scholarly journals Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition

2015 ◽  
Vol 309 (4) ◽  
pp. C239-C250 ◽  
Author(s):  
Karin K. M. Chia ◽  
Chia-Chi Liu ◽  
Elisha J. Hamilton ◽  
Alvaro Garcia ◽  
Natasha A. Fry ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Pump Current ◽  
Reciprocal Relationship ◽  
Ang Ii ◽  
Α Subunit

Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na+-K+ pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na+-K+ pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na+-K+ pump current ( Ip). Coexposure to 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na+-K+ pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47 phox NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22 phox subunit, and it decreased O2·−-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47 phox indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22 phox and p47 phox NADPH oxidase subunits and decrease β1-Na+-K+ pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo.

scholarly journals β3-Adrenoceptor activation relieves oxidative inhibition of the cardiac Na+-K+ pump in hyperglycemia induced by insulin receptor blockade

2015 ◽  
Vol 309 (5) ◽  
pp. C286-C295 ◽  
Author(s):  
Keyvan Karimi Galougahi ◽  
Chia-Chi Liu ◽  
Alvaro Garcia ◽  
Natasha A. Fry ◽  
Elisha J. Hamilton ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nadph Oxidase ◽  
Insulin Receptor ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Ex Vivo ◽  
Pump Current ◽  
Cardiovascular Complications ◽  
Oxidative Modification

Dysregulated nitric oxide (NO)- and superoxide (O2·−)-dependent signaling contributes to the pathobiology of diabetes-induced cardiovascular complications. We examined if stimulation of β3-adrenergic receptors (β3-ARs), coupled to endothelial NO synthase (eNOS) activation, relieves oxidative inhibition of eNOS and the Na+-K+ pump induced by hyperglycemia. Hyperglycemia was established in male New Zealand White rabbits by infusion of the insulin receptor antagonist S961 for 7 days. Hyperglycemia increased tissue and blood indexes of oxidative stress. It induced glutathionylation of the Na+-K+ pump β1-subunit in cardiac myocytes, an oxidative modification causing pump inhibition, and reduced the electrogenic pump current in voltage-clamped myocytes. Hyperglycemia also increased glutathionylation of eNOS, which causes its uncoupling, and increased coimmunoprecipitation of cytosolic p47 phox and membranous p22 phox NADPH oxidase subunits, consistent with NADPH oxidase activation. Blocking translocation of p47 phox to p22 phox with the gp91ds-tat peptide in cardiac myocytes ex vivo abolished the hyperglycemia-induced increase in glutathionylation of the Na+-K+ pump β1-subunit and decrease in pump current. In vivo treatment with the β3-AR agonist CL316243 for 3 days eliminated the increase in indexes of oxidative stress, decreased coimmunoprecipitation of p22 phox with p47 phox, abolished the hyperglycemia-induced increase in glutathionylation of eNOS and the Na+-K+ pump β1-subunit, and abolished the decrease in pump current. CL316243 also increased coimmunoprecipitation of glutaredoxin-1 with the Na+-K+ pump β1-subunit, which may reflect facilitation of deglutathionylation. In vivo β3-AR activation relieves oxidative inhibition of key cardiac myocyte proteins in hyperglycemia and may be effective in targeting the deleterious cardiac effects of diabetes.

Abstract 154: CCDC80 Functions as a Protein Kinase GI Substrate and is Secreted by Cardiac Myocytes

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Robert M Blanton ◽  
Craig Cooper ◽  
Anja Hergruetter ◽  
Mark Aronovitz ◽  
Timothy D Calamaras
Keyword(s):  
Protein Kinase ◽  
Western Blot ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Pressure Overload ◽  
Future Studies ◽  
Transaortic Constriction ◽  
Human Left Ventricle

