with angiotensin II or phenylephrine increased the intracellular Ca 2+ concentration in wild-type but not TG SM-CaMKIIN VSMC. The difference in intracellular Ca 2+ concentration was abolished by pretreatment with nifedipine, an L-type Ca 2+ channel antagonist. In TG SM-CaMKIIN VSMC, the total sarcoplasmic reticulum Ca 2+ content was reduced as a result of diminished sarcoplasmic reticulum Ca 2+ ATPase activity via impaired derepression of the sarcoplasmic reticulum Ca 2+ ATPase inhibitor phospholamban. Despite the differences in intracellular Ca 2+ concentration, CaMKII inhibition did not alter myogenic tone or vasoconstriction of mesenteric arteries in response to KCl, angiotensin II, and phenylephrine. However, it increased myosin light chain kinase activity. These data suggest that CaMKII activity maintains intracellular calcium homeostasis but is not required for vasoconstriction of mesenteric arteries.

An excess of calcium (Ca2+) influx into mitochondria during mitochondrial re-energization is one of the causes of myocardial cell death during ischemic/reperfusion injury. This overload of Ca2+ triggers the mitochondrial permeability transition pore (mPTP) opening which leads to programmed cell death. During the ischemic/reperfusion stage, the activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) enzyme is responsible for Ca2+ influx. To reduce CaMKII-related cell death, sub-micron particles composed of poly(lactic-co-glycolic acid) (PLGA), loaded with a CaMKII inhibitor peptide were fabricated. The CaMKII inhibitor peptide-loaded (CIP) particles were coated with a mitochondria targeting moiety, triphenylphosphonium cation (TPP), which allowed the particles to accumulate and release the peptide inside mitochondria to inhibit CaMKII activity. The fluorescently labeled TPP-CIP was taken up by mitochondria and successfully reduced reactive oxygen species (ROS) caused by Isoprenaline (ISO) in a differentiated rat cardiomyocyte-like cell line. When cells were treated with TPP-CIP prior to ISO exposure, they maintained mitochondrial membrane potential. The TPP-CIP protected cells from ISO-induced ROS production and decreased mitochondrial membrane potential. Thus, TPP-CIP has the potential to be used in protection against ischemia/reperfusion injury.

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

Heart rate increases are a fundamental adaptation to physiological stress, while inappropriate heart rate increases are resistant to current therapies. However, the metabolic mechanisms driving heart rate acceleration in cardiac pacemaker cells remain incompletely understood. The mitochondrial calcium uniporter (MCU) facilitates calcium entry into the mitochondrial matrix to stimulate metabolism. We developed mice with myocardial MCU inhibition by transgenic expression of a dominant-negative (DN) MCU. Here, we show that DN-MCU mice had normal resting heart rates but were incapable of physiological fight or flight heart rate acceleration. We found that MCU function was essential for rapidly increasing mitochondrial calcium in pacemaker cells and that MCU-enhanced oxidative phoshorylation was required to accelerate reloading of an intracellular calcium compartment before each heartbeat. Our findings show that MCU is necessary for complete physiological heart rate acceleration and suggest that MCU inhibition could reduce inappropriate heart rate increases without affecting resting heart rate.

Diabetes, a worldwide epidemic, represents a major public health problem and the vast majority of the patients with diabetes presents with insulin resistance, a fundamental manifestation of this disease. Insulin resistance impairs glucose uptake into skeletal muscle, which takes up about 80% of postprandial glucose in healthy individuals (1). Thus, skeletal muscle plays an indispensable role in maintaining glucose homeostasis. However, the mechanisms underlying the development of insulin resistance remains poorly understood, which perhaps accounts for the lack of effective therapies. Mitochondria, where low levels of superoxide radicals are constitutively generated as a by-product of electron transport, serve as the powerhouse and are also considered a main source for overproduction of reactive oxygen species (ROS) triggered by diabetes (2,3). Oxidative stress is a key pathological signal leading to diabetes complications (4,5). It has been disappointing that broad-spectrum antioxidant therapies have not been effective in improving outcomes in high-risk patients, including patients with diabetes (6), when used as primary prevention, suggesting that detailed knowledge of oxidative injury mechanisms is necessary to develop …

CaMKII is a newly discovered resident of mitochondria in the heart. Mitochondrial CaMKII promotes poor outcomes after heart injury from a number of pathological conditions, including myocardial infarction (MI), ischemia reperfusion (IR), and stress from catecholamine stimulation. A study using the inhibitor of CaMKII, CaMKIIN, with expression delimited to myocardial mitochondria, indicates that an underlying cause of heart disease results from the opening of the mitochondrial permeability transition pore (mPTP). Evidence from electrophysiological and other experiments show that CaMKII inhibition likely suppresses mPTP opening by reducing Ca(2+) entry into mitochondria. However, we expect other proteins involved in Ca(2+) signaling in the mitochondria are affected with CaMKII inhibition. Several outstanding questions remain for CaMKII signaling in heart mitochondria. Most importantly, how does CaMKII, without the recognized N-terminal mitochondrial targeting sequence transfer to mitochondria?

Mutants of Drosophila melanogaster that lack Cu/Zn superoxide dismutase or urate are hypersensitive to reactive oxygen species (ROS) generated in vivo by the redox-cycling agent paraquat. We have subsequently employed paraquat as a selective agent to identify adult viable mutants potentially defective in other, perhaps unknown, components of ROS metabolism. Paraquat screening of ethyl methanesulfonate-induced second- and third-chromosome mutations yielded 24 paraquat hypersensitive mutants. Two mutants were identified as being new alleles of the previously identified doublesex (dsx) and pink (p) genes. The remainder of the mutations identified previously undescribed genes, including one second chromosome paraquat hypersensitive mutant that was found to exhibit shaking legs, abdomen pulsations, and body shuddering under ether anaesthesia. This recessive mutation was mapped to the polytene chromosome region of 48A5–48B2 and defines a new gene we named quiver (qvr). This mutation is similar in phenotype to the Shaker (Sh), ether-a-gogo (eag), and Hyperkinetic (Hk) mutations, all of which affect potassium channel function in D. melanogaster. Key words : Drosophila, paraquat, EMS-mutagenesis, Shaker, oxidative-stress.

