Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained

Nuclear Dbf2-related protein kinases (NDRs) in isolated cardiac myocytes and the myocardium: activation by cellular stresses and by phosphoprotein serine-/threonine-phosphatase inhibitors

The nuclear Dbf2-related protein kinases 1 and 2 (NDR1/2) are closely-related AGC family kinases that are strongly conserved through evolution. In mammals, they are activated inter alia by phosphorylation of an hydrophobic domain threonine-residue [NDR1(Thr-444)/NDR2(Thr-442)] by an extrinsic protein kinase followed by autophosphorylation of a catalytic domain serine-residue [NDR1(Ser-281)/NDR2(Ser-282)]. We examined NDR1/2 expression and regulation in primary cultures of neonatal rat cardiac myocytes and in perfused adult rat hearts. In myocytes, transcripts for NDR2, but not NDR1, were induced by the hypertrophic agonist, endothelin-1. NDR1(Thr-444) and NDR2(Thr-442) were rapidly phosphorylated (maximal in 15-30 min) in myocytes exposed to some phosphoprotein Ser-/Thr-phosphatase 1/2 inhibitors (calyculin A, okadaic acid) and, to a lesser extent, by hyperosmotic shock, low concentrations of H(2)O(2), or chelerythrine. In myocytes adenovirally-transduced to express FLAG-NDR2 (which exhibited a mainly-cytoplasmic localisation), the same agents increased FLAG-NDR2 activity as assessed by in vitro protein kinase assays, indicative of FLAG-NDR2(Ser-282/Thr-442) phosphorylation. Calyculin A-induced phosphorylation of NDR1(Thr-444)/NDR2(Thr-442) and activation of FLAG-NDR2 were inhibited by staurosporine, but not by other protein kinase inhibitors tested. In ex vivo rat hearts, NDR1(Thr-444)/NDR2(Thr-442) were phosphorylated in response to ischaemia-reperfusion or calyculin A. From a pathological viewpoint, we conclude that activities of NDR1 and NDR2 are responsive to cytotoxic stresses in heart preparations and this may represent a previously-unidentified response to myocardial ischaemia in vivo

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

Background: Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results: Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion: The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response

Theorising the ‘African problem’ with governmentality and counter conduct

The ‘African Question’ - why Africa remains trapped in poverty despite its vast natural resources - has been a subject of enduring scholarship of critical accounting. Therein, dynamics of neopatrimonialism, crony capitalism, patronage politics and articulation of two publics have been used as theoretical bases to explore the multitude of accounting and accountability issues through which the African problem has manifested. The dominating role of the World Bank and alike has also been discussed in exploring the ways in which this problem has been attempted through neoliberal economic policies that favour market-oriented solutions and various technomanagerial packages of accounting and good governance. We approach this question from theoretical angles of governmentality and counter-conduct. We argue that the accounting implications of the African problem can be better theorised by contextualising, historicising, and particularising them within the theoretical parameters of governmentality and counter conduct

On the possibility of a relativistic correction to the E and B fields around a current-carrying wire

It is well known that electric and magnetic fields may change when they are observed from different frames of reference. For example, the motion of a charged probe particle moving parallel to a current-carrying wire would be described by utilizing different electric or magnetic fields, depending on from which frame of reference the system is observed and described. To describe the situation in all frames by utilizing the theory of relativity, one has to first describe the situation in one particular frame, and this choice in the case of a current-carrying wire is the topic of this paper. In this study I examine an alternative to the standard choice, and consider its theoretical and experimental validity. An outcome of the alternative approach is that in the rest frame of a wire, running a current introduces also an electric field by giving rise to a minute charge. Present day experimental sensitivities, specifically those of cold ions, may be able to differentiate between the observable signatures predicted by the different approaches

The priming effect of extracellular UTP on human neutrophils: Role of calcium released from thapsigargin-sensitive intracellular stores

