Gemma A. Figtree

Biological markers of oxidative stress: Applications to cardiovascular research and practice.

Oxidative stress is a common mediator in pathogenicity of established cardiovascular risk factors. Furthermore, it likely mediates effects of emerging, less well-defined variables that contribute to residual risk not explained by traditional factors. Functional oxidative modifications of cellular proteins, both reversible and irreversible, are a causal step in cellular dysfunction. Identifying markers of oxidative stress has been the focus of many researchers as they have the potential to act as an “integrator” of a multitude of processes that drive cardiovascular pathobiology. One of the major challenges is the accurate quantification of reactive oxygen species with very short half-life. Redox-sensitive proteins with important cellular functions are confined to signalling microdomains in cardiovascular cells and are not readily available for quantification. A popular approach is the measurement of stable by-products modified under conditions of oxidative stress that have entered the circulation. However, these may not accurately reflect redox stress at the cell/tissue level. Many of these modifications are “functionally silent”. Functional significance of the oxidative modifications enhances their validity as a proposed biological marker of cardiovascular disease, and is the strength of the redox cysteine modifications such as glutathionylation. We review selected biomarkers of oxidative stress that show promise in cardiovascular medicine, as well as new methodologies for high-throughput measurement in research and clinical settings. Although associated with disease severity, further studies are required to examine the utility of the most promising oxidative biomarkers to predict prognosis or response to treatment.

Current state of knowledge on Takotsubo syndrome: a Position Statement from the Taskforce on Takotsubo Syndrome of the Heart Failure Association of the European Society of Cardiology

Takotsubo syndrome is an acute reversible heart failure syndrome that is increasingly recognized in modern cardiology practice. This Position Statement from the European Society of Cardiology Heart Failure Association provides a comprehensive review of the various clinical and pathophysiological facets of Takotsubo syndrome, including nomenclature, definition, and diagnosis, primary and secondary clinical subtypes, anatomical variants, triggers, epidemiology, pathophysiology, clinical presentation, complications, prognosis, clinical investigations, and treatment approaches. Novel structured approaches to diagnosis, risk stratification, and management are presented, with new algorithms to aid decision‐making by practising clinicians. These also cover more complex areas (e.g. uncertain diagnosis and delayed presentation) and the management of complex cases with ongoing symptoms after recovery, recurrent episodes, or spontaneous presentation. The unmet needs and future directions for research in this syndrome are also discussed.

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.

Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism

OBJECTIVES To investigate the potential for plant derived estrogens (phytoestrogens) genistein, phloretin, biochanin A and zearalanone to relax rabbit coronary arteries in vitro and to determine the mechanism(s) of such relaxation. BACKGROUND Epidemiological data suggests a reduction in the incidence of coronary heart disease in humans who have a high intake of phytoestrogens. METHODS Isolated rabbit coronary artery rings were suspended in individual organ baths, precontracted with potassium chloride (30 mM), and the relaxing effects and mechanisms of relaxation to genistein, phloretin, biochanin A and zearalanone were determined by measurement of isometric tension. RESULTS Genistein, phloretin and biochanin A induced significant gender-independent relaxation in rings with and without endothelium. Inhibition of nitric oxide and prostaglandin synthesis with L-NAME and indomethacin had no effect on genistein-induced relaxation. Relaxation was unaffected by the specific estrogen receptor antagonist ICI 182,780, the ATP-sensitive potassium channel inhibitor glibenclamide and the potassium channel inhibitor, barium chloride. Calcium concentration-dependent contraction curves in high potassium depolarization medium were significantly shifted to the right and downward after incubation with genistein and zearalanone. An inhibitory effect of genistein (2 microM) on L-type calcium current in guinea-pig ventricular myocytes confirmed a calcium antagonist relaxing mechanism of action. In healthy volunteers, plasma genistein levels of approximately 2 microM are achieved after ingestion of a commercially available soy protein drink (Supro) containing 37 mg genistein. CONCLUSIONS This study demonstrates that phytoestrogens induce endothelium-independent relaxation of coronary arteries; the mechanism involves calcium antagonism. These mechanisms may contribute to the potential long-term cardiovascular protective effect of these substances.

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.

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.

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.

Glutathionylation mediates angiotensin II-induced eNOS uncoupling, amplifying NADPH oxidase-dependent endothelial dysfunction.

Background Glutathionylation of endothelial nitric oxide synthase (eNOS) “uncouples” the enzyme, switching its function from nitric oxide (NO) to O2•− generation. We examined whether this reversible redox modification plays a role in angiotensin II (Ang II)‐induced endothelial dysfunction. Methods and Results Ang II increased eNOS glutathionylation in cultured human umbilical vein endothelial cells (HUVECs), rabbit aorta, and human arteries in vitro. This was associated with decreased NO bioavailability and eNOS activity as well as increased O2•− generation. Ang II‐induced decrease in eNOS activity was mediated by glutathionylation, as shown by restoration of function by glutaredoxin‐1. Moreover, Ang II‐induced increase in O2•− and decrease in NO were abolished in HUVECs transiently transfected, with mutant eNOS rendered resistant to glutathionylation. Ang II effects were nicotinamide adenine dinucleotide phosphate (NADPH) oxidase dependent because preincubation with gp 91ds‐tat, an inhibitor of NADPH oxidase, abolished the increase in eNOS glutathionylation and loss of eNOS activity. Functional significance of glutathionylation in intact vessels was supported by Ang II‐induced impairment of endothelium‐dependent vasorelaxation that was abolished by the disulfide reducing agent, dithiothreitol. Furthermore, attenuation of Ang II signaling in vivo by administration of an angiotensin converting enzyme (ACE) inhibitor reduced eNOS glutathionylation, increased NO, diminished O2•−, improved endothelium‐dependent vasorelaxation and reduced blood pressure. Conclusions Uncoupling of eNOS by glutathionylation is a key mediator of Ang II‐induced endothelial dysfunction, and its reversal is a mechanism for cardiovascular protection by ACE inhibition. We suggest that Ang II‐induced O2•− generation in endothelial cells, although dependent on NADPH oxidase, is amplified by glutathionylation‐dependent eNOS uncoupling.

Reversible Oxidative Modification: Implications for Cardiovascular Physiology and Pathophysiology

Reminiscent of phosphorylation, cellular signaling can induce reversible forms of oxidative modification of proteins with an impact on their function. Redox signaling can be coupled to cell membrane receptors for hormones and be a physiologic means of regulating protein function, whereas pathologic increases in oxidative stress may induce disease processes. Here we review the role of reversible oxidative modification of proteins in the regulation of their function with particular emphasis on the cardiac Na(+)-K(+) pump. We describe how protein-kinase-dependent activation of redox signaling, mediated by angiotensin receptors and β adrenergic receptors, induces glutathionylation of an identified cysteine residue in the β(1) subunit of the α/β pump heterodimer; and we discuss how this may link neurohormonal abnormalities, increased oxidative stress, and cardiac myocyte Na(+) dysregulation and heart failure with important implications for treatment.