Helge H. Rasmussen

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.

Exaggeration of nonculprit stenosis severity during acute myocardial infarction: Implications for immediate multivessel revascularization

OBJECTIVES This study was designed to assess the prevalence and clinical significance of exaggerated nonculprit lesion stenosis in the setting of acute (<12 h) myocardial infarction (AMI). BACKGROUND Although microvascular spasm may reduce nonculprit artery flow during AMI, it is unknown whether increased tone may exaggerate nonculprit lesion severity. METHODS In patients with additional angiography within nine months of AMI, and significant nonculprit lesions imaged in matching views, stenosis severity was compared between studies in a random blinded fashion using validated quantitative coronary angiography software. Baseline demographics, medications, hemodynamics at each study, and clinical status at follow-up (infarct/unstable angina/stable angina) were used to determine the independent influence of the infarct presentation on stenosis exaggeration. RESULTS From 548 patients with AMI (1/99 to 6/01, 321 with multivessel disease), 112 had additional angiography; of these 48 had 59 lesions suitable for analysis. Between infarct and noninfarct angiograms there was a significant change in minimal lumen diameter (1.53 +/- 0.51 mm vs. 1.78 +/- 0.65 mm, p < 0.001) and percentage stenosis (49.3 +/- 14.5% vs. 40.4 +/- 16.6%, p < 0.0001) of the nonculprit lesion without significant change in reference segment diameter, which was not predicted by changes in medication or hemodynamics. Twenty-one percent of patients had lesions >50% at AMI that were <50% at non-AMI angiography. Infarct versus noninfarct setting was the only significant independent predictor of change in nonculprit stenosis. CONCLUSIONS Significant exaggeration of nonculprit lesion stenosis severity occurs at infarct angiography, which may affect revascularization decision making in an appreciable number of patients.

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.

Apical sparing in tako-tsubo cardiomyopathy.

Background: Tako‐tsubo cardiomyopathy (TTC) is an acute reversible cause of segmental myocardial dysfunction that is poorly understood. We have noted a variant of this condition where a tiny segment at the apex retains some contractile function. This paper delineates the frequency of this variant relative to the classical form and the clinical differences between patients suffering from the two forms.

Hyperaldosteronemia in Rabbits Inhibits the Cardiac Sarcolemmal Na+-K+ Pump

Aldosterone upregulates the Na(+)-K(+) pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na(+)-K(+) pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na(+)-K(+) pump current, arising from the 3:2 Na(+):K(+) exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na(+) in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na(+). Aldosterone had no effect on myocardial Na(+)-K(+) pump concentration evaluated by vanadate-facilitated [(3)H]ouabain binding or by K(+)-dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na(+) activity. The aldosterone-induced decrease in pump current and increased intracellular Na(+) were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na(+) affinity of the Na(+)-K(+) pump, whereas it has no effect on maximal pump capacity.

FXYD proteins reverse inhibition of the Na-K pump mediated by glutathionylation of its β1 subunit

The seven members of the FXYD protein family associate with the Na+-K+ pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na+-K+ pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na+-K+ pump β1 subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β1 subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β1 subunit was increased in myocardium from FXYD1−/− mice. In conclusion, there is a dependence of Na+-K+ pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na+-K+ pump complex. By facilitating deglutathionylation of the β1 subunit, FXYD proteins reverse oxidative inhibition of the Na+-K+ pump and play a dynamic role in its regulation.

Na+ influx and Na+-K+pump activation during short-term exposure of cardiac myocytes to aldosterone

To examine the effect of aldosterone on sarcolemmal Na+ transport, we measured ouabain-sensitive electrogenic Na(+)-K+ pump current (Ip) in voltage-clamped ventricular myocytes and intracellular Na+ activity (alpha iNa) in right ventricular papillary muscles. Aldosterone (10 nM) induced an increase in both Ip and the rate of rise of alpha iNa during Na(+)-K+ pump blockade with the fast-acting cardiac steroid dihydroouabain. The aldosterone-induced increase in Ip and rate of rise of alpha iNa was eliminated by bumetanide, suggesting that aldosterone activates Na+ influx through the Na(+)-K(+)-2Cl- cotransporter. To obtain independent support for this, the Na+, K+, and Cl- concentrations in the superfusate and solution of pipettes used to voltage clamp myocytes were set at levels designed to abolish the inward electrochemical driving force for the Na(+)-K(+)-2Cl- cotransporter. This eliminated the aldosterone-induced increase in Ip. We conclude that in vitro exposure of cardiac myocytes to aldosterone activates the Na(+)-K(+)-2Cl- cotransporter to enhance Na+ influx and stimulate the Na(+)-K+ pump.

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+.

The nitric oxide donor sodium nitroprusside stimulates the Na+–K+ pump in isolated rabbit cardiac myocytes

Nitric oxide (NO) affects the membrane Na+–K+ pump in a tissue‐dependent manner. Stimulation of intrinsic pump activity, stimulation secondary to NO‐induced Na+ influx into cells or inhibition has been reported. We used the whole‐cell patch clamp technique to measure electrogenic Na+–K+ pump current (Ip) in rabbit ventricular myocytes. Myocytes were voltage clamped with wide‐tipped patch pipettes to achieve optimal perfusion of the intracellular compartment, and Ip was identified as the shift in holding current induced by 100 μm ouabain. The NO donor sodium nitroprusside (SNP) in concentrations of 1, 10, 50 or 100 μm induced a significant increase in Ip when the intracellular compartment was perfused with pipette solutions containing 10 mm Na+, a concentration near physiological levels. SNP had no effect when the pump was near‐maximally activated by 80 mm Na+ in pipette solutions. Stimulation persisted in the absence of extracellular Na+, indicating its independence of transmembrane Na+ influx. The SNP‐induced pump stimulation was abolished by inhibition of soluble guanylyl cyclase (sGC) with 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one, by inhibition of protein kinase G (PKG) with KT‐5823 or by inhibition of protein phosphatase with okadaic acid. Inclusion of the non‐hydrolysable cGMP analogue 8pCPT‐cGMP, activated recombinant PKG or the sGC‐activator YC‐1 in patch pipette filling solutions reproduced the SNP‐induced pump stimulation. Pump stimulation induced by YC‐1 was dependent on the Na+ concentration but not the K+ concentration in pipette filling solutions, suggesting an altered sensitivity of the Na+–K+ pump to intracellular 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.