Activation of TRPC Channel Currents in Iron Overloaded Cardiac Myocytes

Abstract

Background -

Arrhythmias and heart failure are common cardiac complications leading to substantial morbidity and mortality in patients with hemochromatosis, yet mechanistic insights remain incomplete. We investigated the effects of iron (Fe) on electrophysiological properties and intracellular Ca²⁺ (Ca²⁺_i) handling in mouse left ventricular cardiomyocytes.

Methods –

Cardiomyocytes were isolated from the left ventricle of mouse hearts and were superfused with Fe³⁺/8-hydroxyquinoline complex (5-100 μM). Membrane potential and ionic currents including transient receptor potential canonical (TRPC) was recorded using the patch-clamp technique. Ca²⁺_i was evaluated by using Fluo-4. Cell contraction was measured with a video-based edge detection system. The role of TRPCs in the genesis of arrhythmias was also investigated by using a mathematical model of a mouse ventricular myocyte with the incorporation of the TRPC component.

Results -

We observed prolongation of the action potential (AP) duration and induction of early and delayed afterdepolarizations (EADs and DADs) in myocytes superfused with 15 μM Fe³⁺/8-hydroxyquinoline (8-HQ) complex. Iron treatment decreased the peak amplitude of the L-type Ca²⁺ current (I_Ca,L) and total K⁺ current (I_K), altered Ca²⁺_i dynamics, and decreased cell contractility. During the final phase of Fe treatment, sustained Ca²⁺_i waves (CaWs) and repolarization failure occurred and ventricular cells became unexcitable. Gadolinium abolished CaWs and restored the resting membrane potential (RMP) to the normal range. The involvement of TRPC activation was confirmed by I_TRPC recordings in the absence or presence of functional TRPC channel antibodies. Computer modeling captured the same AP and Ca²⁺_i dynamics and provided additional mechanistic insights.

Conclusions -

We conclude that iron overload induces cardiac dysfunction that is associated with TRPC channel activation and alterations in membrane potential and Ca²⁺_i dynamics.

Graphical Abstract

Introduction

Iron is essential for life due to its role in a wide range of physiological processes. Primary (hereditary) and secondary (acquired) hemochromatosis are diseases that disrupt the intricate homeostatic mechanism of systemic iron handling resulting in iron overload and oxidative damage to iron-deposited organs ¹. Iron overload cardiomyopathy is a leading cause of morbidity and mortality in patients with hemochromatosis ^{2, 3}.The siderotic (iron overloaded) heart exhibits not only contractile deterioration, but also rhythm disturbances. However, current mechanistic knowledge regarding iron-induced arrhythmogenesis is very limited as electrophysiological studies have demonstrated controversial effects of iron on ionic currents and voltage properties in cardiac myocytes ^{3, 4}.

Recently, a potentially important role for transient receptor potential canonical (TRPC) channels (subtypes 1, 3, and 6) in Ca²⁺ paradox injury and arrhythmogenesis has been reported ^{5, 6}. Inward current through these non-specific cation channels has been demonstrated to be triggered by sarcoplasmic reticulum (SR) Ca²⁺ depletion, a mechanism named store-operated Ca²⁺ entry (SOCE) ^6-8. Additionally, the involvement of TRPC channels in cardiac development ⁹, post-myocardial infarction remodeling ¹⁰, and diastolic Ca²⁺ overload mediated hypertrophic cardiomyopathy ¹¹ have also been studied. However, activation of TRPC channels and their contribution to arrhythmogenesis have never been investigated in iron overload conditions.

In the present study, we used a membrane permeable complex of Fe³⁺ and 8-hydroxyquinoline (8-HQ) and sought to determine the effects of iron overload on left ventricular cardiomyocytes in terms of: (1) cardiac action potential (AP) morphology and resting membrane potential (RMP), (2) transmembrane ionic currents, (3) alterations in the dynamics of intracellular calcium (Ca²⁺_i) handling, and (4) single-cell contractility. We hypothesized that iron overload could induce cellular repolarization abnormalities (i.e. a sustained depolarization of the RMP) and afterdepolarizations by altering the L-type Ca²⁺ current (I_Ca,L), total K⁺ current (I_K), and for the first time the TRPC channel current (I_TRPC) thereby resulting in abnormal calcium transients (CaTs) and calcium wave (CaW) formation with accompanying impairment of cellular contractility.

Methods

Care and use of the animals in all experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Rutgers University-New Jersey Medical School. All procedures conformed to the NIH Guide for the Care and Use of Laboratory Animals. The authors declare that all supporting data are available within the article.

Please refer to the On-line Supplemental Methods for additional details.

Cardiomyocyte isolation

Left ventricular myocytes were enzymatically isolated from hearts of wild-type C57BL/6 mice (male, 2-4 months) as previously described ^{12, 13}. Myocytes were stored at room temperature and used within 8 hours after isolation.

Iron loading and cytosolic iron measurement

To induce iron overload, a membrane-permeable ferric ion (Fe³⁺)/8-hydroxyquinoline (8-HQ) complex (15 μM) was added to the myocyte bathing solution. The level of cytosolic iron was evaluated by the iron-sensitive dye Phen Green SK (Invitrogen by Thermo Fisher Scientific, Grand Island, NY, USA).

Cellular electrophysiological recording

Whole-cell patch-clamp recordings were performed as we have previously described ^14-16. A description on AP and current recordings is available in the On-line Supplemental Methods.

Intracellular Ca²⁺measurement

Ventricular myocytes were incubated with 4 μm Fluo-4 AM (Invitrogen, Grand Island, NY, USA) for 30 min. After washing and de-esterification (30 min), the Ca²⁺ fluorescence was measured as previously described ^{14, 17}.

Measurement of single-cell shortening

Myocytes were placed in a heated chamber (37°C) on an inverted microscope and were subjected to 1-Hz field-pacing using a stimulator (Grass Instruments, West Warwick, Rhode Island, USA). Changes in cell length were monitored by a video-based edge detection system (Crescent Electronics, Sandy, UT, USA). Single-cell shortening was calculated as a percentage of shortening from the baseline cell length in the relaxed state.

