Role of Na+–Ca2+ exchanger in myocardial ischemia/reperfusion injury: evaluation using a heterozygous Na+–Ca2+ exchanger knockout mouse model

doi:10.1016/j.bbrc.2003.12.165

Biochemical and Biophysical Research Communications

Volume 314, Issue 3, 13 February 2004, Pages 849-853

https://doi.org/10.1016/j.bbrc.2003.12.165 Get rights and content

Abstract

We used Na⁺–Ca²⁺ exchanger (NCX) knockout mice to evaluate the effects of NCX in cardiac function and the infarct size after ischemia/reperfusion injury. The contractile function in NCX KO mice hearts was significantly better than that in wild type (WT) mice hearts after ischemia/reperfusion and the infarct size was significantly small in NCX KO mice hearts compared with that in WT mice hearts. NCX is critically involved in the development of ischemia/reperfusion-induced myocardial injury and therefore the inhibition of NCX function may contribute to cardioprotection against ischemia/reperfusion injury.

Section snippets

Materials and methods

NCX KO mice. NCX knockout (KO) mice were generated as described previously [7]. Male heterozygous KO mice and wild type (WT) littermates 12 weeks old were used. All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Electrophysiology. Ventricular cells were prepared from adult mice hearts by standard enzymatic digestion [8]. Whole-cell membrane currents were recorded by the patch-clamp method and the

NCX current density and Western blot analysis

We previously reported that the protein content of NCX in NCX KO mice hearts was ∼50% of that in WT mice hearts [13]. To elucidate the functional activity, we examined NCX current densities from −40 to 40 mV in WT (n=9) and NCX KO ventricular cells (n=6) (Fig. 1). The densities of the reverse mode of NCX at 40 mV in ventricular cells of KO mice (0.57 ± 0.07 pA/pF) were approximately half (55.4%) compared with those of WT mice (1.04 ± 0.14 pA/pF). These results suggest that the functional activity as

Discussion

Myocardial cell injury is induced by a combination of mechanical and chemical stresses during ischemia [14]. Reoxygenation after extended periods of ischemia rapidly induces hypercontracture of cardiomyocytes [15] and aggravates the pre-existing injury [16]. The hypercontracture represents a major cause of acute lethal cell injury in the reperfused myocardium [17], [18]. It has been hypothesized that an increase in intracellular Ca²⁺ levels of cardiomyocytes through NCX induces the

Acknowledgements

We thank to Y. Reien for the current density analysis and R. Kobayashi, E. Fujita, M. Watanabe, M. Iida, and A. Ohkubo for technical assistance. This work was supported in part by grants from Japanese Ministry of Education, Science, Sports and Culture, and Japan Health Sciences Foundation.

References (21)

J.G. Pilitsis et al.
Inhibition of Na⁺/Ca²⁺ exchange by KB-R7943, a novel selective antagonist, attenuates phosphoethanolamine and free fatty acid efflux in rat cerebral cortex during ischemia–reperfusion injury
Brain Res.
(2001)
K. Wakimoto et al.
Targeted disruption of Na⁺/Ca²⁺ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat
J. Biol. Chem.
(2000)
Y. Zou et al.
Both Gs and Gi proteins are critically involved in isoproterenol-induced cardiomyocyte hypertrophy
J. Biol. Chem.
(1999)
C.E. Ganote
Contraction band necrosis and irreversible myocardial injury
J. Mol. Cell. Cardiol.
(1983)
D. Schulze et al.
Sodium/calcium exchanger in heart muscle: molecular biology, cellular function, and its special role in excitation–contraction coupling
Cardiovasc. Res.
(1993)
J.H. Bridge et al.
The relationship between charge movements associated with I_Ca and I_Na–Ca in cardiac myocytes
Science
(1990)
A.G. Kleber
Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global ischemia in isolated perfused guinea pig hearts
Circ. Res.
(1983)
B.N. Eigel et al.
Antisense inhibition of Na⁺/Ca²⁺ exchange during anoxia/reoxygenation in ventricular myocytes
Am. J. Physiol. Heart Circ. Physiol.
(2001)
H. Reuter et al.
Knockout mice for pharmacological screening: testing the specificity of Na⁺/Ca²⁺ exchange inhibitors
Circ. Res.
(2002)
M. Suzuki et al.
Functional roles of cardiac and vascular ATP-sensitive potassium channels clarified by Kir 6.2-knockout mice
Circ. Res.
(2001)

There are more references available in the full text version of this article.

