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 (α2^R/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 α2^R/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.

Abbreviations

ACTH

adrenocorticotropic hormone

Ang II

angiotensin II

AT₁R

angiotensin type‐1 receptor

BP

blood pressure

CNS

central nervous system

CSF

cerebrospinal fluid

C‐Src

C‐Src kinase

CTS

cardiotonic steroid

CV

cardiovascular

DN

dominant negative

DOCA

deoxycorticosterone acetate

EDL

extensor digitorum longus

EF

ejection fraction

ENaC

epithelial Na⁺ channels

EO

endogenous ouabain

HF

heart failure

HH

heart hypertrophy

i.c.v.

intracerebroventricular

jS/ER

junctional sarco‐/endoplasmic reticulum

LV

left ventricular

MBG

marinobufagenin

MI

myocardial infarction

MS

mass spectrometry

NCLX

mitochondrial Na⁺/Ca²⁺ exchanger

NCX

Na⁺/Ca²⁺ exchanger

NKA

Na⁺ pump or Na⁺, K⁺‐ATPase

PLM

phospholemman

PM

plasma membrane

RAAS

renin–angiotensin–aldosterone system

ROS

reactive oxygen species

RyR

ryanodine receptor

s.c.

subcutaneous

S/ER

sarco‐/endoplasmic reticulum

SERCA

sarco‐/endoplasmic reticulum Ca²⁺ pump

SkM

skeletal muscle

SM

smooth muscle

SNA

sympathetic nerve activity

SPM

sub‐PM; SR, sarcoplasmic reticulum

SS

salt‐sensitive

TAC

trans‐aortic constriction

TRPC6

transient receptor potential channel‐6

WT

wild‐type

Introduction

A decade ago, an article in this journal (Zhang et al. 2005), and two contemporary articles (Dostanic et al. 2005; Dostanic‐Larson et al. 2005), supported the hypothesis (Blaustein, 1977) that arterial Na⁺ pumps, their endogenous ouabain‐like ligand, and Na⁺/Ca²⁺ exchangers (NCX), contribute to salt‐sensitive hypertension. The genetically engineered mouse studies implicate the Na⁺ pump catalytic subunit α2 isoform. Here, we review recent reports that substantiate the seminal role of α2 Na⁺ pumps in the pathogenesis of hypertension and also in cardiac hypertrophy and failure. Remarkably, these pathologies can be prevented/attenuated by genetically altered α2 expression and/or ouabain resistant mutation of its binding site. This pinpoints α2 Na⁺ pumps as a key, but largely overlooked, therapeutic target.

Background

Sodium pumps (Na⁺,K⁺‐ATPase or NKA) are expressed in nearly all vertebrate cells. They export three Na⁺ ions and import two K⁺ ions while hydrolysing one ATP molecule during each transport cycle (Blanco & Mercer, 1998). The Na⁺ pumps maintain cell and organism Na⁺ and K⁺ homeostasis and influence numerous physiological processes. They also serve as cellular signal transducers for cardiotonic steroids (CTSs) (Xie & Askari, 2002).

Four Na⁺ pump catalytic subunit isoforms (α1–α4) have been cloned (Shull et al. 1985; Shull & Lingrel, 1987; Woo et al. 1999). Pumps with an α1 isoform (‘α1 Na⁺ pumps’) are expressed in virtually all cells, and are prevalent in most. They maintain the low Na⁺ and high K⁺ concentrations in ‘bulk’ cytoplasm, [Na⁺]_CYT and [K⁺]_CYT, respectively, and the resting membrane potential (e.g. McDonough et al. 1992; He et al. 2001; Radzyukevich et al. 2013), and mediate net Na⁺ transport across epithelia (McDonough et al. 1992; Rajasekaran et al. 2005). α3 is found in neurones, neonatal myocardium, adult human myocardium, and some other tissues; α4 is expressed in sperm (Lingrel, 2010).

α2 Na⁺ pumps

We focus on Na⁺ pumps with an α2 catalytic subunit, which are expressed in the cardiovascular (CV) system (Lingrel, 2010), including the endothelium (Zahler et al. 1996), in skeletal muscle (Radzyukevich et al. 2013) and in the brain (McGrail et al. 1991; Arakaki et al. 2013). In rodent cardiac and vascular smooth muscles, the α1:α2 ratio is ∼4:1 (James et al. 1999; Zhang et al. 2005; Berry et al. 2007; Despa & Bers, 2007); in skeletal muscle the α1:α2 ratio is ∼1:6 (He et al. 2001).

The minimal Na⁺ pump functional unit is an αβ protomer (Blanco & Mercer, 1998). The α subunit contains the Na⁺, K⁺ and Mg‐ATP binding sites, the catalytic machinery, and the CTS binding site. Rodents are unusual, however, because their α1 Na⁺ pumps have very low affinity for CTS (O'Brien et al. 1994). Thus, in rodents, and probably in other orders of mammals too, α2 and α3 Na⁺ pumps are the receptors for picomolar to nanomolar CTS (Song et al. 2013). CTSs selectively inhibit Na⁺ pump‐mediated cation transport (Schatzmann, 1953). Therefore, CTSs, and especially ouabain, which is relatively hydrophilic, are widely employed to study the consequences of Na⁺ pump blockade.

The Na⁺ pump β subunit (there are 3 isosforms) chaperones α, and is essential for the catalytic activity (Shull et al. 1986; Blanco & Mercer, 1998; Lingrel, 2010). β1 is the most prevalent isoform in cardiac muscle and vascular smooth muscle, where it forms both α1β1 and α2β1 protomers (Hundal et al. 1994; Cougnon et al. 2002; Hauck et al. 2009; Dey et al. 2012).

α2 Na⁺ pump localization and its significance

Most arterial (Fig. 1) and cardiac (Fig. 2) myocyte α2 Na⁺ pumps are localized in plasma membrane (PM) microdomains in close proximity to ‘junctional’ elements of the sarco‐/endoplasmic reticulum (jS/ER), i.e. at PM–S/ER junctions (Juhaszova & Blaustein, 1997 a,b; Mohler et al. 2003; Despa & Bers, 2007; Linde et al. 2012). There may, however, be some overlap with α1 in these microdomains (Mohler et al. 2003; Dey et al. 2012). Na⁺/Ca²⁺ exchangers (NCX), too, are localized in the PM–jS/ER microdomains (Figs 1 and 2) (Juhaszova & Blaustein, 1997 a; Berry et al. 2007; Lynch et al. 2008; Davis et al. 2009; Jayasinghe et al. 2009; Kuszczak et al. 2010). In cardiomyocytes, α2 pumps and NCX are found at, or adjacent to (Scriven et al. 2000), PM–S/ER junctions in the transverse (t‐) tubules as well as in the surface membrane (Fig. 2) (Mohler et al. 2003; Berry et al. 2007; Despa & Bers, 2007).

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.

This organization enables privileged communication among the α2 Na⁺ pumps, NCX and S/ER Ca²⁺ pumps (SERCA) through the tiny sub‐PM cytosolic compartment, ‘fuzzy space’, at the junctions (Figs 3 and 4) (Juhaszova & Blaustein, 1997 b; Goldhaber et al. 1999; Poburko et al. 2004; Verdonck et al. 2004; Pritchard et al. 2010; Swift et al. 2010; Aronsen et al. 2013). Consequently, the local α2 pump‐generated Na⁺ electrochemical gradient (Poburko et al. 2007) modulates NCX‐mediated Ca²⁺ transport, and local Ca²⁺ sequestration and Ca²⁺ signalling (Blaustein & Lederer, 1999; Arnon et al. 2000 b; Golovina et al. 2003; Verdonck et al. 2004; Lee et al. 2006; Lingrel, 2010; Despa et al. 2012; Shattock et al. 2015). This α2‐NCX linkage is consistent with the observation that ∼75% knockdown of cardiac NCX1 decreased α2, but not α1, expression by ∼50% (Bai et al. 2013). Also, ∼65% knockdown of α2 decreased NCX1 by ∼65%, but did not affect α1 expression in arteries (Chen et al. 2015 b).