Statistical analysis

Data were presented as mean ± SEM. The Shapiro-Wilk test was used to determine the normality of data distribution. All data except for the incidence of EADs were normally distributed. For the normally distributed data, comparisons between dependent groups were made by two-tailed paired-sample t-test. One-way ANOVA followed by Tukey post-hoc test was used to compare three independent groups. For the non-normally distributed data, the Wilcoxon signed-rank test was used to compare two dependent groups, whereas the Kruskal–Wallis test followed by pairwise Mann–Whitney test with Bonferroni correction was used to compare three independent groups. P< 0.05 was considered statistically significant. All statistical analyses were performed by using the IBM SPSS Statistics 20.0 software (SPSS Inc, Chicago, IL).

Computer model

The mathematical model of a mouse ventricular myocyte used in this study was based on our previous work ¹⁸ where a spatial network of Ca²⁺ release units (CRUs) was incorporated with a full set of membrane ionic currents of mouse ventricular myocytes ¹⁹. To investigate the role of TRPC channels in the genesis of arrhythmias, we incorporated a recently developed TRPC model ²⁰ into our cell model. The formulation of I_TRPC is

$$I_{TRPC}=g_{TRPC}\cdot z_{\infty }([C{a}^{2+}{]}_i)\cdot (V-E_0),$$

where

$$z_{\infty }([C{a}^{2+}{]}_i)={\left[1+e^{-\frac{[C{a}^{2+}{]}_i-C{a}_{half}}{C{a}_{slope}}}\right]}^{-1},$$

E₀ is the Nernst potential and g_TRPC is the conductance of TRPC channels.

All computer programs were coded in CUDA C with single precision. Simulations were carried out on a high throughput computation cluster consisting of Nvidia GeForce GTX 1080 Ti cards.

Results

Cardiac dysfunction under iron overload by means of Fe³⁺/8-HQ complex perfusion

To validate our iron loading protocol, mouse ventricular myocytes were pretreated with the iron-sensitive dye Phen Green SK and then were subjected to continuous superfusion with Tyrode's solution containing 15 μM Fe³⁺/8-HQ, a lipophilic molecular complex capable of rapid membrane permeation. It has been reasoned that Fe³⁺enters the cytoplasm thereby increasing the total amount of intracellular free iron and thus expands the labile iron pool. Various oxidase and reductase enzymes can then catalyze the inter-conversion between Fe³⁺ and Fe^{2+ 21}. Fe²⁺ binds to Phen Green SK quenching the intensity of its fluorescence. As shown in Fig. 1A, a & b, treatment with 15 μM Fe³⁺/8-HQ decreased Phen Green SK fluorescence from 100% at baseline to 41.9 ± 6.8 % (p < 0.05, n = 5) indicating an increase in intracellular Fe²⁺ level. This effect could be reversed by subsequent administration of 5 mM 2,2'-bipyridyl (BPD), a membrane-permeable iron chelator, which increased Phen Green SK fluorescence to 118.4 ± 16.9% at 15 min post perfusion suggesting chelation of basal iron as well (p < 0.01 vs. 15 μM Fe³⁺/8-HQ, n = 5).

Figure 1.

Evaluation of iron overload and its effects on Ca transients (CaTs) and cell contractility. (A) Cytosolic iron loading was achieved by continuous superfusion with 15 μM Fe³⁺/8-HQ. Decreased Phen Green SK fluorescence intensity indicated increased intracellular iron levels. Note that the quenched fluorescence was reversed in the presence of the membrane-permeable iron chelator BPD (*p < 0.05 and **p < 0.01 by paired sample t-test, n = 6 cells). (B) Iron overload resulted in multiphasic alterations of Ca²⁺_i dynamics, as shown in two representative traces (a & b), with elevated diastolic Ca²⁺_i (c), reduced calcium transient (CaT) amplitude (d) (*p < 0.05 by paired sample t-test, n = 8 cells), and induced calcium waves (CaWs) (e) (*p < 0.05 by Fisher’s exact test, the number of cells as indicated on the bars). (C) SR Ca²⁺ content evaluation by using 10 mM caffeine (Caff)-induced transient in baseline (a) and when sustained diastolic Ca²⁺_i elevation occurred after Fe³⁺/8-HQ treatment (b), and summarized data in (c). Note the cells were briefly exposed to 1 mM tetracaine (TTC) to show the basal diastolic Ca²⁺ level (i.e. F₀). *p < 0.05 by paired sample by paired sample t-test, n = 16. (D) Reduced single-cell contractility after Fe³⁺/8-HQ treatment (b vs. control a, summarized in d) (*p < 0.05 by paired sample t-test, n = 5 cells) with episodes of irregular contractions (c).

Next, we assessed how Ca²⁺_i handling was altered under iron overload conditions. As shown in the two representative traces (Fig. 1Ba, b) and the summarized data from 8 myocytes (Fig. 1Bc-e), the iron overloaded cells demonstrated a multiphasic abnormality of Ca²⁺_i dynamics upon Fe³⁺/8-HQ superfusion. Upon an early stage (approximately 4-6 mins) post-Fe³⁺/8-HQ superfusion there was a slight but significant increase in diastolic Ca²⁺_i (from 1.00 to 1.04 ± 0.02, p < 0.05) (Fig. 1Ba-c) while no significant change in CaT amplitudes (Fig. 1Ba, b, d) were observed during early perfusion. Later, either short episodes of CaWs (Fig. 1Ba) or sustained CaWs (Fig. 1Bb) occurred in all cells superfused with Fe³⁺/8-HQ (100% incidence) (Fig. 1Be). Although we observed that CaT amplitudes were transiently increased after the cessation of the short bursts of CaW (as indicated by the arrows in Fig. 1Ba) after these episodes iron-loaded cells exhibited further increases in diastolic Ca²⁺_i (from 1.00 to 1.45 ± 0.09, p < 0.05) which was associated with markedly decreased CaT amplitudes (F/F₀ = 1.92 ± 0.11 at baseline vs. 1.24 ± 0.11 in the final phase (~30 sec before complete loss of CaT) , p < 0.05) (Fig. 1Ba, b, d). As shown in Fig. 1C, we also evaluated SR Ca²⁺ content by rapidly exposing myocytes to 10 mM caffeine. SR Ca²⁺ content was reduced after sustained treatment with 15 μM Fe³⁺/8-HQ (baseline F/F₀ 4.03 ± 0.16 vs. Fe³⁺/8-HQ treatment 2.96 ± 0.18, p < 0.05, n = 16).

