Pivotal role of α2 Na+ pumps and their high affinity ouabain binding site in cardiovascular health and disease

Abstract

Reduced smooth muscle (SM)-specific α2 Na+ pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensin II (Ang II) and dietary NaCl, whilst SM-α2 overexpression lowers basal BP and decreases Ang II/salt sensitivity. Prolonged ouabain infusion induces hypertension in rodents, and ouabain-resistant mutation of the α2 ouabain binding site (α2R/R mice) confers resistance to several forms of hypertension. Pressure overload-induced heart hypertrophy and failure are attenuated in cardio-specific α2 knockout, cardio-specific α2 overexpression and α2R/R mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by Ang II and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous ouabain (EO). Circulating EO modulates ouabain-sensitive α2 Na+ pump activity and Ca2+ transporter expression and, via Na+ /Ca2+ exchange, Ca2+ homeostasis. This regulates sensitivity to sympathetic activity, Ca2+ signalling and arterial and cardiac contraction.

Keywords: artery; cardiac hypertrophy; heart failure; hypertension.

© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Figures

Figure 1. Distribution of α2 Na+ pumps and NCX1 in embryonic mouse (A and B) and human (C and D) artery smooth muscle determined by immunocytochemistry

A, α2 Na+ pumps (green) and the plasma membrane Ca2+ pump (PMCA, red) are not co‐localized (negligible white areas) in this pseudocolour overlay image of a mouse aorta myocyte. B, α2 Na+ pumps (green) and NCX1 (red) exhibit substantial co‐localization (white areas) in a mouse aorta myocyte. C, primary cultured human mesenteric artery smooth muscle cells (hASMCs) were labelled with anti‐α2 polyclonal antibodies (pAb) and anti‐NCX1 monoclonal antibodies (mAb); the SR was then stained with ER‐Tracker, as indicated by the labels. Insets are enlargements of the boxed areas. Pseudocolour images of the enlarged α2 (red) and NCX1 (green) regions, and the overlay, are shown on the right. D, hASMCs were cross‐reacted with anti‐NCX1 mAb and anti‐TRPC6 pAb; the SR was then stained with ER‐Tracker, as indicated. Insets are enlargements of the boxed areas. Pseudocolour images of the enlarged NCX1 (green) and TRPC6 (red) regions, and the overlay, are shown on the right. In C and D, the patterns of staining by both antibodies were very similar to the pattern of ER Tracker (i.e. SR) distribution. Scale bars in C and D = 30 μm. Note that the α2, NCX1 and TRPC6 staining patterns are all very similar to that of ER‐Tracker. This is reflected by the yellow‐orange staining in the C and D overlay panels, and indicates that hASMC α2 Na+ pumps and NCX1 co‐localize (as in the mouse, B) and overlie elements of SR. A and B were kindly provided by Dr Ronald P. Lynch (B is from Lynch et al. 2008 with permission); C and D are from Linde et al. (2012) with permission.

Figure 2. Confocal images of normal adult rat cardiomyocytes immunolabelled with antibodies raised against SERCA2, Na+ pump α1, Na+ pump α2 and NCX1

All four antibodies stained the Z‐line/t‐tubule regions. The surface membrane was stained by anti‐α1, anti‐NCX1 and, to a much lesser extent, anti‐α2 antibodies, but not by anti‐SERCA2. Scale bar = 40 μm. Reproduced from Mohler et al. (2003) with permission.

Figure 3. Diagrams illustrating the acute and chronic effects of EO on Ca2+ homeostasis in arteries: roles of α2 Na+ pumps (NKA), NCX1, SERCA2 and inositol trisphosphate receptors (IP3R)