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, V _m = −35 to −50 mV, is more positive than the NCX1 ‘reversal potential’, E _Na/Ca (Blaustein & Lederer, 1999); i.e. the driving force (V _m − E _Na/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 E _Na/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, r ⁴ (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. 2015 a).

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 V _m, perhaps about −65 to −75 mV, is more negative than E _Na/Ca; i.e. the driving force (V _m − E _Na/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 E _Na/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. 3 B 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. 3 C, D).

The special role of α2 also is evident in skeletal muscle: α2 Na⁺ pumps and NCX are prevalent in t‐tubules, where they may coordinate with junctional sarcoplasmic reticulum (SR) to help regulate the SR Ca²⁺ concentration, [Ca²⁺]_SR, and contraction (Radzyukevich et al. 2013; Altamirano et al. 2014; DiFranco et al. 2015). Skeletal myocytes (Kristensen et al. 2008) (and choroid epithelial cells; Arakaki et al. 2013), are unusual in that most α2 Na⁺ pumps are located in intracellular vesicles and are inactive. When translocated to the PM, triggered, e.g., by insulin, muscle contraction or cyclic stretching, they become active (Therien & Blostein, 2000; Yuan et al. 2007; Benziane & Chibalin, 2008; Kristensen et al. 2008; Zhang et al. 2012).

Regulation of α2 Na⁺ pumps; role of phospholemman

Na⁺ pumps, including α2 pumps, are regulated by multiple factors, including substrates, hormones (e.g. aldosterone, insulin and catecholamines) and protein phosphorylation (Therien & Blostein, 2000; Phakdeekitcharoen et al. 2011). Importantly, the Na⁺ and K⁺ affinities are modulated by the regulatory protein phospholemman (PLM), also called FXYD1 (Crambert et al. 2002; Bibert et al. 2008; Bossuyt et al. 2009; Han et al. 2010; Mishra et al. 2015). Surprisingly, this small molecule with a single transmembrane helix (Geering, 2006) also regulates NCX1 (Wang et al. 2011; Hafver et al. 2016).

Unphosphorylated PLM binds to α2β and reduces α2 affinity for intracellular Na⁺ and extracellular K⁺ (Han et al. 2009; Pavlovic et al. 2013 a). Phosphorylation of cardiac or arterial PLM by protein kinase A or C relieves the pump inhibition by altering PLM‐α2β1 interaction and restoring the Na⁺ high affinity (Bossuyt et al. 2006, 2009; Pavlovic et al. 2007, 2013 a; Han et al. 2010; Dey et al. 2012; Shattock et al. 2015).

Activation of the renin–angiotensin (Ang)–aldosterone system (RAAS), as in hypertension and heart failure (see below), stimulates reactive oxygen species (ROS) generation. This leads to glutathionylation of β1 and pump inhibition (Figtree et al. 2009; Liu et al. 2013). PLM promotes de‐glutathonylation and protects against oxidative inhibition of the pumps in arteries and heart (Liu et al. 2013; Chia et al. 2015).

Endogenous cardiotonic steroids

High affinity CTS binding is observed in all vertebrates (Pressley, 1992; Lingrel, 2010). Ouabain, digoxin and bufalin (a bufadienolide CTS) all block the pump's cation transport pathway (Laursen et al. 2013, 2015). The idea of an endogenous ligand for the Na⁺ pump CTS binding site (Szent‐Gyorgi, 1953) fostered the proposal that an endogenous ouabain‐like compound contributes to the pathogenesis of hypertension (Haddy & Overbeck, 1976; Blaustein, 1977). Studies in mice with an α2 null mutation, α2^+/− and, especially, with ouabain‐resistant α2 Na⁺ pumps, α2^R/R, provide definitive evidence that α2 and its endogenous ligand(s) have a physiological role in mammals (Dostanic et al. 2005; Dostanic‐Larson et al. 2005; Zhang et al. 2005; Lingrel, 2010; Van Huysse et al. 2011; Wansapura et al. 2011).

A CTS was isolated from human plasma and was identified by mass spectrometry (MS) as endogenous ouabain (EO) or a ouabain isomer (Hamlyn et al. 1991; Mathews et al. 1991). Nuclear magnetic resonance (NMR) and MS studies on human, bovine and rodent plasma and tissues verified that the endogenous substance is ouabain (Schneider et al. 1998; Kawamura et al. 1999; Komiyama et al. 2000; Tashko et al. 2010; Jacobs et al. 2012; Hamlyn et al. 2014); reviewed in Hamlyn & Blaustein, 2016). Moreover, recent studies identified two novel EO isomers that are not seen in commercial (plant) ouabain (Jacobs et al. 2012; Hamlyn et al. 2014). The isomers, which may also be present in human plasma (Hamlyn & Blaustein, 2016), apparently are physiologically regulated, but their relative affinities for α2 and their significance are unknown.

Another CTS, marinobufagenin (MBG), was identified in human plasma and urine by immunoassay (Bagrov et al. 1996, 2009). Both EO and MBG reportedly play a role in the pathogenesis of hypertension and heart failure (HF) (Schoner & Scheiner‐Bobis, 2007; Bagrov et al. 2009; Blaustein et al. 2012; Pavlovic, 2014). Prolonged administration of ouabain (Doursout et al. 1992; Yuan et al. 1993; Huang et al. 1994) or MBG (Kennedy et al. 2006) induces hypertension in normal rats, but digoxin and digitoxin do not (Manunta et al. 2000). This implies that Na⁺ pump inhibition per se does not cause the ouabain‐ or MBG‐induced hypertension. Experiments on α2^R/R mice (see Table 1), and studies with fab fragments that immunoneutralize EO and MBG, demonstrate that elevated endogenous CTS levels contribute to CV pathophysiology (Schoner & Scheiner‐Bobis, 2007; Bagrov et al. 2009; Blaustein & Hamlyn, 2010). This review focuses on EO and not MBG because: (i) DigiBind and DigiFab, commercial fab fragments used to immunoneutralize endogenous CTS in vivo, bind ouabain with much higher affinity than MBG (Pullen et al. 2004, 2008); (ii) MBG preferentially binds to α1 rather than α2 subunits (Wansapura et al. 2009); and (iii) several clinical and animal studies on the functions of EO in CV physiology and pathophysiology are backed by analytical (MS) measurements, e.g. Stella et al. (2008), Jacobs et al. (2012) and Hamlyn et al. (2014).

Table 1.

