Original articleMechanisms underlying age-associated manifestation of cardiac sodium channel gain-of-function
Graphical abstract
Introduction
Sodium (Na+) channel gain-of-function (GOF) is a pathological condition associated with cardiac disease, including inherited disorders, such as Long-QT syndrome type 3 (LQT3), as well as acquired diseases, such as ischemia and heart failure [1]. LQT3 is linked to mutations in the SCN5A gene that encodes the α-subunit of the cardiac voltage-gated Na+ channel (NaV1.5) [2]. Na+ channel GOF manifests as a late Na+ current, which can prolong action potential duration (APD) and generate proarrhythmic early afterdepolarizations (EADs) [3,4]. While APD prolongation and EAD formation are reproducible phenomena in isolated myocytes with Na+ channel GOF, in intact tissue, however, this proarrhythmic response can be concealed [5,6], requiring additional perturbations to manifest the disease phenotype [7,8]. As such, LQT3 can be referred to as a “concealed” disease: Patients with LQT3-associated mutations often remain asymptomatic, including a normal QT interval, until later in life, with the risk of first cardiac event occurring primarily after puberty and QT prolongation becoming more prominent and increasing with age [[9], [10], [11]]. While LQT3 accounts for only 5–10% of all patients with long QT syndromes [9], the asymptomatic manifestation in many patients results in high lethality, with Zareba et al. reporting 20% and Schwartz et al. reporting 49% of cardiac events being lethal [12,13]. At the same time, LQT3 can also present earlier in life, with links to sudden infant death syndrome (SIDS) [14,15], and indeed within the same family and specific mutation, both adults and children can present as either symptomatic or asymptomatic [[16], [17], [18]], demonstrating complex age-associated manifestation and individual patient variability.
Recently, we and others have demonstrated that NaV1.5 channels preferentially localize at the intercalated disc (ID), the location of cell-cell junctions in cardiac tissue [[19], [20], [21], [22], [23], [24]]. In silico studies have predicted that Na+ current at the ID can be modulated via Na+ nanodomain signaling localized at the intercellular cleft, modulating cell-cell coupling via a mechanism known as ephaptic coupling (EpC) [19,[25], [26], [27], [28], [29], [30], [31], [32], [33]]. We recently demonstrated that EpC can also be a critical modulator of EAD formation and APD prolongation in tissue comprised of myocytes with LQT3-associated Na+ channel GOF mutations [34,35]. In this paper we consider two primary effects of EpC: Na+ ion depletion and electrical field effects in the intercellular cleft (i.e., the narrow extracellular space between electrically coupled cells at the ID). Briefly, Na+ influx in a depolarizing cell during the cardiac action potential reduces the electrical potential of the intercellular cleft. This intercellular cleft potential reduction then depolarizes the apposing cell from the extracellular, rather than the intracellular, side of the cell membrane. Additionally, Na+ influx reduces the local Na+ concentration of the intercellular cleft ([Na+]cleft), which governs the flux of the Na+ channels at the ID in both cells. Specifically, for a narrow intercellular cleft, both the electric field effects elevating the transmembrane potential (Vmdisc) and local Na+ depletion reducing the Na+ reversal potential (ENadisc) at the ID collectively reduce the Na+ current driving force and thus the Na+ current. This reduction in Na+ current has been termed “self-attenuation,” and has been shown to slow conduction [19,27,28,36].
We recently demonstrated that self-attenuation provides a mechanism that can also suppress APD prolongation and EAD formation in cardiac tissue with Na+ current GOF in tissue simulations and isolated guinea pig heart experiments [34,35]. Specifically, simulations and experiments showed that expanding the intercellular cleft prolonged APD and promoted EAD formation. Further, cleft narrowing promoted localized depletion of [Na+]cleft, which reduced the late Na+ current at the ID, mitigated APD prolongation, and suppressed EADs. Notably, the “concealing” effects of the Na+ channel GOF manifestation required preferential localization of the Na+ channels at the ID, while altered gap junctional (GJ) coupling minimally altered APD prolongation [34].
