Negative inotropic effects of endothelin-1 in mouse cardiomyocytes: evidence of a role for Na⁺-Ca²⁺ exchange

Abstract

Endothelin-1 (ET-1) is a peptide hormone produced within the myocardium which may modulate myocardial contractility in a paracrine-autocrine fashion. In the majority of species, ET-1 has a direct positive inotropic effect on the myocardium that involves both increased myofilament Ca²⁺ sensitivity and increased Ca²⁺ transients. Ca²⁺ entry through reverse-mode Na⁺-Ca²⁺ exchange, involving both indirect effects via elevation of intracellular [Na⁺] and direct activation of the Na⁺-Ca²⁺ exchanger, have been suggested to contribute to the increase in Ca²⁺ transients. Conversely, mouse cardiomyocytes show an exclusively negative inotropic response to ET-1. Here, Nishimaru and colleagues present novel evidence that the negative inotropic effect of ET-1 in mouse cardiomyocytes involves both a reduction in myofilament Ca²⁺ sensitivity and increased Ca²⁺ extrusion, via Na⁺-Ca²⁺ exchange. Data obtained using the selective Na⁺-Ca²⁺ exchange blocker, SEA0400, suggest that a re-assessment of the role of the exchanger in Ca²⁺-handling by mouse cardiomyocytes may be necessary.

It is very nearly two decades since the discovery of endothelin (ET) as a potent vasoconstrictor peptide hormone released by endothelial cells (Yanagisawa et al., 1988). The demonstration of a direct positive inotropic effect (PIE) on the myocardium followed very soon thereafter (Ishikawa et al., 1988). The peptide is thought to be released within the heart, to modulate myocardial function in a paracrine–autocrine fashion (Mebazaa et al., 1993; MacCarthy et al., 2000) and may be involved in the contractile and hypertrophic responses to stretch (Yamazaki et al., 1996; Alvarez et al., 1999). The finding of elevated plasma concentrations of ET in various cardiac disorders and the beneficial effect of ET receptor antagonism in animal models of heart failure have focused attention on ET as a potential therapeutic target (Russell and Molenaar, 2000). Nevertheless, the actions of ET on the heart remain incompletely understood, in part due to the complexity of action of the hormone, involving direct and indirect effects on the myocardium and a multiplicity of signalling pathways, and partly due to species differences in the action of the peptide.

Although ET was originally proposed to act, like certain peptide toxins, by direct interaction with voltage-gated Ca²⁺ channels (Ishikawa et al., 1988; Yanagisawa et al., 1988), this peptide (now renamed ET-1) was demonstrated to act through two subtypes of G-protein-coupled receptor, ET_A and ET_B, ET_A being predominant in cardiomyocytes (Russell and Molenaar, 2000). Although both receptors couple to Gα_q signalling pathways, including phosphatidylinositol hydrolysis and protein kinase C (PKC) mobilization, ET receptors are relatively promiscuous in their coupling with G proteins, and signalling varies between cell types. In cardiomyocytes, not only does ET-1 signal via Gα_q, but ET_A receptors couple to Gα_i pathways, including adenylyl cyclase inhibition (Hilal-Dandan et al., 1992). Thus, there are many reports of the inhibition by ET-1 of currents regulated by β-adrenoceptors, such as the L-type Ca²⁺ current (I_Ca,L) (for example, Tohse et al., 1990; James et al., 1994; Xie et al., 1996). Nevertheless, although transient negative inotropic effects (NIE) are often observed on administration of the peptide, which may involve I_Ca,L inhibition (Woo and Lee, 1999), in the majority of species a more slowly developing, sustained PIE is also observed. The PIE has been associated with an increase in the Ca²⁺ sensitivity of the myofilaments and/or (depending on species) an increase in the Ca²⁺ transients (for example, Krämer et al., 1991; Takanashi and Endoh, 1991; Kelso et al., 1996a). The mechanisms of the increase in myofilament Ca²⁺ sensitivity are controversial but may involve Na⁺–H⁺ exchanger (NHE) activation and intracellular alkalinization (Krämer et al., 1991; Kang and Walker, 2006). Similarly, the basis of the increase in the Ca²⁺ transient is yet to be fully elucidated: With limited cell dialysis, ET-1-mediated increases in I_Ca,L can be observed (see Kelso et al., 1996b; Woo and Lee, 1999; He et al., 2000), demonstrating the sensitivity of ET-1 responses to experimental conditions. NHE may also have a role in increasing Ca²⁺ transients, as stretch activation of NHE via ET-1 has been suggested to increase intracellular Na⁺ concentration ([Na⁺]_i), thereby favouring Ca²⁺ entry via ‘reverse-mode' Na⁺–Ca²⁺ exchange (Alvarez et al., 1999). The Na⁺–Ca²⁺ exchanger (NCX) is an electrogenic transporter (3Na⁺:Ca²⁺) that represents the major route for Ca²⁺ extrusion from cardiomyocytes; the driving force for Ca²⁺ extrusion being the difference between the equilibrium potential for NCX and the membrane potential (Bers, 2002). Thus, NCX can also contribute to Ca²⁺ entry when the driving force for NCX transport reverses (for example, early in the action potential), an event facilitated by elevation of [Na⁺]_i. Results obtained using the NCX blocker, KB-R7943, are consistent with the contribution of reverse-mode NCX in ET-1 responses (Yang et al., 1999; Perez et al., 2001). In addition, ET-1 increases NCX activity directly via PKC-dependent phosphorylation (Zhang et al., 2001).