Cited by (39)

Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1
2021, Cell Calcium
Citation Excerpt :
In healthy myocardium, NCX1 mainly generates an inward Na+ current and extrudes Ca2 (1 Ca2+: 3 Na+) [1,2]. Inappropriate NCX1 activity is associated with a number of severe cardiac pathologies including myocardial infarction [3], heart failure [4] and arrhythmias [5,6]. Multiple regulatory mechanisms control NCX1.
Catalyzed by zDHHC-PAT enzymes and reversed by thioesterases, protein palmitoylation is the only post-translational modification recognized to regulate the sodium/calcium exchanger NCX1. NCX1 palmitoylation occurs at a single site at position 739 in its large regulatory intracellular loop. An amphipathic ɑ-helix between residues 740–756 is a critical for NCX1 palmitoylation. Given the rich background of the structural elements involving in NCX1 palmitoylation, the molecular basis of NCX1 palmitoylation is still relatively poorly understood. Here we found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-PATs, (3) NCX1 is still palmitoylated when it is arrested in either Golgi or ER, indicating that NCX1 is a substrate for multiple zDHHC-PATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcellular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER. We also report that NCX2 and NCX3 are dually palmitoylated, with important implications for substrate recognition and enzyme catalysis by zDHHC-PATs. Our results could support new molecular or pharmacological strategies targeting the NCX1 palmitoylation and depalmitoylation machinery.
Topical review: Shedding light on molecular and cellular consequences of NCX1 palmitoylation
2020, Cellular Signalling
Palmitoylation is the post-translational, covalent and reversible conjugation of a 16C saturated fatty acid to cysteine residues of proteins. The sodium calcium exchanger NCX1 is palmitoylated at a single cysteine residue in its large regulatory intracellular loop. Inactivation, mediated by the NCX1 inhibitory region XIP, is drastically impaired in unpalmitoylatable NCX1. The ability of XIP to bind and inactivate NCX1 is largely determined by NCX1 palmitoylation, which induces local conformational changes in the NCX1 intracellular loop to enable XIP to engage its binding site. Consequently, NCX1 palmitoylation regulates intracellular calcium by changing NCX1 sensitivity to inactivation. NCX1 palmitoylation is a dynamic phenomenon which is catalyzed by the palmitoyl acyl transferase zDHHC5 and reversed by the thioesterase APT1, with the switch between palmitoylated and depalmitoylated states, which has profound effects on NCX1 lipid interactions, influenced by NCX1 conformational poise. Herein we review the molecular and cellular consequences of NCX1 palmitoylation and its physiological relevance and highlight the importance of palmitoylation for NCX1 activity. We discuss the cellular control of protein palmitoylation and depalmitoylation, the relationship between lipid microdomains and lipidated and phospholipid binding proteins, and highlight the important unanswered questions in this emerging field.
Dynamic Palmitoylation of the Sodium-Calcium Exchanger Modulates Its Structure, Affinity for Lipid-Ordered Domains, and Inhibition by XIP
2020, Cell Reports
The transmembrane sodium-calcium (Na-Ca) exchanger 1 (NCX1) regulates cytoplasmic Ca levels by facilitating electrogenic exchange of Ca for Na. Palmitoylation, the only reversible post-translational modification known to modulate NCX1 activity, controls NCX1 inactivation. Here, we show that palmitoylation of NCX1 modifies the structural arrangement of the NCX1 dimer and controls its affinity for lipid-ordered membrane domains. NCX1 palmitoylation occurs dynamically at the cell surface under the control of the enzymes zDHHC5 and APT1. We identify the position of the endogenous exchange inhibitory peptide (XIP) binding site within the NCX1 regulatory intracellular loop and demonstrate that palmitoylation controls the ability of XIP to bind this site. We also show that changes in NCX1 palmitoylation change cytosolic Ca. Our results thus demonstrate the broad molecular consequences of NCX1 palmitoylation and highlight a means to manipulate the inactivation of this ubiquitous ion transporter that could ameliorate pathologies linked to Ca overload via NCX1.
Regulation of NCX1 by palmitoylation
2020, Cell Calcium
Citation Excerpt :
In a setting of ischemia and reoxygenation of ischemic tissue, intracellular Na+ accumulation as a result of Na+/K+-ATPase pump inhibition during ischemia [20,21] generates a driving force for Ca2+ entry via NCX1. Although NCX1 is inhibited by sodium accumulation during ischemia [22], it reactivates rapidly during reperfusion, when reverse mode NCX1 activity drives substantial intracellular Ca2+ overload, leading to necrotic myocyte death [23]. Cardiac-specific deletion of NCX1 protects against ischemia-reperfusion injury [24].
Palmitoylation (S-acylation) is the reversible conjugation of a fatty acid (usually C16 palmitate) to intracellular cysteine residues of proteins via a thioester linkage. Palmitoylation anchors intracellular regions of proteins to membranes because the palmitoylated cysteine is recruited to the lipid bilayer. NCX1 is palmitoylated at a single cysteine in its large regulatory intracellular loop. The presence of an amphipathic α-helix immediately adjacent to the NCX1 palmitoylation site is required for NCX1 palmitoylation. The NCX1 palmitoylation site is conserved through most metazoan phlya. Although palmitoylation does not regulate the normal forward or reverse ion transport modes of NCX1, NCX1 palmitoylation is required for its inactivation: sodium-dependent inactivation and inactivation by PIP2 depletion are significantly impaired for unpalmitoylatable NCX1. Here we review the role of palmitoylation in regulating NCX1 activity, and highlight future questions that must be addressed to fully understand the importance of this regulatory mechanism for sodium and calcium transport in cardiac muscle.
Regulation of cell death in the cardiovascular system
2020, International Review of Cell and Molecular Biology
Citation Excerpt :
During cardiac I/R injury, there are two triggers of cell death: Ca2 + and ROS overload. Inhibition of either trigger reduces cardiomyocyte cell death following I/R injury, which indicates that these death triggers are intertwined (Braunersreuther et al., 2013; Das et al., 1987; Fauconnier et al., 2013; Gardner et al., 1983; Miyamoto et al., 2000; Niwano et al., 2008; Ohtsuka et al., 2004; Sallinen et al., 2007). Both Ca2 + and ROS have the ability to evoke multiple forms of cell death, which should be considered as a continuum instead of individual pathways (Fig. 2 and Table 1) (Konstantinidis et al., 2012).
The adult heart is a post-mitotic terminally differentiated organ; therefore, beyond development, cardiomyocyte cell death is maladaptive. Heart disease is the leading cause of death in the world and aberrant cardiomyocyte cell death is the underlying problem for most cardiovascular-related diseases and fatalities. In this chapter, we will discuss the different cell death mechanisms that engage during normal cardiac development, aging, and disease states. The most abundant loss of cardiomyocytes occurs during a myocardial infarction, when the blood supply to the heart is obstructed, and the affected myocardium succumbs to cell death. Originally, this form of cell death was considered to be unregulated; however, research from the last half a century clearly demonstrates that this form of cell death is multifaceted and employees various degrees of regulation. We will explore all of the cell death pathways that have been implicated in this disease state and the potential interplay between them. Beyond myocardial infarction, we also explore the role and mechanisms of cardiomyocyte cell death in heart failure, myocarditis, and chemotherapeutic-induced cardiotoxicity. Inhibition of cardiomyocyte cell death has extensive therapeutic potential that will increase the longevity and health of the human heart.
Effects of ticagrelor on the sodium/calcium exchanger 1 (NCX1) in cardiac derived H9c2 cells
2019, European Journal of Pharmacology
Ticagrelor is a direct acting and reversibly binding P2Y₁₂ antagonist approved for the prevention of thromboembolic events. Clinical effects of ticagrelor cannot be simply accounted for by pure platelet inhibition, and off-target mechanisms can potentially play a role. In particular, recent evidence suggests that ticagrelor may also influence heart function and improve the evolution of myocardial ischemic injury by more direct effects on myocytes.
The cardiac sodium/calcium exchanger 1 (NCX1) is a critical player in the generation and control of calcium (Ca²⁺) signals, which orchestrate multiple myocyte activities in health and disease. Altered expression and/or activity of NCX1 can have profound consequences for the function and fate of myocytes. Whether ticagrelor affects cardiac NCX1 has not been investigated yet. To explore this hypothesis, we analyzed the expression, localization and activity of NCX1 in the heart derived H9c2-NCX1 cells following ticagrelor exposure. We found that ticagrelor concentration- and time-dependently reduced the activity of the cardiac NCX1 in H9c2 cells. In particular, the inhibitory effect of ticagrelor on the Ca²⁺-influx mode of NCX1 was evident within 1 h and further developed after 24 h, when NCX1 activity was suppressed by about 55% in cells treated with 1 μM ticagrelor. Ticagrelor-induced inhibition of exchanger activity was reached at clinically relevant concentrations, without affecting the expression levels and subcellular distribution of NCX1. Collectively, these findings suggest that cardiac NCX1 is a new downstream target of ticagrelor, which may contribute to the therapeutic profile of ticagrelor in clinical practice.