Cardiovascular and skeletal muscle manifestations of genetically modified mouse α2 Na⁺ pumps

Genetic modification	Vascular effects	Cardiac effects	Skeletal muscle effects	References
Globally reduced α2 (α2+/−)	↑Basal [Ca2+]CYT; ↑myogenic tone; ↑basal BP; i.c.v. Ang I and ↑[Na+]CSF induce hypertension	↑[Ca2+]CYT transients; ↑contractility	↑Contractile force	(James et al. 1999) (He et al. 2001) (Shelly et al. 2004) (Zhang et al. 2005) (Hou et al. 2009)
Smooth muscle α2 dominant negative (α2SM‐DN)	↑Basal BP; ↓myogenic reactivity; ↑pressor response to Ang II + high dietary salt	—	—	(Chen et al. 2015 b)
Smooth muscle α2 transgenic over‐expressor (SM‐α2Tg/Tg)	↓Basal BP; normal myogenic reactivity; ↓pressor response to Ang II ± high dietary salt	—	—	(Pritchard et al. 2007) (Chen et al. 2015 b)
CV α2 null (CV‐α2−/−)	Normal basal BP; No ACTH‐ or ouabain‐induced hypertension	Normal basal cardiac function	—	(Rindler et al. 2011)
Cardiac α2 null (Cardiac‐α2−/−)	Normal basal BP; ACTH induces hypertension	Normal basal cardiac function; attenuated TAC‐induced cardiac hypertophy	—	(Rindler et al. 2013)
Cardiac α2 (Tg) transgenic over‐expressor (Cardiac‐α2Tg)	—	Attenuated TAC‐induced cardiac hypertophy	—	(Correll et al. 2014)
Cardiac α1 (Tg) transgenic over‐expressor (Cardiac‐α1Tg)	—	TAC‐induced cardiac hypertophy	—	(Correll et al. 2014)
Skeletal muscle α2 null (α2SkM−/−)	—	—	↓Contractile force; ↑sensitivity to fatigue	(Radzyukevich et al. 2009) (Radzyukevich et al. 2013)
Global ouabain‐resistant α2 (α2R/R = α1R/R‐α2R/R)	Normal basal BP; no ACTH‐ or ouabain‐induced hypertension; no i.c.v. ouabain‐ or ↑[Na+]CSF‐induced hypertension; ↓BP in 3rd trimester of pregnancy; DOCA + salt induces hypertension	No CTS‐induced cardiotonic effect; attenuated TAC‐induced cardiac hypertophy	↓Sensitivity to fatigue	(Dostanic et al. 2003) (Dostanic et al. 2005) (Dostanic‐Larson et al. 2005) (Lorenz et al. 2008) (Radzyukevich et al. 2009) (Oshiro et al. 2010) (Van Huysse et al. 2011) (Despa et al. 2012) (Lorenz et al. 2012)
Global SWAP (α1S/S‐α2R/R vs. WT = α1R/R‐α2S/S)	—	↓Contractility; ↓sensitivity to CTS‐induced cardiotonic effect (vs.WT); accelerated TAC‐induced cardiac hypertrophy	—	(Dostanic et al. 2004) (Wansapura et al. 2009) (Wansapura et al. 2011) (Despa et al. 2012)

Ouabain‐triggered, Na⁺ transport‐independent cell signalling mediated by Na⁺ pumps

Prolonged treatment with ouabain activates multiple intracellular signalling pathways independent of effects on Na⁺ transport in rat heart and other tissues (Xie & Askari, 2002; Tian & Xie, 2008; Li & Xie, 2009; Zulian et al. 2013). The ouabain‐activated signalling may be mediated by a separate, ‘non‐pumping pool’ of pumps (Liang et al. 2007), perhaps located in caveolae (Liu et al. 2003; Wang et al. 2004; Kristensen et al. 2008). Most investigations employed 1–100 μm ouabain, and emphasized the participation of rodent α1 Na⁺ pumps, which are relatively ouabain resistant (Liang et al. 2006; Tian et al. 2006). The effectiveness of submicromolar ouabain in rat tissues (Liu et al. 2000; Pulina et al. 2010; Zulian et al. 2013), however, implies mediation by ouabain‐sensitive α2 (or α3), and not resistant α1 Na⁺ pumps. Ouabain‐induced cell signalling was not observed in immortalized α1‐deficient cells transfected with rat α2 (Xie et al. 2015), but there is no evidence that α2 was linked to the appropriate signalling molecules in those cells, which do not normally express α2. This requires direct comparison of low dose ouabain in native cells from wild‐type (WT) and α2^R/R mice.

Ouabain‐triggered intracellular signalling involves protein kinase cascades such as C‐Src kinase (C‐Src), ERK1/2, MAPK, phosphatidylinositide 3‐kinase 1A, protein kinase B (Akt) and NF‐κB, and may be cell‐type specific (Xie & Askari, 2002; Li & Xie, 2009; Wu et al. 2013). C‐Src can be activated by ouabain‐induced ROS generation and carbonylation of the pump (Yan et al. 2013).

Functions of α2 Na⁺ pumps in arterial physiology and pathophysiology

Arterial myocyte α2 Na⁺ pumps, modulation of vasoconstriction and blood pressure

Most, if not all, α2 pumps in vascular smooth muscle co‐localize with NCX1 at the PM–jS/ER where, together, they help regulate Ca²⁺ homeostasis and influence Ca²⁺ signalling and vasoconstriction (Juhaszova & Blaustein, 1997 a; Lynch et al. 2008; Linde et al. 2012). Normally, most arteries maintain myogenic or vasoconstrictor‐induced (mainly sympathetic nerve‐mediated) ‘basal’ tone (Hill et al. 2001; Zhang et al. 2010 a). Myocyte membrane potential is in the order of −35 to −50 mV (Knot & Nelson, 1998), and the electrochemical driving force on NCX (Blaustein & Lederer, 1999) favours net Ca²⁺ entry (Iwamoto et al. 2004; Zhang et al. 2010 b; Wang et al. 2015) (Fig. 3 A). Consequently, reduced α2 Na⁺ pump activity (e.g. ouabain inhibition or reduced expression) should raise the local, sub‐PM Na⁺ concentration ([Na⁺]_SPM) and promote net Ca²⁺ gain via NCX, thereby enhancing Ca²⁺ stores and signalling, and increasing vascular tone and BP (Fig. 3 B) (Zhang et al. 2005, 2009; Chen et al. 2015 b).

In fact, ouabain induces hypertension in most strains of rats; the few negative reports (Ghadhanfar et al. 2014) are consistent with the evidence that ouabain sensitivity is genetically controlled (Aileru et al. 2001). Ouabain also induces hypertension in WT, but not α2^R/R mice (Dostanic et al. 2005). Further, α2^R/R mice are resistant to adrenocorticotropic hormone (ACTH)‐induced hypertension, and ACTH hypertension is prevented by DigiBind and by the NCX antagonist KB‐R7942 (Dostanic‐Larson et al. 2005; Lorenz et al. 2008). Mice in which CV α2 pumps are selectively knocked out (CV‐α2^−/− mice) are also resistant to ACTH‐induced hypertension (Rindler et al. 2011). Because ACTH stimulates EO secretion (Laredo et al. 1994), the implication is that EO‐induced inhibition of α2 raises [Na⁺]_SPM and promotes NCX1‐mediated net gain of Ca²⁺ and increased arterial constriction.

Mice with genetically reduced α2 pump expression, whether global α2 heterozygous null mutants, α2^+/− (Zhang et al. 2005), or smooth muscle (SM)‐specific dominant negative (DN) knockdown, α2^SM‐DN (Chen et al. 2015 b), also have elevated BP. (Global α2^−/− is embryonic lethal; James et al. 1999.) The α2^SM‐DN mice have increased BP sensitivity to subcutaneous (s.c.) Ang II and high dietary salt (vs. WT; not tested in α2^+/− mice), presumably because there are fewer available α2 EO receptors and a larger fraction are inhibited by the elevated EO (Blaustein et al. 2015; Chen et al. 2015 b). Conversely, mice with SM‐specific α2 overexpression (α2^SM‐Tg) and excess α2 EO binding sites, have low basal BP (Pritchard et al. 2007; Chen et al. 2015 b) and reduced BP sensitivity to s.c. Ang II and high dietary salt (Chen et al. 2015 b).