P2Y2 receptors, which are equally responsive to ATP and UTP, can trigger intracellular signaling events, such as intracellular calcium mobilization and mitogen-activated protein (MAP) kinase phosphorylation in polymorphonuclear leukocytes (PMN). Moreover, extracellular nucleotides have been shown to prime chemoattractant-induced superoxide production. The aim of our study was to investigate the mechanism responsible for the priming effect of extracellular nucleotides on reactive oxygen species (ROS) production induced in human neutrophils by two different chemoattractants: formyl-methionyl-leucyl-phenylalanine (fMLP) and interleukin-8 (IL-8). Nucleotide-induced priming of ROS production was concentration- and time-dependent. When UTP was added to neutrophil suspensions prior to chemoattractant, the increase of the response reached the maximum at 1 min of pre-incubation with the nucleotide. UTP potentiated the phosphorylation of p44/42 and p38 MAP kinases induced by chemoattractants, however the P2 receptor-mediated potentiation of ROS production was still detectable in the presence of a SB203580 or U0126, supporting the view that MAP kinases do not play a major role in regulating the nucleotide-induced effect. In the presence of thapsigargin, an inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca2+-ATPases in mammalian cells, the effect of fMLP was not affected, but UTP-induced priming was abolished, suggesting that the release of calcium from thapsigargin-sensitive intracellular stores is essential for nucleotide-induced priming in human neutrophils

Quantum bits with Josephson junctions

Already in the first edition of this book (Barone and Paterno, "Fundamentals and Physics and Applications of the Josephson Effect", Wiley 1982), a great number of interesting and important applications for Josephson junctions were discussed. In the decades that have passed since then, several new applications have emerged. This chapter treats one such new class of applications: quantum optics and quantum information processing (QIP) based on superconducting circuits with Josephson junctions. In this chapter, we aim to explain the basics of superconducting quantum circuits with Josephson junctions and demonstrate how these systems open up new prospects, both for QIP and for the study of quantum optics and atomic physics.Comment: 30 pages, 10 figures. Book chapter for a new edition of Barone and Paterno's "Fundamentals and Physics and Applications of the Josephson Effect". Final versio

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the “RAF paradox”) may have the same effect. BRAF was upregulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a “RAF paradox” effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the “RAF paradox”. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function

A dose- rather than delivery profile-dependent mechanism regulates the "muscle-full" effect in response to oral essential amino acid intake in young men

Background: The anabolic response of skeletal muscle to essential amino acids (EAAs) is dose dependent, maximal at modest doses, and short lived, even with continued EAA availability, a phenomenon termed “muscle-full.” However, the effect of EAA ingestion profile on muscle metabolism remains undefined.Objective: We determined the effect of Bolus vs. Spread EAA feeding in young men and hypothesized that muscle-full is regulated by a dose-, not delivery profile–, dependent mechanism.Methods: We provided 16 young healthy men with 15 g mixed-EAA, either as a single dose (“Bolus”; n = 8) or in 4 fractions at 45-min intervals (“Spread”; n = 8). Plasma insulin and EAA concentrations were assayed by ELISA and ion-exchange chromatography, respectively. Limb blood flow by was determined by Doppler ultrasound, muscle microvascular flow by Sonovue (Bracco) contrast-enhanced ultrasound, and phosphorylation of mammalian target of rapamycin complex 1 substrates by immunoblotting. Intermittent muscle biopsies were taken to quantify myofibrillar-bound 13C6-phenylalanine to determine muscle protein synthesis (MPS).Results: Bolus feeding achieved rapid insulinemia (13.6 μIU · mL−1, 25 min after commencement of feeding), aminoacidemia (∼2500 μM at 45 min), and capillary recruitment (+45% at 45 min), whereas Spread feeding achieved attenuated insulin responses, gradual low-amplitude aminoacidemia (peak: ∼1500 μM at 135 min), and no detectable capillary recruitment (all P < 0.01 vs. Bolus). Despite these differences, identical anabolic responses were observed; fasting fractional synthetic rates of 0.054% · h−1 (Bolus) and 0.066% · h−1 (Spread) increased to 0.095% and 0.104% · h−1 (no difference in increment or final values between regimens). With both Spread and Bolus feeding strategies, a latency of at least 90 min was observed before an upswing in MPS was evident. Similarly with both feeding strategies, MPS returned to fasting rates by 180 min despite elevated circulating EAAs.Conclusion: These data do not support EAA delivery profile as an important determinant of anabolism in young men at rest, nor rapid aminoacidemia/leucinemia as being a key factor in maximizing MPS. This trial was registered at clinicaltrials.gov as NCT01735539