Excessive activation of the beta-adrenergic, angiotensin II (Ang II) and aldosterone signaling pathways promotes mortality after myocardial infarction, and antagonists targeting these pathways are core therapies for treating this condition. Catecholamines and Ang II activate the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), the inhibition of which prevents isoproterenol-mediated and Ang II-mediated cardiomyopathy. Here we show that aldosterone exerts direct toxic actions on myocardium by oxidative activation of CaMKII, causing cardiac rupture and increased mortality in mice after myocardial infarction. Aldosterone induces CaMKII oxidation by recruiting NADPH oxidase, and this oxidized and activated CaMKII promotes matrix metalloproteinase 9 (MMP9) expression in cardiomyocytes. Myocardial CaMKII inhibition, overexpression of methionine sulfoxide reductase A (an enzyme that reduces oxidized CaMKII) or NADPH oxidase deficiency prevented aldosterone-enhanced cardiac rupture after myocardial infarction. These findings show that oxidized myocardial CaMKII mediates the cardiotoxic effects of aldosterone on the cardiac matrix and establish CaMKII as a nodal signal for the neurohumoral pathways associated with poor outcomes after myocardial infarction.

Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the beta(2)-adrenergic receptor (beta(2)AR), the trimeric G(s) protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA-type glutamate receptor subunit GluR1, which is linked to the beta(2)AR through stargazin and PSD-95 and their homologues. Only GluR1 associated with the beta(2)AR is phosphorylated by PKA on beta(2)AR stimulation. Peptides that interfere with the beta(2)AR-GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic beta(2)AR-cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins.

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a 'voltage clock' and a Ca(2+) dependent process, or 'Ca(2+) clock.' The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (I(f)) is thought to be particularly important. A Ca(2+) dependent process triggers APs when sarcoplasmic reticulum (SR) Ca(2+) release activates inward current carried by the forward mode of the electrogenic Na(+)/Ca(2+) exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca(2+) clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective I(f) antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest ( approximately 14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca(2+) release, but did not affect basal or isoproterenol-enhanced I(f). Taken together, these results indicate that voltage and Ca(2+) dependent automaticity mechanisms coexist in canine SAN cells, and suggest that I(f) and SR Ca(2+) release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.

SummaryCalcium/calmodulin (Ca2+/CaM)-dependent protein kinase II (CaMKII) couples increases in cellular Ca2+ to fundamental responses in excitable cells. CaMKII was identified over 20 years ago by activation dependence on Ca2+/CaM, but recent evidence shows that CaMKII activity is also enhanced by pro-oxidant conditions. Here we show that oxidation of paired regulatory domain methionine residues sustains CaMKII activity in the absence of Ca2+/CaM. CaMKII is activated by angiotensin II (AngII)-induced oxidation, leading to apoptosis in cardiomyocytes both in vitro and in vivo. CaMKII oxidation is reversed by methionine sulfoxide reductase A (MsrA), and MsrA−/− mice show exaggerated CaMKII oxidation and myocardial apoptosis, impaired cardiac function, and increased mortality after myocardial infarction. Our data demonstrate a dynamic mechanism for CaMKII activation by oxidation and highlight the critical importance of oxidation-dependent CaMKII activation to AngII and ischemic myocardial apoptosis.

Ca2+–Calmodulin (CaM) kinase II (CaMKII) activation depends predominantly on modifications of amino acids within the regulatory domain on this multidomain kinase. A number of mechanisms of activation have been described since the original mode of activation, via Ca2+–CaM binding and phosphorylation of a threonine residue in the regulatory domain of CaMKII, was demonstrated. Gutierrez et al .1 propose a mechanism of CaMKII activation by nitrosation. CaMKII serves as a link between β-adrenergic stimuli with subsequent changes in Ca2+ levels to the cellular response in cardiomyocytes. CaMKII phosphorylates dozens of substrates in many cell types. When activated by binding of Ca2+–CaM, CaMKII subunits can autophosphorylate neighbouring subunits at Threonine-287 (Thr287) within the CaMKII holoenzyme.2 Autophosphorylation at Thr287 results in autonomous kinase activity, converting to an activation state independent of Ca2+–CaM binding. A number of studies have shown that CaMKII can achieve sustained activity even in the absence of phosphorylation at Thr287. Mechanisms to attain CaMKII sustained include substrate binding,3–6 oxidation of the paired methionine residues (281/282) proximal to the Thr287,7 and now nitrosation of cysteine residues.1 Modifications that activate CaMKII occur primarily in the regulatory domain ( Figure 1 ). These events prevent the catalytic domain from binding to the regulatory …

Cumulative oxidative damages to cell constituents are considered to contribute to aging and age-related diseases. The enzyme peptide methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized methionine in proteins by reducing methionine sulfoxide back to methionine. However, whether MSRA plays a role in the aging process is poorly understood. Here we report that overexpression of the msrA gene predominantly in the nervous system markedly extends the lifespan of the fruit fly Drosophila . The MSRA transgenic animals are more resistant to paraquat-induced oxidative stress, and the onset of senescence-induced decline in the general activity level and reproductive capacity is delayed markedly. The results suggest that oxidative damage is an important determinant of lifespan, and MSRA may be important in increasing the lifespan in other organisms including humans.