Background: Protein kinase G I alpha (PKGIa) inhibits cardiac hypertrophy, remodeling, and dysfunction. Downstream PKGI substrates remain incompletely understood and represent potential novel therapeutic targets for myocardial disease. We previously identified through a molecular screen that PKGIa binds and phosphorylates the protein coiled-coiled domain containing 80 (Ccdc80; also termed SSG1 and URB) in vascular smooth muscle cells. Previous work also identified that Ccdc80 is secreted from adipocytes. However, the expression and secretion of Ccdc80 from the cardiac myocyte has not been investigated. The current study tested the hypothesis that Ccdc80 is expressed in and secreted from the cardiac myocyte. Results: In cultured rat cardiac myocytes (CM), we detected Ccdc80 by western blot. Western blot for Ccdc80 also detected a band of the predicted Ccdc80 molecular weight present in media from these cells, but not in uncultured media. Ccdc80 could be detected in the human left ventricle (LV), though expression did not differ between hearts of normal controls and patients with hypertrophic cardiomyopathy. In the setting of LV pressure overload induced by transaortic constriction (TAC), we observed an increase in Ccdc80 expression in 1 week TAC LVs, compared with sham LVs (5.0 +/- 0.3 arbitrary densitometric units in sham versus 9.6 +/- 0.9 in TAC; n=4 per group). Conclusion: Taken together, our findings identify that the PKGIa substrate Ccdc80 expresses in cardiac myocytes, becomes secreted from CMs, resides in the human heart, and increases in expression in the mouse LV in response to pressure overload. Given the anti-remodeling role of PKGIa, these findings support future studies to understand the in vivo role of Ccdc80 in the cardiovascular system. Future studies will also explore the significance of Ccdc80 secretion from the CM and its potential regulation by PKG.

Angiotensin II inhibits the Na+-K+ pump via PKC-dependent activation of NADPH oxidase

2009 ◽  
Vol 296 (4) ◽  
pp. C693-C700 ◽  
Author(s):  
Caroline N. White ◽  
Gemma A. Figtree ◽  
Chia-Chi Liu ◽  
Alvaro Garcia ◽  
Elisha J. Hamilton ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Cardiac Myocyte ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Oxidase Inhibitor ◽  
Ang Ii ◽  
Inhibitory Peptide ◽  
Myocyte Function ◽  
Nadph Oxidase Activation

The sarcolemmal Na+-K+ pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (ANG II). Since ANG II activates NADPH oxidase, we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/l ANG II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD), by the NADPH oxidase inhibitor apocynin, and by myristolated inhibitory peptide to ε-protein kinase C (εPKC), previously implicated in ANG II-induced Na+-K+ pump inhibition. A role for εPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of εPKC with the receptor for the activated kinase and with the cytosolic p47 phox subunit of NADPH oxidase. ANG II decreased electrogenic Na+-K+ pump current in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase, and by εPKC inhibitory peptide. Since colocalization should facilitate NADPH oxidase-dependent regulation of the Na+-K+ pump, we examined whether there is physical association between the pump subunits and NADPH oxidase. The α1-subunit coimmunoprecipitated with caveolin 3 and with membrane-associated p22 phox and cytosolic p47 phox NADPH oxidase subunits at baseline. ANG II had no effect on α1/caveolin 3 or α1/p22 phox interaction, but it increased α1/p47 phox coimmunoprecipitation. We conclude that ANG II inhibits the Na+-K+ pump via PKC-dependent NADPH oxidase activation.

Protein kinase Cɛ contributes to regulation of the sarcolemmal Na+-K+ pump

2001 ◽  
Vol 281 (3) ◽  
pp. C1059-C1063 ◽  
Author(s):  
Kerrie A. Buhagiar ◽  
Peter S. Hansen ◽  
Nerida L. Bewick ◽  
Helge H. Rasmussen
Keyword(s):  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Enzyme Inhibitor ◽  
Pump Current ◽  
Ang Ii ◽  
Converting Enzyme ◽  
Kinase C

A reduction in angiotensin II (ANG II) in vivo by treatment of rabbits with the angiotensin-converting enzyme inhibitor, captopril, increases Na+-K+ pump current ( I p) of cardiac myocytes. This increase is abolished by exposure of myocytes to ANG II in vitro. Because ANG II induces translocation of the ɛ-isoform of protein kinase C (PKCɛ), we examined whether this isozyme regulates the pump. We treated rabbits with captopril, isolated myocytes, and measured I p of myocytes voltage clamped with wide-tipped patch pipettes. I p of myocytes from captopril-treated rabbits was larger than I p of myocytes from controls. ANG II superfusion of myocytes from captopril-treated rabbits decreased I p to levels similar to controls. Inclusion of PKCɛ-specific blocking peptide in pipette solutions used to perfuse the intracellular compartment abolished the effect of ANG II. Inclusion of ψɛRACK, a PKCɛ-specific activating peptide, in pipette solutions had an effect on I p that was similar to that of ANG II. There was no additive effect of ANG II and ψɛRACK. We conclude that PKCɛ regulates the sarcolemmal Na+-K+ pump.

