Karin K.M. Chia

Reversible Oxidative Modification. A Key Mechanism of Na+-K+ Pump Regulation

Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na+-K+ pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of β1, but not α1, subunits in rabbit ventricular myocytes at baseline. β1 Subunit glutathionylation was increased by peroxynitrite (ONOO−), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased α1/β1 subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with β1 subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na+-K+ pump current (Ip). Cysteine (Cys46) of the β1 subunit was the likely candidate for glutathionylation. We expressed Na+-K+ pump α1 subunits with wild-type or Cys46-mutated β1 subunits in Xenopus oocytes. ONOO− induced glutathionylation of β1 subunit and a decrease in Na+-K+ pump turnover number. This was eliminated by mutation of Cys46. ONOO− also induced glutathionylation of the Na+-K+ ATPase β1 subunit from pig kidney. This was associated with a ≈2-fold decrease in the rate-limiting E2→E1 conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na+-K+ pump occurs via glutathionylation of its β1 subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na+ levels.

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

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 epsilon-protein kinase C (epsilonPKC), previously implicated in ANG II-induced Na(+)-K(+) pump inhibition. A role for epsilonPKC was also supported by an ANG II-induced increase in coimmunoprecipitation of epsilonPKC 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 epsilonPKC 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 alpha(1)-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 alpha(1)/caveolin 3 or alpha(1)/p22(phox) interaction, but it increased alpha(1)/p47(phox) coimmunoprecipitation. We conclude that ANG II inhibits the Na(+)-K(+) pump via PKC-dependent NADPH oxidase activation.

β3 Adrenergic Stimulation of the Cardiac Na+-K+ Pump by Reversal of an Inhibitory Oxidative Modification

Background— Inhibition of L-type Ca2+ current contributes to negative inotropy of &bgr;3 adrenergic receptor (&bgr;3 AR) activation, but effects on other determinants of excitation-contraction coupling are not known. Of these, the Na+-K+ pump is of particular interest because of adverse effects attributed to high cardiac myocyte Na+ levels and upregulation of the &bgr;3 AR in heart failure. Methods and Results— We voltage clamped rabbit ventricular myocytes and identified electrogenic Na+-K+ pump current (Ip) as the shift in holding current induced by ouabain. The synthetic &bgr;3 AR agonists BRL37344 and CL316,243 and the natural agonist norepinephrine increased Ip. Pump stimulation was insensitive to the &bgr;1/&bgr;2 AR antagonist nadolol and the protein kinase A inhibitor H-89 but sensitive to the &bgr;3 AR antagonist L-748,337. Blockade of nitric oxide synthase abolished pump stimulation and an increase in fluorescence of myocytes loaded with a nitric oxide–sensitive dye. Exposure of myocytes to &bgr;3 AR agonists decreased &bgr;1 Na+-K+ pump subunit glutathionylation, an oxidative modification that causes pump inhibition. The in vivo relevance of this was indicated by an increase in myocardial &bgr;1 pump subunit glutathionylation with elimination of &bgr;3 AR–mediated signaling in &bgr;3 AR−/− mice. The in vivo effect of BRL37344 on contractility of the nonfailing and failing heart in sheep was consistent with a beneficial effect of Na+-K+ pump stimulation in heart failure. Conclusions— The &bgr;3 AR mediates decreased &bgr;1 subunit glutathionylation and Na+-K+ pump stimulation in the heart. Upregulation of the receptor in heart failure may be a beneficial mechanism that facilitates the export of excess Na+.

Activation of cAMP-dependent signaling induces oxidative modification of the cardiac Na+-K+ pump and inhibits its activity

Cellular signaling can inhibit the membrane Na+-K+ pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the β1 subunit of the pump α/β heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47phox and p22phox, required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na+-K+ pump β1 subunit and decreased its co-immunoprecipitation with the α1 subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the α1/β1 subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na+-K+ pump current (arising from the 3:2 Na+:K+ exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the ϵ-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na+-K+ pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump β1 subunit.

Frequency of Late Drug-Eluting Stent Thrombosis With Non-Cardiac Surgery

Drug-eluting stents (DES) are highly effective in reducing restenosis but have a small but significant risk for late stent thrombosis (LAST). Cessation of antiplatelet drugs for noncardiac surgery has been implicated in precipitating LAST, prompting surgery to be done on antiplatelet therapy, with all the attendant bleeding risks, or deferred until 12 months after DES implantation, despite limited data defining the risk for LAST. Using billing data from 2 large health funds, members who had DES insertion (n = 9,321) with subsequent noncardiac surgery (n = 4,126) were mailed a questionnaire regarding their noncardiac procedures, antiplatelet use, and subsequent coronary events. From 1,086 returned, 710 were suitable for inclusion, identifying 11 patients (1.5%) with perioperative myocardial infarctions confirmed by medical records. Angiography showed that only 2 had stent thromboses, while 7 had new culprit lesion (2 patients did not undergo angiography). Before their noncoronary procedures, 66% were receiving dual-antiplatelet therapy, and 30% were taking single agents. Surgery was performed on dual therapy in 18%, on single agents in 23%, and with no antiplatelet therapy in 59%. The mean time to surgery from stent implantation was 348 days, with 56% <12 months. In conclusion, noncardiac surgery after DES implantation is frequent and appears to have low cardiac morbidity despite variable antiplatelet cessation. Perioperative myocardial infarctions occur because of narrowings in nonstented coronary arteries rather than from LASTs.

Ventricular Tachycardia in Ischemic Heart Disease

Ventricular arrhythmias are a significant cause of morbidity and mortality in patients with ischemic structural heart disease. Endocardial and epicardial mapping strategies include scar characterization channel identification, and recording and ablation of late potentials and local abnormal ventricular activities. Catheter ablation along with new technology and techniques of bipolar ablation, needle catheter, and autonomic modulation may increase efficacy in difficult to ablate ventricular arrhythmias. Catheter ablation of ventricular arrhythmias seem to confer mortality and morbidity benefits in patients with ischemic heart disease.