ATP-sensitive K + (K ATP ) channels are present in the sarcolemma of cardiac myocytes where they link membrane excitability with the cellular bioenergetic state. These channels are in vivo composed of Kir6.2, a pore-forming subunit, SUR2A, a regulatory subunit, and at least four accessory proteins. In the present study, real-time RT-PCR has demonstrated that of all six sarcolemmal K ATP channelforming proteins, SUR2A was probably the least expressed protein. We have generated mice where the SUR2A was under the control of a cytomegalovirus promoter, a promoter that is more efficient than the native promoter. These mice had an increase in SUR2A mRNA/protein levels in the heart whereas levels of mRNAs of other channel-forming proteins were not affected at all. Imunoprecipitation/Western blot and patch clamp electrophysiology has shown an increase in K ATP channel numbers in the sarcolemma of transgenic mice. Cardiomyocytes from transgenic mice responded to hypoxia with shortening of action membrane potential and were significantly more resistant to this insult than cardiomyocytes from the wild-type. The size of myocardial infarction in response to ischemia-reperfusion was much smaller in hearts from transgenic mice compared to those in wild-type. We conclude that overexpression of SUR2A generates cardiac phenotype resistant to hypoxia/ischemia/reperfusion injury due at least in part to increase in levels of sarcolemmal K ATP channels. KeywordsSUR2A; K ATP channel; cardioprotection; hypoxia; heart Throughout a lifetime, the heart is subjected to a variety of metabolic stresses, including ischemia/hypoxia. Work carried out over the last 15 years has demonstrated that powerful cardioprotective signaling pathways exist in cardiomyocytes that, when activated, enable cardiac cells to function under adverse conditions (1,2).Sarcolemmal ATP-sensitive K + (K ATP ) channels, ion channels that couple metabolic status of the cell with membrane excitability (3), have been implicated in cardioprotective signaling (4,5). These channels are selectively permeable to K + ions and are closed by high intracellular ATP levels (3). It has been suggested that the opening of K ATP channels protects against myocardial infarction, mediates ischemic preconditioning (a phenomenon when brief periods of ischemia/reperfusion protect the heart against sustained ischemia, 2), and promotes survival of cardiomyocytes exposed to different kinds of metabolic stresses (4,5). Conditions associated 1Correspondence: Maternal and Child Health Sciences, Ninewells Hospital & Medical School, University of Dundee, Dundee, DD1 9SY Scotland, UK. E-mail: a.jovanović@dundee.ac.uk. UKPMC Funders GroupAuthor Manuscript UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscriptwith an increase in expression of sarcolemmal K ATP channels, such as estrogen treatment and chronic hypoxia, are associated with increased cardiac resistance to stress (6-8). Also, ischemic preconditioning seems to be mediated by recruitment and an increase in number ...

Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that is complex and yet to be fully defined. AMP-activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the a2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC-1, a mitochondrial membrane potential-sensitive dye, and laser confocal microscopy. In contrast, experiments with di-8-ANEPPS, a sarcolemmal-potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning-induced shortening of the action membrane potential. The preconditioning-induced activation of sarcolemmal K ATP channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal K ATP channels opening, inhibited preconditioning-induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioninginduced recruitment of sarcolemmal K ATP channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal K ATP channels without being a part of signaling pathway that regulates mitochondrial membrane potential.

Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that are yet to be fully defined. 3′-phosphoinositide-dependent kinase-1 (PDK1) has been implicated in numerous cellular processes. However, the involvement of PDK1 in preconditioning has yet to be elucidated. Studying PDK1 is not as straightforward as it is for the majority of kinases, due to the lack of a specific inhibitor of PDK1. Therefore, we have taken advantage of PDK1 hypomorphic mutant mice with reduced expression of PDK1 to study the role of PDK1 in preconditioning. Whole heart and single cell models of preconditioning demonstrated that the hearts and cardiac cells from PDK1 hypomorphic mice could not be preconditioned. The cardioprotective effect of PDK1 was not related to the effect that preconditioning has on sarcolemmal membrane action potential as revealed by di-8-ANEPPS, a sarcolemmal-potential sensitive dye, and laser confocal microscopy. In contrast, experiments with JC-1, a mitochondrial membrane potentialsensitive dye, has demonstrated that intact PDK1 levels were required for preconditioning-mediated regulation of mitochondrial membrane potential. Western blotting combined with functional experiments have shown that intact PDK1 levels were required for preconditioning-induced phosphorylation of protein kinase B (PKB), glycogen synthase kinase-3β (GSK-3β), and cardioprotection. We conclude that PDK1 mediates preconditioning in the heart by regulating activating PKB-GSK-3β to regulate mitochondrial but not sarcolemmal membrane potential. 3′ phosphoinositide-dependent kinase-1 (PDK1) is essential for ischemic preconditioning of the myocardium. Keywords GSK-3; hypoxia; ischemia; mitochondria; cardioprotectionThe recently discovered 3′-phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine protein kinase that phosphorylates several members of the automatic gain control (AGC) family of protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum-and glucocorticoid-induced protein kinase (SGK), and protein kinase C (PKC) (1). So far, it has been shown that the PDK1/automatic gain control kinase-signaling pathway regulates diverse cellular processes, including those relevant to cell survival, proliferation, and metabolic responses to insulin (2). Inadequate regulation of AGC kinase members could contribute to many diseases. A deficiency in the activation of AGC kinases may be a primary cause of the insulin-resistant form of diabetes (3), neuronal cell death following a stroke (4), 1Correspondence: Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK. E-mail: a.jovanovic@dundee.ac.uk. UKPMC Funders Group UKPMC Funders Group Author Manuscript UKPMC Funders Group Author Manuscriptand heart failure (5). However, hyperactivation of this pathway has been implicated ...

Type I restriction-modification (R-M) systems encode multisubunit/multidomain enzymes. Two genes (M and S) are required to form the methyltransferase (MTase) that methylates a specific base within the recognition sequence and protects DNA from cleavage by the endonuclease. The DNA methyltransferase M.AhdI is a 170 kDa tetramer with the stoichiometry M2S2 and has properties typical of a type I MTase. The M.AhdI enzyme has been prepared with deuterated S subunits, to allow contrast variation using small-angle neutron scattering (SANS) methods. The SANS data were collected in a number of 1H:2H solvent contrasts to allow matching of one or other of the subunits in the multisubunit enzyme. The radius of gyration (Rg) and maximum dimensions (Dmax) of the M subunits in situ in the multisubunit enzyme (50 Å and 190 Å, respectively) are close of those of the entire MTase (51 Å and 190 Å). In contrast, the S subunits in situ have experimentally determined values of Rg = 35 Å and Dmax = 110 Å, indicating their more central location in the enzyme. Ab initio reconstruction methods yield a low-resolution structural model of the shape and subunit organization of M.AhdI, in which the Z-shaped structure of the S subunit dimer can be discerned. In contrast, the M subunits form a much more elongated and extended structure. The core of the MTase comprises the two S subunits and the globular regions of the two M subunits, with the extended portion of the M subunits most probably forming highly mobile regions at the outer extremities, which collapse around the DNA when the MTase binds.

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.

Histone 2A increases glucose-6-phosphatase activity in liver microsomes. The effect has been attributed either to the conformational change of the enzyme, or to the permeabilization of microsomal membrane that allows the free access of substrate to the intraluminal glucose-6-phosphatase catalytic site. The aim of the present study was the critical reinvestigation of the mechanism of action of histone 2A. It has been found that the dose-effect curve of histone 2A is different from that of detergents and resembles that of the pore-forming alamethicin. Inhibitory effects of EGTA on glucose-6-phosphatase activity previously reported in histone 2A-treated microsomes have been also found in alamethicin-permeabilized vesicles. The effect of EGTA cannot therefore simply be an antagonization of the effect of histone 2A. Histone 2A stimulates the activity of another latent microsomal enzyme, UDP-glucuronosyltransferase, which has an intraluminal catalytic site. Finally, histone 2A renders microsomal vesicles permeable to non-permeant compounds. Taken together, the results demonstrate that histone 2A stimulates glucose-6-phosphatase activity by permeabilizing the microsomal membrane.

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