The fact that CV‐α2^−/− mice have normal basal BP despite the nearly complete absence of arterial SM α2 Na⁺ pumps (Rindler et al. 2011) seems inconsistent with these other reports. In CV‐α2^−/− mice, however, the α2‐NCX1 coupling at PM–S/ER junctions is disrupted and NCX1‐mediated Ca²⁺ transport is stabilized by overexpression of α1 Na⁺ pumps (Rindler et al. 2011) which maintain a constant, low global [Na⁺]_CYT and are resistant to ouabain/EO.

Digibind lowers BP in deoxycorticosterone acetate (DOCA)–salt hypertensive rats (Krep et al. 1995), and their arteries overexpress the Ca²⁺ transporter transient receptor potential channel‐6 (TRPC6) (NCX1 and SERCA2 were not tested) (Bae et al. 2007), suggesting that EO is involved. However, α2^R/R mice develop DOCA–salt hypertension (Lorenz et al. 2012), implying that EO and MBG are not involved. Whether this is a species or technical difference is unknown.

Collectively, the above reports demonstrate that arterial SM α2 Na⁺ pumps and EO, along with arterial NCX1, modulate arterial tone and BP, and play an important role in some forms of hypertension (Table 1 and Fig. 3).

Brain α2 Na⁺ pumps and hypertension

The role of the central nervous system (CNS) in the pathogenesis of essential hypertension and salt‐sensitive (SS‐) hypertension is well documented, albeit incompletely understood. There is broad agreement that central sympathetic drive is a major contributor to BP elevation (Fisher & Fadel, 2010; Allen, 2011; Gabor & Leenen, 2012; Stocker et al. 2015). In addition, EO in the hypothalamus (‘brain ouabain’) and brain α2 Na⁺ pumps play a role in the pathogenesis of rodent SS‐hypertension: In Wistar rats (Leenen, 2010; Gabor & Leenen, 2012) and WT mice, but not in α2^R/R mice (Van Huysse et al. 2011), prolonged intracerebroventricular (i.c.v.) infusion of Na⁺‐rich cerebrospinal fluid (CSF) or very low dose ouabain elevates BP. These effects are augmented in α2^+/− mice (Hou et al. 2009), presumably because there are fewer available α2 EO receptors and a larger fraction are inhibited. Further, i.c.v. infusion of anti‐ouabain, but not control, fab fragments prevents the Na⁺‐rich CSF‐induced BP elevation (Huang et al. 2006; Van Huysse et al. 2011). Clearly, α2 Na⁺ pumps, their CTS binding site, and the endogenous ligand are all critical for SS‐hypertension.

Salt‐sensitive hypertension is also attenuated by i.c.v. infusion of the epithelial Na⁺ channel (ENaC) inhibitor benzamil (Gomez‐Sanchez et al. 1996; Leenen, 2010; Gabor & Leenen, 2012; Van Huysse et al. 2012; Osborn et al. 2014). Brain ENaCs are expressed in neurones and glia, and in the choroid plexus and ependyma (Amin et al. 2005; Leenen, 2010; Miller & Loewy, 2013; Miller et al. 2013; Oshima et al. 2013). Knockout of the ubiquitin ligase Nedd4‐2, a regulator of ENaC expression, enhances ENaC activity in the kidney and brain, and Nedd4‐2^−/− mice develop moderate SS‐hypertension (Shi et al. 2008; Van Huysse et al. 2012). When crossed with α2^R/R mice, the double mutants (Nedd4‐2^−/−‐α2^R/R mice) had a markedly attenuated BP elevation, compared to Nedd4‐2^−/− mice, in response to either Na⁺‐rich CSF (i.c.v.) or high dietary salt (Leenen et al. 2015). Thus, both arterial and brain α2 Na⁺ pumps, and their endogenous ligand, contribute to SS‐hypertension. The locus of the relevant brain α2 pumps is unknown, but α2 is expressed in meningeal capillary endothelia, in the choroid epithelial cell cytoplasm (Arakaki et al. 2013), and in neurones and glia (McGrail et al. 1991; Moseley et al. 2003). The cytoplasmic pumps may be cycled to the PM (Benziane & Chibalin, 2008) under conditions yet to be determined.

Ouabain‐triggered, α2‐mediated cell signalling and hypertension

Prolonged exposure to nanomolar ouabain increases expression of several arterial Ca²⁺ transporters, both in rats in vivo (i.e. during hypertension induction), and in primary cultured human and rat artery myocytes. Ca²⁺ signalling is then augmented even after ouabain washout (Pulina et al. 2010; Linde et al. 2012; Zulian et al. 2013). The same proteins, most notably NCX1 and SERCA2, are also up‐regulated in several rodent hypertension models, including Dahl‐salt‐sensitive, Milan, and spontaneously hypertensive rats, and the Ang II, Ang II + salt and DOCA + salt models (Blaustein et al. 2012, 2015; Pulina et al. 2013). This is consistent with the idea that circulating EO is elevated in these models and that it initiates these changes in protein expression. Arterial NCX1 is also up‐regulated in human primary pulmonary hypertension (Zhang et al. 2007). Prolonged ouabain/EO–α2 interaction triggers arterial myocyte C‐Src phosphorylation, reduces ERK1/2 phosphorylation, and leads to the Ca²⁺ transporter reprogramming (Fig. 3 C) (Zulian et al. 2013). Importantly, both the acute and chronic actions of EO and ouabain augment arterial Ca²⁺ entry and signalling. Both should therefore foster vasoconstriction and BP elevation in essential hypertension, primary aldosteronism, and other forms of hypertension in which plasma EO is elevated (Fig. 3 B–D) (Blaustein & Hamlyn, 2010; Blaustein et al. 2012).

CTS–α2 Na⁺ pump interactions are more complex than anticipated. All CTSs inhibit α2 (and α3) Na⁺ pumps, augment Ca²⁺ signalling (Song et al. 2013) and have similar acute vasotonic effects (Song et al. 2014). In contrast, ouabain‐like CTSs (e.g. Strophanthus steroids), but not digoxin‐like CTSs (e.g. Digitalis steroids), also activate downstream signalling cascades that modify protein expression (Zulian et al. 2013). These chronic ouabain‐induced effects are blocked by digoxin (Zulian et al. 2013), which is a ouabain antagonist (Huang et al. 1999; Manunta et al. 2000; Song et al. 2014). This suggests that novel digoxin analogues might block the actions of ouabain without inhibiting Na⁺ transport; such agents might be therapeutically useful. Indeed, one such agent, rostafuroxin, was synthesized from digoxigenin (Quadri et al. 1997). It blocks the actions of ouabain at concentrations that do not inhibit the Na⁺,K⁺‐ATPase (Ferrari et al. 1998; Song et al. 2014), and lowers BP in ouabain hypertensive rats and Milan hypertensive rats (Ferrari et al. 1998, 1999). Unfortunately, rostafuroxin's affinity for the vascular myocyte Na⁺ pump may be too low to be clinically useful (Song et al. 2014).

Functional linkage of arterial α2 Na⁺ pumps and NCX1

The above findings emphasize the functional (and structural) linkage between α2 and NCX1 in arterial myocytes. This cross‐talk probably occurs via alterations in [Na⁺]_SPM, which may also be influenced by other adjacent channels and transporters such as TRPC6 (Fig 1 D) (Arnon et al. 2000 a; Poburko et al. 2007, 2008).