To link changes in Ca²⁺_i handling to cardiac contractile function, we also evaluated the effect of iron overload on single-cell shortening. Consistent with the observed decrease in CaT amplitude, single cell contractility (shortening) was decreased in iron overloaded ventricular myocytes (5.92 ± 0.59% in vehicle vs. 4.01 ± 1.55% in 15 μM Fe³⁺/8-HQ, p < 0.05, n = 5; Fig. 1Da-b & d). Episodes of irregular contractions, which were associated with the occurrence of CaWs, were also observed in all cells as demonstrated in Fig. 1Dc.

Iron overload induced afterdepolarizations, prolonged action potential duration, and repolarization failure

In order to further understand the cellular basis for the aforementioned dysfunction in cardiac contractility and rhythm, we next performed whole-cell patch clamp experiments. Fig. 2A shows representative continuous AP recordings (with expanded regions labeled a-d) from a myocyte superfused with Tyrode's solution (Fig. 2Aa, baseline), 15μM 8-HQ (Fig. 2Ab, vehicle), and 15 μM Fe³⁺/8-HQ (Fig. 2Ac, d). While regular APs with normal morphology were elicited during the baseline condition and with 15 μM 8-HQ superfusion (Fig. 2Aa & b, respectively), during 15 μM Fe³⁺/8-HQ superfusion, however, afterdepolarizations and triggered activities were noticed occasionally in the early phase (Fig. 2Ac) and their occurrence increased in frequency in the late phase (Fig. 2Ad). We have counted an event as an early afterdepolarization (EAD) if occurring in phase 2 or 3 of the AP, and labeled an event as a delayed afterdepolarization (DAD) if occurring in phase 4, although we did not test the rate dependency properties of the observed afterdepolarizations ²². Fig. 2B and 2C summarize the incidence of EADs and DADs, as well as accompanying triggered activities (TAs). Compared to baseline, both types of afterdepolarizations were increased by 15 μM Fe³⁺/8-HQ superfusion (EAD 0.00 ± 0.00% vs. 45.00 ± 14.96% and DAD 4.33 ± 1.35% vs. 27.00 ± 7.04%, p < 0.05, n = 5).

Figure 2.

Electrophysiological effect of iron overload on action potential (AP) morphology and generation of EADs, DADs, and TAs. (A) A representative continuous AP recording obtained from a ventricular myocyte successively superfused with normal Tyrode's solution (a), 15 μM 8-HQ (b) and 15 μM Fe³⁺/8-HQ (c and d). Expanded traces in the boxed areas (a-d) are shown beneath. EADs, DADs, and TAs in c & d are indicated by the symbols "◆", "◼", and "●" respectively. (B & C) Summarized data of EADs/TAs and DADs/TAs incidence in iron-loaded myocytes (*p < 0.05 by Wilcoxon signed rank test and paired sample t-test in panels B and C, respectively, n = 5 cells). (D) Representative AP traces demonstrating effect of iron overload on AP morphology. (E-I) Summarized data demonstrating effect of iron overload on AP amplitude, resting membrane potential (RMP), and action potential duration at 30/50/90% repolarization (APD₃₀, APD₅₀, and APD₉₀) (*p < 0.05 by paired sample t-test, n = 5 cells). Note the parameters in E-I were measured during the 1-min window period prior to the onset of sustained depolarization state

As shown in the late phase of Fig. 2A, all cells eventually failed to repolarize to the original RMP (−68.9 ± 0.9 mV). Instead, the cells remained at a depolarized membrane potential which was much less negative (−25.0 ± 3.7 mV). At this depolarized secondary RMP, the cells lost excitability. Only stimulation artifact spikes were noted.

In addition, we further analyzed the effect of iron treatment on AP morphology as presented in Fig. 2D-I. AP amplitude (Fig. 2E), RMP (Fig. 2F), or AP duration (APD) at 30%, 50%, and 90% repolarization (APD₃₀, APD₅₀, and APD₉₀) (Fig. 2, G-I) were evaluated during a 1-min time window prior to the cell entering a sustained depolarized state. As shown in Fig. 2I, APD₉₀ was significantly prolonged compared to vehicle (50.2 ± 5.9 ms vs. 203.6 ± 63.4 ms, p < 0.05, n = 5), while other parameters (i.e. AP amplitude, RMP, APD₃₀, APD₅₀) remained unchanged.

Effect of iron overload on I_Ca,L, total I_K, and late I_Na in mouse ventricular myocytes

To determine the ionic basis for APD₉₀ prolongation, afterdepolarizations, and persistent depolarization of the RMP, we carried out more detailed voltage clamp experiments. I_Ca,L, total I_K, and late I_Na which are major determinants of APD in mouse ventricular myocytes were measured by whole-cell voltage clamp mode. As shown in Fig. 3A-C, cellular iron overload caused reduction of the peak I_Ca,L density (test voltage at 0 mV) over time (−16.5 ± 1.7 pA/pF in vehicle vs. −11.4 ± 1.3 pA/pF in 15 μM Fe³⁺/8-HQ; ~ 5 min after superfusion, p < 0.05, n=5). Meanwhile, it also reduced the peak I_K density at a test voltage of +60 mV over time (57.9 ± 3.1 pA/pF in vehicle vs. 50.4 ± 3.0 pA/pF in 15 μM Fe³⁺/8-HQ; ~ 5 min after perfusion, p < 0.05, n = 5) (Fig. 3D-F). Note that both I_Ca,L and I_K densities were decreased at various test potentials as can be seen in the current-voltage relationship curves (Fig. 3A&D). On the contrary, treatment with 15 μM Fe³⁺/8-HQ did not cause any alteration in late I_Na (Fig. 3G&H). The density of integrated late I_Na was −22.1 ± 6.3 pC/nF at baseline and −21.7 ± 4.5 pC/nF after superfusion with 15 μM Fe³⁺/8-HQ for ~ 5 min (no significant difference p = 0.93 by paired sample t-test, n = 8).

Figure 3.

