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Rationale: Cardiotonic steroids (CTS), such as digoxin, have been used to treat heart failure (HF) for over 200 years. They inhibit the sodium-potassium pump (NKP), and increase cardiac contractility by inhibiting efflux of sodium through the pump (“digitalis hypothesis”). CTS possess three structural components: a saturated/unsaturated lactone ring, steroid core, and sugar moiety, each of which may be involved in NKP inhibition/stimulation. It is now known that inhibition of the NKP in patients with HF increases mortality, and all major beneficial treatments increase its activity. Endogenous circulating CTS such as ouabain are generally thought to inhibit the NKP, despite studies sporadically reporting ouabain-induced pump stimulation. This study aims to identify whether ouabain-induced pump stimulation occurs, and if so, which structural components are involved in causing pump stimulation. Methods & Results: Cardiac myocytes were isolated from male New Zealand White rabbits, placed in a Tyrode’s solution, and whole-cell patch clamped. They were exposed to 0-30nM ouabain, 0-50nM dihydroouabain (ouabain with a saturated lactone ring) or 0-500nM ouabagenin (ouabain lacking a sugar moiety) for 1 min, followed by a potassium-free solution, with the difference in current yielding the NKP current. Compared to the 0.47±0.05 pA/pF Tyrode’s solution control (n=11), 5nM ouabain significantly increased NKP current to 0.69±0.09 pA/pF ( P P Conclusion: Low-dose ouabain uniquely stimulates NKP activity. Low-dose dihydroouabain and ouabagenin do not, suggesting that a sugar moiety and unsaturated lactone ring are required for pump stimulation. Ouabain in its unaltered form may be a potential treatment for HF.

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 β 1 , but not α 1 , subunits in rabbit ventricular myocytes at baseline. β 1 Subunit glutathionylation was increased by peroxynitrite (ONOO − ), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased α 1 /β 1 subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with β 1 subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na + -K + pump current ( I p ). Cysteine (Cys46) of the β 1 subunit was the likely candidate for glutathionylation. We expressed Na + -K + pump α 1 subunits with wild-type or Cys46-mutated β 1 subunits in Xenopus oocytes. ONOO − induced glutathionylation of β 1 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 β 1 subunit from pig kidney. This was associated with a ≈2-fold decrease in the rate-limiting E 2 →E 1 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 β 1 subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na + levels.

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.

We enjoyed reading the recently published review by Burgoyne et al1 titled “Redox Signaling in Cardiac Physiology and Pathology” that provides an excellent overview of the expanding number of protein targets in the cardiovascular system undergoing redox regulation by direct posttranslational oxidative modification. The importance of this mode of signaling in both health and disease is well described. We were honored to see the inclusion of our recent work identifying the molecular mechanism for …