Genetic engineering studies illustrate a crucial difference between primary alteration of α2 expression and of NCX1 (and SERCA2) expression. Reduction of α2 by heterozygous null mutation elevates cell Ca²⁺ and induces hypertension and secondary reduction of NCX1 and SERCA2 expression; the latter is, presumably, a compensatory effect (Zhang et al. 2005; Chen et al. 2015 b). Conversely, transgenic overexpression of SM‐α2 lowers BP and causes a secondary increase in NCX1 and SERCA2 expression, probably to compensate, partially, for the BP reduction which may be due to a fall in [Ca²⁺]_CYT (Pritchard et al. 2007; Chen et al. 2015 b). In contrast, primary SM‐NCX1 overexpression increases [Ca²⁺]_CYT and elevates BP (Iwamoto et al. 2004), whilst SM‐specific knockout of NCX1 lowers [Ca²⁺]_CYT and BP (Zhang et al. 2010 b; Wang et al. 2015). We infer that the EO‐induced, α2‐mediated increase in arterial NCX1 and SERCA2 expression, observed in many types of hypertension (Blaustein & Hamlyn, 2010; Blaustein et al. 2012; Pulina et al. 2013), contributes directly to the elevation of BP (Chen et al. 2015 b).

How the brain talks to the arteries

In many forms of hypertension, including salt‐sensitive hypertension, a central angiotensinergic pathway (‘brain RAAS’) is activated (Allen, 2011; Gabor & Leenen, 2012; Takahashi, 2012). Circulating Ang II, which is elevated in some forms of hypertension, also stimulates the brain RAAS via circumventricular organs such as the subfornical organ (SFO) (Huang et al. 2010; Biancardi et al. 2014; Ufnal & Skrzypecki, 2014). This increases CNS driven arterial sympathetic nerve activity (SNA) and α‐adrenergic arterial constriction (Fink & Bruner, 1985; Osborn et al. 2007, 2011; Gabor & Leenen, 2012; Leenen, 2014), and contributes to BP elevation (Wang et al. 2013). Persistent activation of this central angiotensinergic mechanism appears to depend upon a novel neurohumoral pathway that is triggered by high dietary salt/Na⁺‐rich CSF (Huang et al. 2006), as well as Ang II (Huang et al. 2010). The hypothalamic component of the neurohumoral pathway involves local aldosterone production, mineralocorticoid receptors, ENaCs, local EO release and α2 Na⁺ pumps (Huang & Leenen, 1999; Van Huysse & Hou, 2004; Leenen, 2010; Gabor & Leenen, 2012; Van Huysse et al. 2012; Takahashi, 2012). This ‘brain EO’ enhances hypothalamic Ang type 1 receptor (AT₁R) signalling (Huang et al. 2011).

Sustained neurohumoral pathway activation raises circulating EO, which increases arterial expression of NCX1 and SERCA2 (Hamlyn et al. 2014); this should enhance arterial responses to sympathetic drive. Elevation of plasma EO and up‐regulation of arterial Ca²⁺ transporters, as well as the elevation of BP, are prevented by directly blocking the central neurohumoral pathway (Hamlyn et al. 2014). This implies that the increased SNA and the neurohumoral pathway that enhances arterial Ca²⁺ signalling operate jointly to raise BP chronically when the brain angiotensinergic mechanisms are activated.

α2 Na⁺ pumps and cardiac function

α2 Na⁺ pumps mediate the cardiotonic response to CTS

The positive inotropic effect of CTS on the heart (Fig. 4 A, B and E), analogous to the previously mentioned vasotonic effect, requires both ouabain‐sensitive Na⁺ pumps (see Table 1) and NCX1 (Reuter et al. 2002; Dostanic et al. 2003, 2004; Altamirano et al. 2006). Inhibition of cardiac Na⁺ pumps by CTS, the presumed consequent rise in [Na⁺]_SPM (see Swift et al. 2010), and the Na⁺‐dependent, NCX‐mediated net gain of intracellular Ca²⁺ and enhanced Ca²⁺ signalling (Swift et al. 2007, 2010) are widely accepted as the basis of the cardiotonic response. Both α1 and α2 Na⁺ pumps are located in rodent cardiac muscle t‐tubules at or near PM–SR junctions (Mohler et al. 2003; Dostanic et al. 2004; Berry et al. 2007), but which isoform mediates this cardiotonic response? To obtain a definitive answer, engineered ‘SWAP’ mice (with ouabain‐sensitive α1 and resistant α2 pumps, α1^S/S‐α2^R/R) and WT mice (with ouabain‐resistant α1 and sensitive α2 pumps, α1^R/R‐α2^S/S) were compared. SWAP mice exhibited a positive inotropic response to ouabain that was mediated by the mutated, ouabain‐sensitive α1 pumps (Dostanic et al. 2004). Nevertheless, comparable inhibition (∼25%) of total Na⁺ pump activity by low dose ouabain in WT and SWAP mice demonstrates that the α2 isoform preferentially modulates SR Ca²⁺ release and Ca²⁺ transients in cardiomyocytes (Fig. 4 A and B) (Despa et al. 2012). Swift and colleagues came to the same conclusion by showing that cardiac α2 pumps and NCX1 are functionally coupled via [Na⁺]_SPM (Swift et al. 2007, 2010). Importantly, despite this compelling evidence, low dose ouabain‐induced elevation of [Na⁺]_SPM (Fig. 4 B) has not yet been measured (Swift et al. 2010). Furthermore, these observations imply that Na⁺ diffusion between the sub‐PM microdomains and bulk cytosol is restricted (Wendt‐Gallitelli et al. 1993; Arnon et al. 2000 b; Silverman et al. 2003; Poburko et al. 2007; Swift et al. 2010; Aronsen et al. 2013), but definitive data are still lacking.

Cardiac hypertrophy and failure induced by pressure overload: role of α2 Na⁺ pumps

Mouse models with genetically engineered α2 Na⁺ pumps (Table 1) or altered pump regulation elucidate the link between Na⁺ pump expression/activity and cardiac function, and provide new clues to the pathogenesis of heart hypertrophy (HH) and HF. Pressure overload induced by trans‐aortic constriction (TAC) is a common model for inducing HH and HF. TAC induces progressive HH and left ventricular (LV) dysfunction in WT mice that depends on the extent and duration of the TAC (Liao et al. 2002). Cardio‐specific knockout of α2 delays the development of TAC‐induced cardiac dysfunction, i.e. increased end‐diastolic and systolic volumes, and decreased ejection fraction (EF) (Rindler et al. 2013). However, cardio‐specific α2, but not α1, overexpression also attenuates TAC‐induced HH (Correll et al. 2014). How can we reconcile these contradictory results?