Effects of iron overload on L-type Ca²⁺ current (I_Ca,L) and total K⁺ current (I_K). (A & D) Current-voltage (I-V) relationship of I_Ca,L and I_K (*p < 0.05 vs. baseline and ^#p < 0.05 vs. vehicle by paired sample t-test, n = 8 cells for I_Ca,L in A and 7 cells for I_K in D) with (B & E) representative tracings in the insets in baseline, vehicle (HQ 15 μM) and iron treatment (Fe/HQ 15 μM) for ~ 5 min. (C & F) Summarized data for peak I_Ca,L and I_K (*p < 0.05 by paired sample t-test, n = 8 cells for peak I_Ca,L in C and 7 cells for peak I_K in F). (G) Representative traces showing late Na current (I_Na at baseline and after superfusion with 15 μM Fe³⁺/8-HQ for ~ 5 min. (H) Summarized data for late I_Na integrated over the last 50 ms, and normalized to the cell capacitance (pC/nF) (NS: no significant difference by paired sample t-test, n = 8). Voltage clamp protocol for eliciting the current setting is indicated in the inset for panel B, E, and G, respectively.

Afterdepolarizations and repolarization failure in iron overloaded myocytes were suppressed by blocking Gd³⁺-sensitive TRPC1- and TRPC6-mediated inward current

In our recent work ⁶, we have revealed that activation of TRPC channels induces failure of cellular membrane repolarization, induces intracellular Ca²⁺ mishandling as well as EADs and DADs, all of which were ameliorated by the TRPC inhibitor Gd³⁺ and functional TRPC antibodies. To examine the role of I_TRPC in the pathophysiology of iron overloaded myocytes, we next examined the effects of Gd³⁺ on iron-treated cells and also measured the Gd³⁺-sensitive inward current. As shown in Fig. 4A & B, superfusion with 15 μM Fe³⁺/8-HQ resulted in persistent depolarization with loss of excitability of myocytes as described above. The original normal RMP (R₁) was shifted to a secondary depolarized level, R₂. The average values were R₁= −69.4 ± 0.4 mV and R₂ = −29.6 ± 4.8 mV (p < 0.01, n = 6). Application of 1 mM Gd³⁺ (in the presence of Fe³⁺/8-HQ) effectively abolished the persistent depolarized state causing the depolarized RMP to shift back to a normal RMP range (R₁-Gd = −59.8 ± 1.8 mV compared to R₂ = −29.6 ± 4.8 mV, p < 0.01), while the excitability of the myocytes was also restored. In addition, Fe³⁺/8-HQ treatment-induced APD prolongation was also restored by inhibiting TRPC channels with 1 mM Gd³⁺ (Fig. 4C & D).

Figure 4.

Involvement of TRPC current (I_TRPC) in iron-induced cardiac electrophysiological disturbance. (A) Membrane potential traces showing that normal RMP (R₁) was depolarized to R₂ after iron treatment. Gd³⁺superfusion caused RMP to shift from R₂ back to a more negative value, R₁-Gd. (B) Summarized data for R₁, R₂ and R₁-Gd (*p < 0.05 by paired sample t-test, n = 6). (C) Representative traces of the action potential at baseline, during 15 μM Fe³⁺/HQ superfusion, and after 1 mM Gd³⁺. (D) Summarized APD₉₀ values in the three groups (*p < 0.05 by paired sample t-test, n = 5). (E) Current-voltage relationship of the Gd³⁺-sensitive current (presumably I_TRPC), demonstrating that this current was increased by 15 μM Fe³⁺/8-HQ and suppressed by 1 mM Gd³⁺. (F) Summarized values of the inward currents at −100 mV (*p < 0.05 by paired sample t-test, n = 6). (G & H) Iron treatment had no effect on the Gd³⁺-sensitive inward current in myocytes pre-incubated with TRPC1 or TRPC6 functional antibodies, respectively (NS: no significant difference, n = 6). (I & J) Effect of TRPC1 and TRPC6 blockade on incidence of EADs/TAs (*p < 0.05 by Mann-Whitney test, n = 5) and DADs/TAs (NS, n = 5) in iron overload condition.

Furthermore, as shown in Fig. 4E & F, whole-cell voltage clamp recordings revealed an inward current that was increased by iron treatment (−1.35 ± 0.06 pA/pF in vehicle vs. −2.75 ± 0.34 pA/pF in 15 μM Fe³⁺/8-HQ, p < 0.05, n = 6). This inward current was sensitive to blockade by 1 mM Gd³⁺ (decreased to −1.27 ± 0.10 pA/pF in Gd³⁺, p < 0.05 vs. 15 μM Fe³⁺/8-HQ; n = 6). In contrast, in myocytes pre-incubated with TRPC1 or TRPC6 antibodies, iron treatment failed to activate this inward current (Fig. 4G & H). TRPC1 and TRPC6 antibodies also reduced the incidence of EADs and associated TAs from 45.0 ± 15% to 11.2 ± 2.3% and 7.0 ± 3.6%, respectively (p < 0.05, n = 5) (Fig. 4I), while the incidence of DADs and associated TAs were not significantly suppressed (Fig. 4J).

Failure of the cell to repolarize (persistent depolarization), which was the final phase of iron-induced voltage abnormality, was further investigated by simultaneous recording of the membrane potential and Ca²⁺_i handling (Fig. 5). When the normal RMP (R₁, Fig. 5Aa, baseline) was shifted to a depolarized level (R₂, Fig. 5Ba, with iron treatment), Ca²⁺_i accumulated and remained at a persistently elevated level and was associated with a complex Ca²⁺ handling behavior as shown by the pseudo-linescan images (Fig. 5C). In some cases at an early stage of iron treatment (e.g. in Fig. 5B & C), cells would spontaneously regain the original RMP and excitability (Fig. 5Ba), and the CaT amplitude was found to be increased immediately after cessation of the depolarized state (Fig. 5Bb, Cc; also see the arrows in Fig. 1Ba) indicating more Ca²⁺ had fluxed into the cell across the plasma membrane presumably via TRPCs. This resulted in greater SR Ca²⁺ uptake and release. In cells where spontaneous recovery of the RMP did not occur, neither nifedipine (Fig. 5 Da, b) nor SEA-400 (Fig. 5 Ea, b) suppressed the depolarized state in iron-loaded cells. However, application of 1 mM Gd³⁺ could effectively abolish the persistent depolarized state causing the depolarized RMP (R₂) to shift back to a normal RMP range (R₁-Gd) (Fig. 5 Ea). A transient increase in CaT amplitude was also observed in Gd³⁺-treated cells right at the time of recovery of the R₁ phase (Fig. 5Eb). This result suggests that the Ca²⁺ entry during this state was mediated by a Gd³⁺-sensitive current, most likely I_TRPC, but not I_Ca,L or I_NCX .