It is against this background that Nishimaru et al. (in press) have investigated the actions of ET-1 in mouse cardiomyocytes, which appears on pages XX–YY of this volume of the journal. In contrast to most other species, the adult mouse shows exclusively a NIE to ET-1 (Sekine et al., 1999; Sakurai et al., 2002). Using epifluorescence and videometric methods, the authors investigated the effects of ET-1 on Ca²⁺ transients and cell shortening in isolated mouse cardiomyocytes loaded with the ratiometric Ca²⁺ fluorophore, Indo-1. Their conclusions contrast beautifully with the extant literature on ET-1-mediated PIE; the NIE of mouse cardiomyocytes involves reduction of myofilament Ca²⁺ sensitivity and activation of NCX. ET-1 concentration-dependently reduced both Ca²⁺ transients and cell shortening, although the percentage reductions in cell shortening were greater than those in the Ca²⁺ transient. Elevation of extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the presence of ET-1 restored the amplitude of cell shortening and of the Ca²⁺ transient. Plotting cell shortening against Indo-1 fluorescence ratio during a single twitch in control and in the presence of ET-1 plus elevated [Ca²⁺]_o provided key evidence for the reduction in Ca²⁺ sensitivity; the slope of this relationship was less steep in the presence of ET-1, despite similar amplitudes of the Ca²⁺ transient and contraction. The mechanisms for this reduction in myofilament sensitivity remain unclear, although phosphorylation of troponin I by ET-1 has been suggested to reduce myofilament Ca²⁺ sensitivity (Cuello et al., 2007). As ET-1-induced phosphorylation of troponin I also occurs in species other than mouse (Damron et al., 1995), presumably additional mechanisms account for the increased myofilament Ca²⁺ sensitivity in these species.

The selective NCX blocker, 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline (SEA0400) provided evidence for the involvement of NCX in the NIE through reduction in Ca²⁺ transients: blockade of NCX completely abolished the ET-1-induced reduction in Ca²⁺ transient, suggesting a role for increased Ca²⁺ extrusion via NCX. On the other hand, inhibition of I_Ca,L was not required for the NIE as the action of ET-1 on Ca²⁺ transients was unaffected by L-type blockade. At first sight, this result may seem surprising since evidence from transgenic models suggests that NCX plays only a minor role in the recovery of Ca²⁺ transients in mouse (reviewed in Bers, 2002). Although the evidence in the present study is largely pharmacological, the authors were wise in their selection of NCX blocker: in contrast to KB-R7943, which blocks I_Ca,L, SEA0400 is relatively selective for NCX (Birinyi et al., 2005). Indeed, the increase in Ca²⁺ transients produced by SEA0400 is consistent with a significant role for NCX in Ca²⁺ extrusion. So how can increased NCX activity be involved in both the NIE of ET-1 in mouse and the PIE in other species? The answer may lie, at least partially, in species differences in the driving force for NCX-mediated Ca²⁺ transport during the cardiac cycle (Bers, 2002). In mice, the action potential is relatively short, with no clear plateau phase, and [Na⁺]_i is comparatively high (Bers, 2002). As a consequence, although the driving force for Ca²⁺ extrusion via NCX at the resting membrane potential during diastole is small, it is markedly increased by the Ca²⁺ transient during the repolarization phase of the action potential. In contrast, the longer action potential, with a clear plateau, and lower [Na⁺]_i of other species (for example, rabbit, human) lead to a greater Ca²⁺ extrusion during diastole and less during repolarization (Bers, 2002). Thus, ET-1-induced [Na⁺]_i increase reduces diastolic Ca²⁺ extrusion and favours reverse-mode NCX in these species (Aiello et al., 2005). By extension, one might speculate that in mouse cardiomyocytes perhaps ET-1 does not increase [Na⁺]_i and thus NCX operates predominantly in the forward mode, extruding Ca²⁺ during repolarization of the action potential.

In summary, this elegant study not only demonstrates the existence of two parallel mechanisms for the ET-1-induced NIE in mouse, but also provides important insights into species differences in hormone action. Further studies are warranted to elucidate the underlying mechanisms of ET-1 action in the mouse. Importantly, although the evidence is limited, the study suggests that a re-assessment of the role of NCX in Ca²⁺ handling by mouse cardiomyocytes may be necessary.

Abbreviations

[Ca²⁺]_o

extracellular Ca²⁺ concentration

ET

endothelin

ET_A, ET_B

A- and B-subtypes of endothelin receptor

I_Ca,L

L-type Ca²⁺ current

KB-R7943, an NCX blocker

2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate

[Na⁺]_i

intracellular Na⁺ concentration

NCX

Na⁺–Ca²⁺ exchanger

NHE

Na⁺–H⁺ exchanger

NIE

negative inotropic effect

PIE

positive inotropic effect

PKC

protein kinase C

SEA0400

a selective NCX blocker, 2-[4-[(2,5-difluorophenyl) methoxy]phenoxy]-5-ethoxyaniline