Other Ca2+ transporters such as L‐type voltage‐gated Ca2+ channels and plasma membrane (PM) Ca2+ pumps (PMCA) are omitted for simplicity. A, basal conditions. In arteries with tone, myocyte NCX1 operates primarily in the Ca2+ entry mode because the membrane potential, Vm = −35 to −50 mV, is more positive than the NCX1 ‘reversal potential’, ENa/Ca (Blaustein & Lederer, 1999); i.e. the driving force (Vm − ENa/Ca) is positive. B, acute exposure of arteries to low dose ouabain or EO inhibits (a fraction of) arterial myocyte α2 Na+ pumps, raises [Na+] in the sub‐PM restricted cytosolic space between the PM and SR (shaded area; i.e. [Na+]SPM)*, thereby increasing ENa/Ca and the driving force for NCX1‐mediated Ca2+ entry. The consequent rise in [Ca2+]CYT and Ca2+ sequestered in the SR augments Ca2+ signalling and contraction (the vasotonic effect), thereby increasing vascular tone and BP. C, sustained exposure of arterial myocytes to low dose ouabain or EO, in addition to its acute effects, activates an α2 Na+ pump‐mediated protein kinase (PK) signalling cascade that leads to increased expression of Ca2+ transporters including NCX1 and SERCA (green dotted line and ‘+’ sign). This promotes long‐term arterial Ca2+ gain and sequestration in the SR; via increased Ca2+ signalling, this leads to long‐term elevation of BP. D, comparison of approximate acute and chronic EO‐induced relative changes in NCX1 and SERCA2 expression and contraction, and anticipated [Na+]SPM * and [Ca2+]CYT *. The α2 Na+ pump–NCX1 functional coupling acts as an amplifier: small increases in [Na+]SPM translate to large increases in [Ca2+]CYT and contraction because of the 3 Na+:1 Ca2+ stoichiometry of NCX1 (Blaustein & Lederer, 1999). Furthermore, arterial resistance is inversely related to the fourth power of the radius, r4 (Poiseuille's law), so small decreases in the radii of resistance arteries will greatly increase peripheral vascular resistance and BP. *Note: [Na+]SPM has not been measured in arterial myocytes, nor have the acute and chronic effects of EO/ouabain on [Ca2+]CYT been compared; thus, the relative changes shown in the figure are speculative. The anticipated [Na+]SPM changes are consistent with NCX1‐mediated Ca2+ entry during chronic high EO (Iwamoto et al. 2004) and with the evidence that immuno‐neutralization of EO rapidly decreases BP in mice with chronic Ang II + salt‐induced hypertension (Chen et al. 2015a).

Figure 4. Diagrams illustrating the acute and chronic effects of EO on Ca2+ homeostasis in the heart: roles of α2 Na+ pumps (NKA), NCX1, SERCA2 and ryanodine receptors (RyR)

A, basal conditions. In cardiac myocytes, during the major part of the cardiac cycle the NCX1 operates in the Ca2+ exit mode because the diastolic Vm, perhaps about −65 to −75 mV, is more negative than ENa/Ca; i.e. the driving force (Vm − ENa/Ca) is negative. B, acute exposure of the heart to low dose ouabain or EO inhibits cardiac myocyte α2 Na+ pumps and raises [Na+]SPM. This increases ENa/Ca, but reduces the driving force for Ca2+ extrusion and elevates [Ca2+]SPM. Thus, the net effect, as in arteries, is enhanced Ca2+ signalling and contraction (i.e. the cardiotonic effect). C and D, sustained exposure of cardiac myocytes to low dose ouabain or EO also, as in arteries, activates an α2 Na+ pump‐mediated protein kinase (PK) signalling cascade. In the heart, however, this leads to increased NCX1 expression, but decreased SERCA expression (green and red dotted lines and ‘+’ and ‘−’, respectively). Thus, initially, the cytosolic and SR [Ca2+] are elevated, the cardiotonic effect prevails, increased cardiac contraction is sustained (as in ‘B’), and the heart may hypertrophy from the increased workload. Eventually, however, the sustained Na+ pump inhibition and [Na+]CYT/[Na+]SPM elevation will maintain an elevated diastolic [Ca2+]CYT (C) despite the up‐regulated NCX1. The decreased SERCA2 expression and leakage of Ca2+ from the SR via RyR, however, reduces SR Ca2+ sequestration and [Ca2+]CYT transients (D); thus, cardiac contraction decreases, and the heart fails. E, summary of the acute and chronic EO‐induced approximate relative changes in NCX1 and SERCA2 expression, [Na+]SPM (postulated; see Fig. 3 legend), [Ca2+]CYT and contraction. Note that the acute vasotonic (Fig. 3B and D) and cardiotonic effects of EO are similar, whereas the chronic effects of EO in the heart (C–E) differ greatly from those in arteries (Fig. 3C, D).