Figure 5.

Involvement of TRPCs in repolarization failure and Ca²⁺_i dysregulation in iron overloaded myocytes. (A & B) Simultaneous membrane potential (MP, a) and Ca²⁺_i recording (F/F₀, b) at the baseline (A) and after iron treatment (B). The line scan imaging in the indicated time windows are shown in panel C, a-c, respectively. (D-E) Simultaneous membrane potential (a) and Ca²⁺_i recordings (b) from cells treated with iron exhibiting sustained depolarization. Neither 10 μM nifedipine (Nif, an I_Ca,L inhibitor) (Da, b) nor 2 μM SEA-400 (an NCX inhibitor) (Ea, b) could suppress the sustained depolarization and the accompanying Ca²⁺_i elevation, while 1 mM Gd³⁺ could effectively suppress the sustained depolarization state induced by iron treatment (Ea) and accompanying Ca²⁺_i elevation (Eb).

Computer simulation study: Activation of TRPC channels induced afterdepolarizations and repolarization failure

To gain further mechanistic insights into the effect of TRPC activation on cardiac electrophysiology and arrhythmogenesis, we performed computer simulations using a mouse ventricular AP model with detailed spatiotemporal Ca²⁺ cycling. We incorporated a TRPC model into this AP model (see Methods). Fig. 6A shows a simulation in which the maximum conductance of TRPC channels (g_TRPC) was first set at zero (control) from t = 0 to t = 10 s, increased linearly from zero to 0.2 nS/pF from t = 10 s to t = 85 s, and then kept at a constant, i.e., g_TRPC = 0.2 nS/pF for the remainder of the time. During the ramping phase of increasing g_TRPC, Ca²⁺ entry via TRPC increased the Ca²⁺ load in the cell which led to DADs, triggered activity, as well as EADs. Once g_TRPC reached 0.073 nS/pF repolarization failure occurred. During the repolarization failure phase, blocking I_Ca,L (by 50%) had little effect on either membrane potential or Ca²⁺ transient amplitude. In another simulation shown in Fig. 6B, we blocked I_NCX by 50% in the repolarization failure phase. This had little effect on the RMP, but elevated diastolic Ca²⁺ levels. Blocking I_TRPC within 30 s recovered the cellular AP and diastolic Ca²⁺ back to control levels. These behaviors agree well with the experimental observations (Figs. 2, 4 & 5). To reveal the underlying mechanisms by which TRPCs can impact complex AP configurations and Ca²⁺ cycling dynamics, we performed additional computer simulations as described below.

Figure 6.

Induction of afterdepolarizations and repolarization failure by I_TRPC activation in a computer model of mouse ventricular myocytes. (A) Voltage and cytosolic Ca²⁺ traces under a protocol simulating the experimental data shown in Fig. 5D & E: g_TRPC = 0 for t = 0 to 10 s; from 10 s to 85 s, g_TRPC increased from 0 to 0.2 nS/pF; from t = 85 s, g_TRPC remained at 0.2 nS/pF. In the indicated time window, DADs (arrow), DAD-mediated triggered APs (*), EADs (filled circle), and repolarization failure occurred, agreeing with the experimental observations shown in Figs. 2, 4, and 5. DADs and the triggered APs are caused by spontaneous Ca²⁺ release induced Ca²⁺ waves as indicated by the line scan of Ca²⁺ (the lower panel). In the repolarization failure phase, cytosolic Ca²⁺remained high and Ca waves with fast oscillation frequencies occurred, agreeing with the experimental observations shown in Fig. 5C. In the constant g_TRPC phase, we blocked I_Ca,L by gradually reducing its maximum conductance from the control value to 50% and then gradually increasing it back to the control value as indicated. (B) Voltage and cytosolic Ca²⁺ traces under a protocol simulating the experimental data shown in Fig. 5E: g_TRPC = 0.2 nS/pF for t = 0 to 140 s, and gradually changed to 0 ns/pF from t = 140 s to 190 s. From t = 40 s to 90 s, we blocked I_NCX by gradually reducing its maximum activity to 50% control value and then gradually increasing it back to the control value as indicated. Note: In the computer model, NCX is the only mechanism to pump Ca²⁺ out of the cell. Therefore, the elevation of cytosolic Ca²⁺ appeared to be higher than the experimental recording when NCX was blocked.

Fig. 7A shows the relation between RMP and g_TRPC. When g_TRPC was small, the RMP was at ~ −80 mV, i.e. a normal RMP. When g_TRPC was large, the RMP became more positive, i.e. a more depolarized state occurs (~ −20 mV). In the middle range (region III in Fig.7A), the RMP could be either low or high. This is a typical bistable behavior widely seen in complex biological systems ^{23, 24}. The AP behaviors can be characterized in four regions. When g_TRPC was small (Region I in Fig. 7A), APs were normal (top panel, Fig. 7Ba). As g_TRPC increased (Region II in Fig. 7A), Ca²⁺ waves, DADs, DAD-triggered APs, and EADs occurred. In region III, bistability occurred. The middle two panels in Fig. 7B demonstrate two kinds of AP behaviors. The upper trace (Fig. 7Bb) shows complex AP behaviors, but the myocyte always repolarized to the normal RMP. However, when the membrane potential was clamped at a higher value, e.g., −10 mV for a certain time (~ 5 s) (Fig. 7Bc), the modeled cell failed to repolarize. Therefore, in this region depending on the initial conditions, such as different initial Ca²⁺ loads, the myocyte can exhibit two distinct behaviors. In region IV, repolarization failure occurred (bottom trace, Fig. 7Bd), which is independent of the initial conditions.

Figure 7.