Importantly, several properties related to the self-attenuation mechanism are known to change with age: Na+ channel and GJ expression are both age-dependent. Neonatal cells have immature, i.e. not fully formed, IDs, and Na+ channels and GJs are primarily diffusively distributed throughout the sarcolemma. Cai et al. and Cordeiro et al. both report that pediatric cardiomyocytes produce less Na+ current than adult cardiomyocytes [37,38], consistent with reduced Na+ current expression. Additionally, Na+ channel and GJ distribution change significantly with age. Studies found that the primary ventricular GJ protein, connexin 43 (Cx43), remains almost undetectable until 23 weeks in utero and that GJs are sporadically distributed on the sarcolemma in neonatal cardiomyocytes [[39], [40], [41], [42]]. Vreeker et al. noted that Cx43 gradually lateralizes at 5 months postnatal and begins to preferentially localize at the ID around 2.5 to 5 years old, with full preferential localization occurring around 7 years of age [39]. Similarly, NaV1.5 channels are primarily on the lateral membrane in neonatal cells, and begin to preferentially localize at the ID around 5 months postnatal [39]. Thus, Na+ channels are primarily localized at the ID at an earlier developmental stage than GJs [39,43]. Additionally, cell size, which broadly influences all electrical activity by altering cell volume, surface area, ion channel expression, membrane capacitance, etc., also increases with age, as studies have shown that adult cardiomyocytes are larger than neonatal cardiomyocytes [38,39,44,45].
Based on this proposed mechanism for concealment of the LQT3 phenotype, we hypothesize that the age-associated manifestation of LQT3 may be directly linked to changes in cellular and tissue properties that occur with development. In this paper, we perform a wide parameter investigation, varying age-associated parameters including Na+ channel localization at the ID (IDNa), cell size (S), Na+ channel density (ρNa), and gap junctional conductance (fgap), and measure APD in simulated guinea pig cardiac tissue with LQT3-associated Na+ channel GOF.
To our knowledge, only one study has investigated the relationship between intercellular cleft width and age in any setting; our prior work found a positive correlation between intercellular cleft width and age in atrial tissue in atrial fibrillation and control patients later in life (approximately 40–80 years old) [46]. However, no studies have investigated intercellular cleft width in ventricular tissue in either the setting of control or LQT3 tissue and the early developmental changes, i.e., neonatal to adult, relevant to the LQT3 concealment hypothesis. Thus, in our study, we investigate a range for intercellular cleft width values consistent with our prior measurements [20,35]. In addition to cleft width, simulations predict that total Na+ current conductance is a critical factor in EAD formation. We find that for early development stages, EADs are suppressed; however, the LQT3 phenotype can be “unmasked” by multiple possible structural or cellular changes, including increases in cell size and Na+ channel density, with minimal dependence on cleft width. In contrast, for adult stages, EADs form for all conditions, unless suppressed by narrow cleft width, consistent with our prior studies in adult myocardium [34].
Section snippets
Methods
Full details of the computational model are provided in Supporting Material. Briefly, we simulate a 50-cell cable of guinea pig ventricular myocytes [47], incorporating a model of either a wild-type (WT) or LQT3-associated mutated (Y1795C) Na+ channel [48] as seen in Fig. 1. Na+ channel dynamics were governed by a 13-state Markov model [48] previously shown to produce a late Na+ current in the mutant channel, with channel states representing two modes of gating: a baseline background gating
Mechanisms of EAD formation in mutant cardiac tissue
We simulate cardiac tissue comprised of myocytes with mutant (Y1795C) Na+ channels and modulate cell size (S), Na+ channel density (ρNa), Na+ channel localization at the ID (IDNa), and intercellular cleft width (w). Motivated by our previous findings that intercellular cleft width is a key regulator of APD changes [34,35] and our hypothesis that cell size is a critical factor in age-dependent regulation, we first investigate the mechanism for APD modulation via intercellular cleft width (w) and
Discussion
In this study, we demonstrate that in guinea pig cardiac tissue model with a Na+ channel GOF, age-associated increases of total Na+ current conductance are a critical factor governing APD prolongation and EAD formation, in addition to previously identified regulation by intercellular cleft width and Na+ channel localization [34,35]. However, regulation by total Na+ current conductance is complex and depends on both cell size and Na+ channel density, such that simulations predict that increases
Disclosures
None.
Acknowledgements
This study was supported by funding from the National Institutes of Health, grant numbers R01HL138003 (SHW, SP) and R01HL102298 (SP).
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