First, consider the effects of TAC in mice with altered Na⁺ pump ouabain sensitivity. SWAP (α1^S/S‐α2^R/R) mice are more susceptible to HH following TAC than are WT (α1^R/R‐α2^S/S) or α1^R/R‐α2^R/R mice, even though the latter two lines have higher LV systolic pressures (Wansapura et al. 2011). Heart weight was greatly increased in SWAP mice, but only modestly in WT and α1^R/R‐α2^R/R mice, after 4 weeks of TAC. SWAP mice also had substantial LV enlargement, and a reduced EF, indicating cardiac decompensation (HF), i.e. the pathophysiological processes were accelerated. Remarkably, banded α1^R/R‐α2^R/R mice had no LV enlargement and no echocardiographic evidence of cardiac dysfunction (vs. sham) after 4 weeks of TAC (Wansapura et al. 2011). Clearly, TAC‐induced HH and HF depend, in part, upon ouabain sensitivity. Further, the cardiac changes are attenuated by anti‐ouabain fab fragments (Wansapura et al. 2011). Thus, Na⁺ pumps and their endogenous ligand contribute to the pathogenesis of HH and HF. More rapid TAC‐induced cardiac dysfunction is therefore anticipated in SWAP mice because low CTS concentrations inhibit only cardiac α2 pumps in WT mice, and ouabain‐sensitive α1 Na⁺ pumps in SWAP mice, and the α1:α2 ratio is ∼4:1 in both strains (James et al. 1999; Berry et al. 2007; Despa & Bers, 2007). Thus, at submaximal EO, more pumps will be inhibited in SWAP than in WT mice. In other words, the TAC‐induced cardiac dysfunction correlates with the proportion of Na⁺ pumps that is EO sensitive. These considerations also explain why both cardiac‐α2 knockout (Rindler et al. 2013) and overexpression (Correll et al. 2014) delay/attenuate TAC‐induced cardiac dysfunction. Neither α2 nor its ouabain receptor is expressed in knockouts. In over‐expressors, more ‘reserve’ α2 pumps/EO receptors are available to keep [Na⁺]_SPM low when a fraction is blocked by EO.

The pressure overload data suggest that EO, via its cardiotonic effect, contributes to HH with preserved, or even enhanced, cardiac performance, e.g. increased EF (Wansapura et al. 2011). Human and rodent HF data infer, however, that the impaired contractility and reduced EF also are linked to high plasma EO (Gottlieb et al. 1992; Pitzalis et al. 2006; Stella et al. 2008; Blaustein et al. 2015). How can this be reconciled?

The fact that prolonged ouabain treatment activates protein kinase cascades that modulate cardiac protein expression (Tian & Xie, 2008; Li & Xie, 2009) suggests an explanation. In cultured cardiomyocytes, 30–100 μm ouabain (24–48 h) increases NCX1 expression (Vemuri et al. 1989; Müller‐Ehmsen et al. 2003); indeed, 50 nm ouabain (72 h) is sufficient, but 100 nm digoxin is ineffective (Blaustein et al. 2015). Increased cardiac NCX1 and decreased SERCA2 expression, which are common findings in human HF and animal models (Studer et al. 1994; O'Rourke et al. 1999), contribute to the reduced SR Ca²⁺ stores and attenuated Ca²⁺ signals (Bers & Despa, 2006; Lehnart et al. 2009). Therefore, while its acute effect is cardiotonic, chronically elevated ouabain/EO and the enhanced NCX1 and reduced SERCA2 expression should accelerate Ca²⁺ extrusion, and promote [Ca²⁺]_SR decline and progression to hypocontractility and HF (Fig. 4 B and C) (Rodriguez et al. 2014). Indeed, partial NCX inhibition restores Ca²⁺ signalling in myocytes from failing hearts (Hobai et al. 2004). Importantly, these conclusions need to be tested in other HH and HF models, e.g. coronary artery ligation/myocardial infarction (MI), in α2^R/R and SWAP mice.

Regulation of α2 pumps in HH and HF

In some forms of HF, expression of α1, α2 and PLM, and Na⁺ pump activity, are all reduced, and [Na⁺]_CYT is elevated, in left ventricular myocytes (Bossuyt et al. 2005; Pavlovic et al. 2013 a), although PLM transcription is up‐regulated (Gronich et al. 2010). Also, in HF, oxidative stress (Burgoyne et al. 2012) inhibits cardiomyocyte Na⁺ pumps by inducing β1 subunit glutathionylation; this can be reversed by phosphorylated PLM (Bibert et al. 2011).

The link between α2 activity and cardiac pathophysiology is affirmed by two models of PLM dysregulation. In PLM knockout mice, total Na⁺,K⁺‐ATPase activity and α2 Na⁺ pump expression are reduced by 50–60% (Jia et al. 2005). This should sustain a high [Na⁺]_SPM and a large cardiotonic effect (Golovina et al. 2003) to account for the hypertrophic hearts and increased LV EF (Jia et al. 2005).

In the second model, mice with non‐phosphorylatable PLM (PLM^35A) have normal cardiac function under basal conditions. Following TAC, however, PLM^35A mice exhibit accelerated cardiac hypertrophy and dysfunction with increased NCX and decreased SERCA2a expression (Boguslavskyi et al. 2014). The inability to phosphorylate PLM and augment pump‐mediated Na⁺ extrusion and, thus, NCX‐mediated Ca²⁺ extrusion, when the heart is stressed (e.g. by TAC) enhances Ca²⁺ dysregulation and accelerates the cardiomyopathy. These models reinforce the view that cardiac α2 Na⁺ pumps and NCX1 conjointly contribute to the pathogenesis of HH and HF. An important caveat, however, is that in the rat heart in HF, expression of α2 declines and α3, the fetal isoform, increases (Semb et al. 1998; Verdonck et al. 2003), but the significance of this isoform switch and the localization of α3 are unknown.

Myocardial [Na⁺]_CYT and NCX1 in HF

Elevated myocardial [Na⁺]_CYT (Pogwizd et al. 2003; Murphy & Eisner, 2009; Bay et al. 2013; Pavlovic et al. 2013 a) fosters the NCX‐mediated Ca²⁺ dysregulation in HF (Bers & Despa, 2006; Despa & Bers, 2013; Shattock et al. 2015). Multiple mechanisms may contribute to the high [Na⁺]_CYT, including: (i) reduced α2 pump expression; (ii) Na⁺ pump dysregulation due to reduced PLM expression (Bossuyt et al. 2005); (iii) increased late Na⁺ current due to altered CaMKII regulation of cardiac Na⁺ channels (Grandi & Herren, 2014); (iv) direct inhibition of α2 by the elevated plasma EO (Gottlieb et al. 1992; Pitzalis et al. 2006; Stella et al. 2008; Hamlyn & Manunta, 2015); (v) increased Na⁺ entry via Na⁺/H⁺ exchange (Baartscheer et al. 2003; Karmazyn et al. 2008); (vi) dysregulation of the Ca²⁺‐dependent, nitric oxide (NO)‐mediated mechanism that stimulates Na⁺ pumps by phosphorylating PLM (Pavlovic et al. 2013 b); and (vii) increased oxidative stress and ROS generation (Munzel et al. 2015; Zuo et al. 2015) that not only reduces NO availability and PLM phosphorylation, but also increases β1 subunit glutathionylation (Figtree et al. 2009); both mechanisms depress pump‐mediated cation transport.

Elevated [Na⁺]_CYT promotes Ca²⁺ export by the mitochondrial Na⁺/Ca²⁺ exchanger, NCLX, which lowers intra‐mitochondrial [Ca²⁺] and increases oxidation of mitochondrial NADH (Murphy & Eisner, 2009; Liu et al. 2010; De Marchi et al. 2014; Nita et al. 2015). Thus, elevated [Na⁺]_CYT and/or [Ca²⁺]_CYT (which should limit NCLX‐mediated Ca²⁺ export) can not only enhance Ca²⁺ signalling, but also increase oxidative stress and ROS production (Li et al. 2014) and further depress Na⁺ pump function (Figtree et al. 2009).

The preceding two paragraphs focus on ‘global’ [Na⁺]_CYT. However, [Na⁺]_SPM, which apparently modulates cardiac [Ca²⁺]_CYT transients and excitation–contraction coupling, may be independently affected (Su et al. 2001; Verdonck et al. 2004; Swift et al. 2010; Aronsen et al. 2013). Further, [Na⁺]_SPM may also be modified by Na⁺ channels associated with these microdomains (Verdonck et al. 2004; Aronsen et al. 2013).