Abstract 177: Promotion Of Nitroso-redox Balance By Beta 3 Adrenoceptor Agonism: Therapeutic Implications For Cardiovascular Complications Of Diabetes

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Keyvan Karimi Galougahi ◽  
Chia-chi Liu ◽  
Alvaro Garcia ◽  
Natasha A Fry ◽  
Clare L Hawkins ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Cardiac Myocytes ◽  
Vascular Dysfunction ◽  
Pump Current ◽  
Redox Balance ◽  
Cardiovascular Complications ◽  
Oxidative Modification ◽  
Oxidation Products ◽  
Therapeutic Implications ◽  
Endothelial Nitric Oxide

Rationale: Disrupted balance between NO and O2.- is central in pathobiology of diabetes-induced cardiomyopathy and vascular dysfunction. We examined if stimulation of β3 adrenergic receptors (β3 ARs), coupled to endothelial nitric oxide synthase (eNOS) activation, would re-establish NO/O2.- balance, relieve oxidative inhibition of key caveolar proteins and protect against diabetes-induced cardiovascular dysfunction. Methods/Results: A hyperglycemic, hyperinsulinemic state was established in male White New Zealand rabbits by infusion of the insulin receptor antagonist S961 (12 μg/kg/h). Diabetes induced NADPH oxidase-dependent glutathionylation (GSS-) of the caveolar proteins Na+-K+ pump’s β1 subunit and eNOS in cardiac myocytes and aorta, an oxidative modification that inhibits the pump and uncouples eNOS. Consistent with this, diabetes was associated with reduced electrogenic Na+-K+ pump current in voltage-clamped cardiac myocytes and impaired endothelium-dependent vasorelaxation. Selective β3 AR agonist CL316243 (CL, 40 μg/kg/h) restored NO levels analysed by spin-trapping of NO-Fe(DETC)2 complexes; decreased diabetes-induced elevation in O2.- measured by HPLC analysis of dihydroethidium oxidation products, improved endothelium-dependent vasorelaxation, and restored the Na+-K+ pump function in cardiac myocytes. These effects were mediated by CL abolishing diabetes-induced increase in eNOS-GSS and β1-GSS through a decrease in forward reaction rate for glutathionylation by suppressing diabetes-induced NADPH oxidase activation, which was further amplified by promotion of de-glutathionylation via enhancement in association of glutaredoxine-1, the enzyme catalysing de-glutathionylation, with eNOS and Na+-K+ pump. Conclusion: β3 AR activation re-established nitroso-redox balance and relieved oxidative inhibition of key caveolar proteins in diabetes. β3 AR agonists are promising in treatment of diabetes-induced cardiovascular complications.

Abstract 99: Hypotonic Swelling Promotes Nitric Oxide Release in Cardiac Ventricular Myocytes

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Luis Gonano ◽  
Malena Morell ◽  
Juan I Burgos ◽  
Martin Vila Petroff
Keyword(s):  
Nitric Oxide ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Contractile Function ◽  
Cell Swelling ◽  
Contractile Dysfunction ◽  
Ischemia And Reperfusion ◽  
Hypotonic Solution ◽  
No Release ◽  
Hypotonic Swelling

Cardiac myocyte swelling occurs in multiple pathological situations and in particular contributes to the deleterious effects of ischemia and reperfusion by promoting contractile dysfunction. We investigated whether hypotonic swelling promotes nitric oxide (NO) release in cardiac myocytes and if so, whether it impacts on swelling induced contractile dysfunction. Perfusing rat cardiac myocytes, loaded with the NO sensor DAF-FM, with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient amplitude and significantly increased DAF-FM fluorescence. When cells were exposed to the HS supplemented with 2.5 mM of the NO synthase inhibitor L-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the NOS1 inhibitor, Nitroguanidine. In addition, Colchicine (an inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either L-NAME, Nitroguandine or the guanylate cyclase inhibitor, ODQ, suggesting that NOS1-derived NO provides contractile support via a GMP-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and the HS-induced increment in ryanodine receptor (RyR2) phosphorylation at site Ser2808 suggesting that in the context of hypotonic swelling, cGMP may contribute to preserve contractile function by enhancing SR Ca2+ release. Our findings suggest a novel mechanism for NO release in cardiac myocytes with putative pathophysiological relevance in the context of ischemia and reperfusion, where it may be cardioprotective by reducing the extent of contractile dysfunction associated with hypotonic swelling.

scholarly journals Evidence for a role of protein kinase C in hypoxic pulmonary vasoconstriction