Mechanistic insights into arrhythmogenic role of TRPCs from computer simulations. (A) Bistable resting membrane potential (RMP) caused by TRPC activation. Black dots are membrane potentials when g_TRPC increased from lower to higher values and red squares are membrane potentials when g_TRPC decreased from higher to lower values. As g_TRPC increased, the transition from the low RMP to the high RMP occurred at g_TRPC = 0.073 nS/pF (dashed black arrow), while as g_TRPC decreased the transition from the high RMP to the low RMP occurred at g_TRPC = 0.065 nS/pF(dashed red arrow), forming a hysteresis loop. Therefore, a bistability state existed between g_TRPC = 0.065 and 0.073 nS/pF where the cell could either exhibit the low or high (depolarized) RMP. (B) Different AP behaviors induced by TRPCs. (Ba) g_TRPC = 0 with normal APs. (Bb and c) g_TRPC = 0.07 nS/pF with the bistability state. In the first simulation (Bb), DADs, triggered APs and EADs occurred during pacing and after pacing stopped, but the APs always repolarized to the low RMP. (Bc) Starting from the same initial conditions as in Bb, the voltage was then clamped at −10 mV for 5 s as indicated and repolarization failure occurred (red line). (Bd) g_TRPC = 0.08 nS/pF predicted repolarization failure. (C) Membrane potential (Voltage), cytosolic Ca²⁺, and SR Ca²⁺ following a sudden turnoff of I_TRPC. Note different Ca²⁺ levels at arrows 1-3 in Cc. (D) Effect of I_TRPC on voltage dynamics under three cases: (Da) typical I_TRPC (electrogenic I_TRPC with Ca²⁺ as a charge carrier, g_TRPC = 0.074 nS/pF; (Db) I_TRPC was set to only bring Ca²⁺ into the cell, but is non-electrogenic; (Dc) I_TRPC was set to only be electrogenic, but do not bring Ca²⁺ into the cell.

Fig. 7C shows a simulation in which I_TRPC were suddenly turned off and the effect on SR Ca²⁺ dynamics. When I_TRPC was still on, the voltage was at a depolarized RMP and cytosolic Ca²⁺ remained persistently at a much higher level than the normal resting diastolic Ca²⁺ level (Fig. 7Cb). However, the SR Ca²⁺ load showed a much lower level (arrow 1) than the control SR load (arrow 3) (Fig. 7Cc). This is because high cytosolic Ca²⁺ results in a high open probability for RyR via Ca²⁺ induced Ca²⁺ release (CICR) which reduces the Ca²⁺ content in the SR. After I_TRPC was turned off, the membrane potential immediately repolarized to the normal RMP and the SR Ca²⁺ load was transiently increased to a level (arrow 2) higher than the control level (arrow 3). This resulted in a much larger Ca²⁺ transient following the turning off of I_TRPC. After several beats, the amplitude of Ca²⁺ transient reached a lower steady-state level. The change in Ca²⁺ transient amplitude after blocking I_TRPC is a result of a dynamic rebalance of Ca²⁺ in the SR and cytosol.

Since the opening of TRPCs results in a non-selective inward current under negative potentials and brings Ca²⁺ into the cytosol, it is not surprising that the inward current would promote EADs and the increased cytosolic Ca²⁺ would promote DADs and DAD-mediated triggered APs. Since some TRPCs (e.g., TPPC4 and 5) are Ca²⁺-activated ^{25, 26}, I_TRPC and Ca²⁺ form a positive feedback loop that may play a vital role in promoting complex AP dynamics. In Fig. 7Da, we show a typical simulation recording where TRPCs were activated and DADs, triggered APs, and EADs were all exhibited. In Fig. 7Db, TRPCs were set to be non-electrogenic, but only to bring Ca²⁺ into the cytosol. In this case, the APs were almost normal although small-amplitude DADs occurred without triggering APs. In Fig. 7Dc, TRPCs were set to be only electrogenic, but without bringing Ca²⁺ into the cytosol. In this case, the APs were slightly prolonged compared to the normal control (Fig. 7Ba), but no EADs occurred. These simulations demonstrate that the positive feedback between I_TRPC and Ca²⁺ is a key to the genesis of complex AP dynamics.

Discussion

In order to understand the molecular/ionic mechanism(s), electrophysiological studies on the effects of iron have been conducted by numerous investigators. However, the results remain controversial thus far ²⁷. One reason for such controversies may be due to the administration of different forms of iron, such as ferrous chloride (FeCl₂), ferric ammonium citrate (FAC), or iron dextran (chronic injection). In addition, whether Fe²⁺ is taken up efficiently into myocytes remains questionable given that only a limited iron current via L-type Ca²⁺ channels (LTCCs) has been observed even in the presence of a very high iron concentration (15 mM) ²⁸. In the present study, we exclusively used Fe³⁺/8-HQ, which is a membrane-permeable complex. After this lipophilic Fe³⁺/8-HQ complex enters into the cell, Fe³⁺ undergoes rapid intracellular reduction to Fe^{2 +21}. Cytosolic Fe³⁺ and Fe²⁺can then become part of the pool of labile iron within the cell, i.e. chelatable and redox-active ²⁹. The major findings of the present study are: 1) Intracellular iron overload induced afterdepolarizations (EADs and DADs) and eventually a secondary depolarized RMP. 2) Activation of TRPC channel currents plays an important role in causing the failure in membrane repolarization. 3) Subsequent abnormal Ca²⁺_i handling such as enhanced diastolic Ca²⁺ level, reduced CaT amplitude, and the occurrence of CaWs accompanied impaired cellular contractility. 4) Computer simulation which included a TRPC component for the first time provided supportive evidence for our findings and data interpretation.