View all citing articles on Scopus

View full text

Role of Na+–Ca2+ exchanger in myocardial ischemia/reperfusion injury: evaluation using a heterozygous Na+–Ca2+ exchanger knockout mouse model

Abstract

Section snippets

Materials and methods

NCX current density and Western blot analysis

Discussion

Acknowledgements

Brain Res.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Cell. Cardiol.

Sodium/calcium exchanger in heart muscle: molecular biology, cellular function, and its special role in excitation–contraction coupling

Cardiovasc. Res.

The relationship between charge movements associated with ICa and INa–Ca in cardiac myocytes

Science

Resting membrane potential, extracellular potassium activity, and intracellular sodium activity during acute global ischemia in isolated perfused guinea pig hearts

Circ. Res.

Antisense inhibition of Na+/Ca2+ exchange during anoxia/reoxygenation in ventricular myocytes

Am. J. Physiol. Heart Circ. Physiol.

Knockout mice for pharmacological screening: testing the specificity of Na+/Ca2+ exchange inhibitors

Circ. Res.

Functional roles of cardiac and vascular ATP-sensitive potassium channels clarified by Kir 6.2-knockout mice

Circ. Res.

Role of Na⁺–Ca²⁺ exchanger in myocardial ischemia/reperfusion injury: evaluation using a heterozygous Na⁺–Ca²⁺ exchanger knockout mouse model

The relationship between charge movements associated with I_Ca and I_Na–Ca in cardiac myocytes

Antisense inhibition of Na⁺/Ca²⁺ exchange during anoxia/reoxygenation in ventricular myocytes

Knockout mice for pharmacological screening: testing the specificity of Na⁺/Ca²⁺ exchange inhibitors