Genetically induced cardiac NCX1 overexpression in mice, itself, accelerates HH and HF induced by stresses (TAC, intense exercise, or pregnancy) known to activate the RAAS (Roos et al. 2007). The NCX1 over‐abundance and enhanced Ca²⁺ removal are manifested by reduced SR Ca²⁺, Ca²⁺ transients, and excitation–contraction coupling gain (Reuter et al. 2004; Ottolia et al. 2013), also observed in myocytes from failing human and rat hearts (Gomez et al. 1997; Piacentino et al. 2003). In contrast, genetically reduced (50%) cardiac NCX1 expression confers tolerance to pressure overload and attenuates HF development, perhaps by reducing Ca²⁺ overload (Takimoto et al. 2002; Jordan et al. 2010). Nevertheless, ∼20% of WT NCX1 is essential for cardiac function: nearly complete knockout causes HH and accelerates stress‐induced progression to HF (Jordan et al. 2010), presumably because of Ca²⁺ overload due to impaired Ca²⁺ clearance. NCX1 apparently also plays a role in some other HH and HF models. For example, cardiac‐specific NCX1 knockout (by 80–90%) mitigates chronic intermittent hypoxia‐induced LV hypertrophy and contractile dysfunction in mice (Chen et al. 2010).

Cardiomyocyte Ca²⁺ dysregulation in HF

In HF, cardiac Ca²⁺ dysregulation is usually manifested by elevated diastolic [Ca²⁺]_CYT but reduced SR Ca²⁺ content (Fig. 4 C–E) (Lehnart et al. 2009; Reuter & Schwinger, 2012). The latter, which may be the result of reduced SERCA2 expression and activity (Lehnart et al. 2009) and increased Ca²⁺ leakage through ryanodine receptors (RyRs) (Marx & Marks, 2013), probably explains the attenuated peak systolic [Ca²⁺]_CYT transients and cardiac hypocontractility (Fig. 4 D and E). The elevated diastolic (quasi‐steady state) [Ca²⁺]_CYT can be attributed largely to the previously mentioned high [Na⁺]_CYT and reduced driving force for Ca²⁺ extrusion via NCX, although reduced SR Ca²⁺ uptake and increased RyR leak may also contribute. The high [Na⁺]_CYT and thus [Ca²⁺]_CYT may help explain the impaired relaxation and increased stiffness of cardiac muscle in HF (Kass et al. 2004; Louch et al. 2010; Li et al. 2012).

Central and peripheral mechanisms in the progression from hypertrophy to failure

In HH and HF, as in hypertension, central angiotensinergic mechanisms are usually stimulated, and sympathetic drive is increased (Yu et al. 2008; Westcott et al. 2009; Lymperopoulos et al. 2013; Zucker et al. 2014). Blockers of these mechanisms are therefore used to treat both hypertension and HF (Leenen et al. 2012; Krum & Driscoll, 2013; James et al. 2014). The angiotensinergic mechanisms activate the neurohumoral pathway that elevates circulating EO (Hamlyn et al. 2014): e.g. both MI and s.c. Ang II + high dietary salt raise plasma EO and increase both cardiac and arterial NCX1 expression (Blaustein et al. 2015). Because arterial NCX1 operates primarily in the Ca²⁺ entry mode, both acute and chronic high EO should enhance vasoconstriction and foster hypertension, but why do high EO and increased NCX1 also lead to cardiac hypocontractility and failure? The main cardiac Ca²⁺ extrusion mechanism, NCX1, exports Ca²⁺ during most of the cardiac cycle because, during diastole, the membrane potential is about −65 to −75 mV (Eisner et al. 2013; Eisner, 2014). Acutely elevated plasma EO therefore induces ‘classic’ positive inotropy, but markedly increased cardiac NCX1 expression, due to chronically elevated EO, promotes Ca²⁺ extrusion, reduces [Ca²⁺]_CYT and causes negative inotropy (Fig. 4 B–D).

Comparison of WT, α2^R/R and SWAP mouse data (Wansapura et al. 2011) lead us to postulate that TAC activates the brain RAAS–neurohumoral pathway, raises plasma EO, and in WT, and even more so in SWAP mice (α2^R/R mice are EO‐resistant), induces a positive inotropic response. This is initially amplified as NCX1 expression increases. The consequent, sustained hypercontractility, as well as other, possibly EO‐triggered, changes in protein programming contribute to hypertrophy and, at least initially, to enhanced cardiac performance. With progressive increase in NCX1 and decrease in SERCA2 expression, however, the NCX1‐mediated Ca²⁺ extrusion mode starts to prevail, and [Ca²⁺]_CYT falls, thereby reducing cardiac performance and leading to HF; i.e. the cardiac changes in HH and HF are a continuum. Indeed, HF with preserved EF (Kamimura et al. 2012; Gladden et al. 2014; Sharma & Kass, 2014; Zuo et al. 2015) might be an intermediate stage in this continuum. Further, following MI, even the RAAS‐stimulated initial tendency to induce a positive inotropic response may be circumvented rapidly if there is much damaged and unresponsive myocardium. The altered NCX1 and SERCA2 expression may then dominate early on, leading rapidly to a negative ionotropic response and HF (Fig. 4 D).

Exercise intolerance in HF: role of skeletal muscle α2 Na⁺ pumps in fatigue resistance

Skeletal muscle (SkM) α2 plays a negligible role in quiescent muscle, but is activated by the rise in t‐tubule [K⁺] and [Na⁺]_CYT during exercise, and is needed to attenuate fatigue (DiFranco et al. 2015; Manoharan et al. 2015). As in the heart, phosphorylation of SkM PLM enhances Na⁺ pump activity, but PLM knockout mice show that PLM is not needed for acute exercise‐induced SkM α2 activation (Manoharan et al. 2015). Nevertheless, intense exercise increases PLM phosphorylation, and α2 pump expression and activity in human type II (fast twitch, fatigable) SkM fibres, which express more α2 than do type I (slow twitch, fatigue‐resistant) fibres (Kristensen et al. 2008; Thomassen et al. 2010, 2013; Benziane et al. 2011). Reduced α2 Na⁺ pump expression in ageing humans may decrease muscle strength and increase fatigability (Chibalin et al. 2012).

Mice with targeted knockout of SkM α2 pumps, α2^SkM−/−, fatigue faster than WT mice on a treadmill (Radzyukevich et al. 2013). Also, extensor digitorum longus (EDL) muscles isolated from SkM‐α2^−/− mice have reduced twitch and tetanic force compared to WT EDL. Selective block of α2 by ouabain in WT EDL mimics the results in α2^SkM−/− EDL (Radzyukevich et al. 2013). Resistance to fatigue apparently is due in part to the rapid increase in α2‐mediated cation transport triggered by the rise in t‐tubule [K⁺] during stimulation (DiFranco et al. 2015).

α2 ouabain binding sites and EO play a role in SkM: α2^R/R mice exhibit fewer exercise failures on a treadmill than do WT mice (Radzyukevich et al. 2009). Also, ⁸⁶Rb (K⁺ surrogate) uptake is reduced following high frequency contractile activation (vs. rest) in EDL from WT mice. In contrast, following muscle stimulation, ⁸⁶Rb uptake is increased in EDL from α2^R/R mice and in EDL from WT mice pre‐infused with DigiBind prior to euthanasia (Radzyukevich et al. 2009).