Proarrhythmic effect of iron overload at the cellular level

Arrhythmias and heart failure are well-documented cardiac complications under iron overload conditions (see ² and ³ for clinical reviews). Clinical entities of arrhythmias found in siderotic patients are heterogeneous, ranging from sinoatrial node dysfunction and various types of conduction block to lethal tachyarrhythmias ⁴. This heterogeneity suggests that multiple patho-electrophysiological processes exist at different structural levels, i.e. from iron-laden subcellular compartments to the remodeled failing heart with inter-organ interactions. However, current insight even at the most fundamental levels is still far from being comprehensive. The present study focuses on mechanistic investigation of iron-induced arrhythmogenicity at the molecular and cellular levels. It was revealed that APD prolongation and afterdepolarizations/triggered activities occurred in iron overloaded ventricular myocytes. Therefore, to determine the underlying ionic mechanism, we further assessed major transsarcolemmal ionic currents that are physiologically activated during cellular repolarization, i.e. I_Ca,L, total I_K, and late I_Na

Interestingly, both I_Ca,L and I_K were found to be decreased with iron overload. Increased transient outward K⁺ current (I_to) has been reported in cultured neonatal rat ventricular myocytes subjected to 24–72 hours incubation with FAC (40–80 μg Fe/ml), but the explanation regarding the mechanism by which iron potentiates I_to is still lacking ³⁰. Also, the effect of iron on delayed rectifier K⁺ currents (I_Ks and I_Kr) which are abundant in human hearts, but absent in mice ³¹, are not known. It should be pointed out that we measured total I_K and did not distinguish its components. There are at least three time- and voltage-dependent outward K⁺ current components in the mouse heart that impact AP repolarization. These include: 1) a Ca²⁺-independent transient outward K⁺ current (I_to), 2) an ultra-rapid delayed rectifier K⁺ current (I_Kur), and 3) a slowly activating and non-inactivating (steady-state) outward K⁺ current (I_ss) ³². Although Fe³⁺/8-HQ treatment decreased the total I_K by a small extent, it would be interesting to determine which potassium currents were decreased by iron overload in future studies.

Decreased outward I_K could at least partially be responsible for the observed prolongation in APD. However, the concurrent decrease in inward I_Ca,L could produce a counteracting effect, i.e. blunting the APD-prolonging effect of decreased I_K. In addition, we did not observe any changes in late I_Na. Because we could observe markedly prolonged APD and frequent EADs in iron overloaded ventricular myocytes, other inward currents may contribute to this obvious repolarization defect. Moreover, the increased incidence of DADs and CaWs indicates that intracellular Ca²⁺ overload occurred in iron overloaded ventricular myocytes. We therefore proposed that Ca²⁺ could be the charge carrier of the inward currents noted, which we assumed to be mediated by TRPC channels.

A previous study has reported iron-induced I_Ca,L attenuation ²⁸. The chemical form of iron used in that study was Fe²⁺ co-administered with ascorbic acid. Competitive permeation between Ca²⁺ and Fe²⁺ through LTCCs was suggested to be the cause of the reduced I_Ca,L ²⁸. In contrast, the membrane-permeable Fe³⁺/HQ complex used in our study, due to its larger molecular radius and lipophilic property, is unlikely to be capable of permeating through the aqueous pores of LTCCs. Thus, other mechanism(s) of iron-induced I_Ca,L suppression may exist. It is still unproven whether cytosolic iron can bind to and inhibit LTCC in a manner similar to Ca²⁺-induced inactivation. Oxidative damage of LTCCs due to iron-catalyzed reactive oxygen species (ROS) production, also known as Fenton reaction, is another potential cause of impaired I_Ca,L ^{33, 34}. CaMKII oxidation/activation-induced activation of I_Ca,L ¹⁴ seemed not likely to be involved.

Roles of TRPC channels in iron-induced cell dysfunction and arrhythmogenesis

TRPC channels (subtypes 1 and 3-7) are non-selective cation channels expressed in the heart ³⁵. Ionic current through cardiac TRPC6 channels has been recorded ³⁶ and together with TRPC1 and TRPC3 has been shown to mediate Ca²⁺ entry into ventricular myocytes ⁶. Hyperforin-induced TRPC6 channel hyperactivation is arrhythmogenic ⁶. Furthermore, anti-arrhythmic effects of Gd³⁺, a nonspecific TRP channel inhibitor, has been demonstrated in a stretch-induced atrial fibrillation model ³⁷. Redox regulation of channels in the TRPC family has also been reported ³⁸. Thus, abnormal activation of TRPC channels may occur and contribute to arrhythmogenesis in ventricular myocytes suffering from iron-induced oxidative injury. Several studies including our recent paper ^39-41 have revealed that Fe treatment increases cellular and/or mitochondrial ROS, which likely play an essential role in TRPC activation. Future comprehensive studies that aim to elucidate the detailed mechanisms for TRPC channel activation and Ca²⁺_i mishandling in iron overload cardiomyocytes are warranted.

Our present study provides convincing evidence of I_TRPC involvement in iron-induced arrhythmogenesis. Cellular iron overload increased a Gd³⁺-sensitive inward current in ventricular myocytes. In addition, using more specific inhibitors of TRPC1 and TRPC6 which are pore-blocking antibodies, the inward current as well as iron-induced EADs were suppressed. Furthermore, the persistent depolarized state (secondary RMP) in iron overloaded cells was effectively suppressed by Gd³⁺, but not by I_Ca,L blockade (nifedipine) or I_NCX inhibition (SEA-400). It has been shown that certain pathological conditions can activate various sustained inward currents, which together with the inward rectification property of the background inward rectifier potassium current (I_K1) can generate two possible values for RMP on an N-shaped current-voltage relationship ⁴². The sustained depolarization phase found in iron overloaded cells could be a manifestation of this bistable behavior of the RMP and its sensitivity to suppression by Gd³⁺ suggests that I_TRPC could be the underlying cause of this phenomenon.

Iron overload induces abnormal Ca²⁺_i dynamics and contractile dysfunction

Ca²⁺ is a key mediator of cardiac excitation-contraction coupling ⁴³ via CICR and Ca²⁺ binding to troponin complexes followed by turning on of the contractile function of cardiac myofilaments. Thus, systolic [Ca²⁺]_i and Ca²⁺-troponin C affinity determine the strength of myocardial contraction ⁴³. To allow cardiac relaxation, low diastolic [Ca²⁺]_i is achieved by Ca²⁺re-sequestration into the SR via the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) and Ca²⁺ extrusion to the extracellular space via the Na⁺-Ca²⁺ exchanger (NCX) and sarcolemmal Ca²⁺ ATPase. Other minor mechanisms of cytosolic Ca²⁺ removal, for instance, Ca²⁺ uptake into mitochondria, also exist ⁴³. Siderotic hearts clinically exhibit both diastolic and systolic dysfunction ^{2, 3}, but effects of iron overload on cardiac Ca²⁺ cycling remains incompletely understood ^{44, 45}.