1999 ◽  
Vol 276 (1) ◽  
pp. L90-L95 ◽  
Author(s):  
Norbert Weissmann ◽  
Robert Voswinckel ◽  
Thorsten Hardebusch ◽  
Simone Rosseau ◽  
Hossein Ardeschir Ghofrani ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
Superoxide Anion ◽  
Hypoxic Pulmonary Vasoconstriction ◽  
Ang Ii ◽  
Pkc Inhibitor ◽  
Pulmonary Vasoconstriction ◽  
Pressor Responses ◽  
Hypoxic Vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation, thus optimizing gas exchange. NADPH oxidase-related superoxide anion generation has been suggested as part of the signaling response to hypoxia. Because protein kinase (PK) C activation can occur during hypoxia and PKC activation is known to be critical for NADPH oxidase stimulation in different cell types, we probed the role of PKC in hypoxic vasoconstriction in intact rabbit lungs. Control vasoconstrictor responses were elicited by angiotensin II (ANG II) and the stable thromboxane analog U-46619. Portions of the experiments were performed while NO synthesis and prostanoid generation were blocked with N G-monomethyl-l-arginine and acetylsalicylic acid to avoid confounding effects due to interference with these vasoactive mediators. The PKC inhibitor H-7 (10–50 μM) caused dose-dependent inhibition of HPV, but this agent lacked specificity because ANG II- and U-46619-induced vasoconstrictions were correspondingly suppressed. In contrast, low concentrations of the specific PKC inhibitor bisindolylmaleimide I (BIM; 1–15 μM) strongly inhibited the hypoxic vasoconstriction without any interference with the responses to the pharmacological agents. Superimposable dose-inhibition curves were also obtained for BIM when lung NO synthesis and prostanoid generation were blocked throughout the experiments. Under either condition, BIM did not affect normoxic vascular tone. The PKC activator farnesylthiotriazole (FTT), ascertained to stimulate rabbit NADPH oxidase by provocation of alveolar macrophage superoxide anion generation in vitro, caused rapid-onset, transient pressor responses in normoxic lungs. After FTT, the hypoxic vasoconstrictor response was totally suppressed, in contrast to the largely maintained pressor responses to ANG II and U-46619. The lungs became refractory even to delayed hypoxic challenges after FTT application. In conclusion, these data support the concept that activation of PKC is involved in the transduction pathway forwarding pulmonary vasoconstriction in response to alveolar hypoxia.

C-myc protooncogene modulates cardiac hypertrophic growth in transgenic mice

1992 ◽  
Vol 262 (2) ◽  
pp. H590-H597 ◽  
Author(s):  
R. J. Robbins ◽  
J. L. Swain
Keyword(s):  
Transgenic Mice ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Fold Increase ◽  
Cardiac Mass ◽  
Hypertrophic Growth ◽  
Basal Expression ◽  
Pharmacological Stimulation ◽  
Beta Adrenergic Agonist

Protooncogenes such as c-myc have been implicated in the transduction of growth signals in the cardiac myocyte. We examined whether increases in c-myc expression occur in murine heart in vivo as a generalized response to the pharmacological stimulation of myocyte growth. Both triiodothyronine (T3) and the beta-adrenergic agonist isoproterenol were demonstrated to induce a rapid and transient increase in cardiac c-myc mRNA abundance, which preceded an increase in cardiac mass. We then examined whether myocyte growth could be modulated by selectively altering cardiac c-myc expression. The model system used was a strain of transgenic mice exhibiting a 20-fold increase in cardiac c-myc expression. Although in nontransgenic mice the administration of T3 and isoproterenol resulted in similar increases in cardiac mass, in transgenic mice the degree of myocardial growth induced with T3 was significantly greater than that induced with isoproterenol (P less than 0.001). This study demonstrates that increasing the basal expression of c-myc in cardiac myocytes alters the growth response of the heart in vivo to certain hypertrophic stimuli and implicates the c-myc protooncogene in the transduction of selective hypertrophic growth signals in differentiated cardiac myocytes.

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

2005 ◽  
Vol 288 (5) ◽  
pp. H2412-H2421 ◽  
Author(s):  
Markus Frederich ◽  
Li Zhang ◽  
James A. Balschi
Keyword(s):  
Protein Kinase ◽  
Metabolic Inhibitors ◽  
Protein Kinase Activity ◽  
Α Subunit ◽  
Rat Hearts ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

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