Clearly, susceptibility to fatigue is inversely related to skeletal muscle α2 Na⁺ pump expression/activity and is modulated by EO, but why? A clue is that knockout of the predominant NCX isoform in SkM, NCX3, also reduces endurance and increases fatigue, although it increases both twitch and tetanus tension (Sokolow et al. 2004). We postulate that reduced NCX3‐mediated Ca²⁺ extrusion, due to decreased NCX3 expression or diminished Na⁺ extrusion by α2 pumps when exercise elevates t‐tubule [K⁺], enhances fatigability.

The above findings imply that the high EO levels observed in hypertension and HF contribute to the reduced SkM α2 Na⁺ pump activity (contrast Barr et al. 2005), increased fatigability (Carlsen et al. 1996; Helwig et al. 2003; Okita et al. 2013; Tzanis et al. 2014) and reduced hand grip strength (Mainous et al. 2015). Exercise may enhance SkM α2 Na⁺ pump activity by increasing α2 expression or translocation to the sarcolemma, or PLM phosphorylation, and thereby reduce fatigability and improve muscle strength (Thomassen et al. 2010; Rasmussen et al. 2011).

Summary and conclusions

Mice with genetically engineered α2 Na⁺ pumps, PLM and NCX1 provide novel insight into the central role of α2 and its endogenous ligand, EO, in regulating Ca²⁺ homeostasis and the function of cardiac, skeletal and vascular muscles. The juxtaposition of these findings enables us to recognize the striking similarities and key differences between the mechanisms involved in the pathogenesis of hypertension, HH and HF. In all three situations, brain angiotensinergic mechanisms are activated; this triggers the CNS rapid sympathetic and slower neurohumoral (EO‐mediated) pathways. Acutely increased nerve frequency is often attenuated by self‐tuning (Greengard, 2001; Turrigiano, 2008), but EO may potentiate peripheral synaptic transmission and sympathetic nerve responses (Aileru et al. 2001). Also, the chronic, protein kinase cascade‐mediated effects of elevated plasma EO on arteries and heart may amplify the cardiac and vascular responses to sympathetic drive. Initially, the cardio‐ and vasotonic actions of EO enhance Ca²⁺ signalling and contractility and thus elevate BP and heighten cardiac function, and lead to hypertrophy. Slow, EO‐mediated up‐regulation of NCX1 (and SERCA2) in arteries favours Ca²⁺ entry and further fosters vasoconstriction. In the heart, however, EO‐mediated NCX1 up‐regulation (and SERCA2 decline) eventually tips the balance toward Ca²⁺ exit, hypocontractility and HF. It is worth emphasizing that, when the brain RAAS is activated in hypertension, the elevated plasma EO is expected to influence cardiac function simultaneously. Likewise, when an MI activates the brain RAAS, simultaneously altered arterial function is expected (Blaustein et al. 2015). Thus, elevated plasma EO is likely to contribute to the increased peripheral vascular resistance often observed in HF post‐MI (Zelis et al. 1968; Ledoux et al. 2003).

Despite this compelling evidence for the key roles of α2 and EO, numerous challenges remain. First of all, details of the CNS pathways are poorly understood. For example, brain α2 pumps are important in SS‐hypertension (Leenen et al. 2015), but the cellular location of the relevant pumps is unknown. Also, the proposed role of brain α2 in HH and HF must be verified. Further, while EO is synthesized in the brain, and is a critical link in both hypertension and HF (Leenen et al. 1995; Huang et al. 2010), precisely where in the CNS pathways it participates is unresolved.

Circulating EO comes from the adrenals (Hamlyn et al. 1991; Boulanger et al. 1993; Manunta et al. 2010), but what is the biosynthetic pathway? Also, how does brain RAAS regulate plasma EO? Is it via increased sympathetic traffic to the adrenals (Shah et al. 1998), or some other mechanism? And, what role, if any, does ACTH play (Laredo et al. 1994)?

We suggest that α2 pumps mediate both the acute and chronic effects of nanomolar ouabain/EO in rodents (Dostanic et al. 2005; Despa et al. 2012; Zulian et al. 2013), but others suggest that α1 pumps are responsible for the chronic effects (Liu & Xie, 2010; Xie et al. 2015). Comparison of the acute and chronic effects of nanomolar ouabain on WT and α2^R/R cardiac and arterial myocytes could resolve this controversy. Further, since human α1 is ouabain sensitive, do human α2 pumps play the same key role as in rodents? A clue is that human, like rodent, arterial α2 is localized in PM microdomains at PM–S/ER junctions (Linde et al. 2012).

We postulate that the [Na⁺]_SPM at PM–S/ER junctions is a crucial factor in the EO‐dependent modulation of Ca²⁺ signalling and contractility in the arteries and heart (Figs 3 and 4). More precise information about the structural organization of the junctions and their resident transporters, e.g. using super‐resolution imaging, should improve our understanding of ion regulation in these regions. Critically, direct measurement of the effects of nanomolar ouabain on [Na⁺]_SPM with Na⁺‐sensitive fluorochromes and, e.g., ‘total internal reflection fluorescence’ (TIRF) imaging, is needed to validate our inferences.

The effect of sustained ouabain/EO exposure on signalling cascades is established, but the precise time course of these responses, and all of the contributors (e.g. the complete range of affected Ca²⁺ transporter and signalling molecules), are unknown. For example, does ouabain/EO, per se, trigger down‐regulation of cardiac SERCA2 expression? Measurement of ouabain/EO‐ and disease‐dependent gene activation (quantitative PCR analysis of mRNA) or changes in protein expression (MS) would provide important new clues to underlying pathogenic mechanisms.

Finally, a fundamental implication of the work reviewed above is that novel agents that interfere with the biosynthesis, release and/or peripheral actions of EO should be therapeutically beneficial in hypertension, HH and HF. Such agents might also be useful in attenuating the renal damage, often linked to these CV diseases, that has been attributed to elevated circulating EO (Bignami et al. 2013; Ferrandi et al. 2014; Hamlyn & Manunta, 2015). Indeed, application of these agents would provide a critical test of many of the ideas summarized here.

Additional information

Competing interests

None declared.

Funding

This work was supported by NIH grants HL‐45215 and HL‐107555 to M.P.B. and J.M.H., AM‐063710 to J.B.L., HL‐091969 and OD‐018315 to W.G.W., and HL‐107654 to J.Z., by Canadian Institutes of Health Research grant FRN:MOP‐74432 to F.H.H.L., and by American Heart Association Grant‐in‐Aid 24940022 to J.Z.

Biographies

Baltimore group (left to right; key mentors in parentheses): Mordecai Blaustein, a discoverer of Na⁺/Ca²⁺ exchange (NCX), studies arterial Ca²⁺ regulation, arterial tone and hypertension (Daniel Tosteson and Alan Hodgkin).

Ling Chen investigates cardiovascular mechanisms of hypoxia and ischaema (Morris Karmazyn and Steven Scharf).

John Hamlyn identified (with Blaustein) endogenous ouabain and its role in hypertension and heart failure (Thomas Duffy and Alan Senior).

Gil Wier, pioneer of cardiovascular Ca²⁺ signalling in vitro, in situ, and in awake mice in vivo (John Blinks).

Jin Zhang performed seminal studies on arteries from genetically altered arterial α2 and NCX mice (Xiaoliang Wang and Blaustein). Individual photos. Frans Leenen (Ottawa, left) discovered brain ouabain's role in the novel slow neuromodulatory pathway in hypertension and heart failure (Wybren deJong and Alvin Shapiro). Jerry Lingrel (Cincinatti), who cloned the Na⁺ pump isoforms, studies their physiological roles in genetically engineered mice (Harry Bosook and John Gurdon).