Our present study demonstrated that iron overload caused aberrant Ca²⁺ handling in ventricular myocytes. Increased diastolic [Ca²⁺]_i preceded CaW generation and further sustained elevation in [Ca²⁺]_i with decreased CaT amplitudes then occurred. Potential mechanisms responsible for the elevated diastolic [Ca²⁺]_i may include iron-induced inhibition of SERCA ^{40, 46} and/or inhibition of the sarcolemmal Ca²⁺ ATPase ⁴⁷ as well as impaired Ca²⁺ extrusion through the NCX ⁴⁸. Decreased CaT amplitude has been demonstrated previously in rats subjected to chronic iron overload ⁴⁹. In our acute study, iron overloaded ventricular myocytes showed a consistent finding (decreased CaT amplitude) during the final phase of [Ca²⁺]_i alteration. This could be the result of attenuated CICR due to competitive binding of Fe²⁺ at the Ca²⁺-binding site of RyRs as previously reported ⁵⁰ as well as a decrease in I_Ca,L. Consistent with the observed decrease in the CaT amplitude in the late phase of Fe³⁺/8-HQ treatment, we found reduced SR Ca²⁺ content (Fig. 1 C) which may be caused by extra Ca²⁺ efflux from the SR as a result of the formation of Ca waves⁵¹ and/or a potential inhibition of Ca²⁺ uptake by SERCA^{40, 46}. Functionally, these abnormal features of [Ca²⁺]_i regulation resulted in decreased cellular contractility and irregular contractions.

Interestingly, the reversible abnormality in Ca²⁺ handling and failure in the RMP to repolarize and loss of excitability caused by iron overload are reminiscent of myocardial “stunning”, a status of transient reversible myocardial contractile dysfunction often induced by acute ischemia ⁵². Multiple mechanisms including ROS generation, SR dysfunction, and Ca²⁺ overload have been proposed to account for myocardial stunning ⁵². Our present study suggests that activation of TRPC channels and subsequent elevation of diastolic [Ca²⁺]_i and repolarization failure likely play important roles in causing myocardial stunning. This notion is supported by the recent observation that the mRNA level and activation of TRPC channels are increased after myocardial infarction ¹⁰.

Insights obtained from computer simulations

By using the computer simulation approach incorporating a TRPC component to our previously published ventricular AP model, we have obtained additional novel findings. TRPCs are non-selective cation channels thus their activation evokes not only Ca²⁺ influx that increases [Ca²⁺]_i, but also generates an inward current that depolarizes the cell membrane. Either of these two consequences by itself may account for the generation of EADs, DADs, and arrhythmias ^{14, 22, 53}. However, our computer simulation study (Fig. 7D) suggests a positive feedback between [Ca²⁺]_i and this inward current that plays a key role in generating the complex AP dynamics (including repolarization failure). This feedback loop seems to be both necessary and possible since it has been shown that certain subtypes of TRPCs (e.g. TRPC4 and 5) are activated by intracellular Ca^{2+ 25, 26}. Furthermore, functional interactions between TRPC and other Ca²⁺-permeable channels (e.g. LTCC) ⁵⁴ need to be taken into account under various pathophysiological conditions.

One limitation of our experimental study is that one cannot monitor SR Ca²⁺ content in a dynamic manner. However, we took advantage of our mathematical model that incorporates a spatial network of Ca²⁺ release units (CRUs) and used it to analyze cytosolic and SR Ca²⁺ behaviors during TRPC activation by Fe³⁺/8-HQ and especially during persistent cytosolic Ca²⁺ elevation (and simultaneous repolarization failure). We determined that the SR Ca²⁺ load was transiently increased by TRPC activation (Fig. 7C, SR Ca²⁺ was higher at arrow 2 than arrow 3 after I_TRPC was turned off) which is consistent with the overall increased cellular Ca²⁺ loading through TRPC entry and transiently enhanced CaT amplitude after repolarization was restored. It is also interesting to note that the SR Ca²⁺ content was lower when repolarization failure occurred (Fig. 7C, SR Ca²⁺ was lower at arrow 1 than arrow 2). Thus iron-induced TRPC activation causes cytosolic and SR Ca²⁺ mishandling, which is arrhythmogenic and may cause myocardial “stunning”.

Limitations and future research

Hemochromatosis is caused by increased deposition of Fe in parenchymal organs including the heart. Fe overload cardiomyopathy is manifested as systolic or diastolic cardiac dysfunction and may lead to congestive heart failure. While atrial and ventricular tachyarrhythmias and even sudden cardiac death are often observed in patients with cardiac hemochromatosis ^{55, 56}, it is possible that severe cardiac arrhythmias arise secondarily from the dilated cardiomyopathic or heart failure state rather than directly from electrical alterations primarily or directly caused by the Fe overload state itself. Although the complete loss of excitability we observed under acute Fe treatment seems to represent a much more dramatic phenomenon than what is seen in many patients with hemochromatosis whose cardiac contractility may be reduced, our present findings have provided valuable mechanistic insights by highlighting the potential pathogenic role of TRPC activation in Fe overload-induced cardiac contractile dysfunction and arrhythmias.

A very recent study⁴⁰ has established a secondary iron overload cardiomyopathy model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) treated with 100 μM Fe²⁺ for 5 days. Although there is a discrepancy as to whether diastolic Ca²⁺ concentration is altered in their model, their findings are in agreement with our findings in adult mouse ventricular myocytes with regard to contractile dysfunction, oxidative stress, mitochondrial dysfunction, the incidence of EADs and arrhythmias, as well as the dysregulation of Ca kinetics ^{57, 41}. It should be noted that there are scientific concerns regarding the immaturity of iPSC-CMs, therefore, future comprehensive studies in Fe-overloaded human cardiomyocytes are warranted.

What Is Known?

• Iron overload cardiomyopathy occurs in patients with hemochromatosis.

• Arrhythmias and heart failure are complications leading to substantial morbidity and mortality in such patients, however, the underlying mechanisms are not well understood.

What the Study Adds?

• Iron overload activates transient receptor potential canonical (TRPC) channels in ventricular myocytes.

• Iron overload induces contractile dysfunction via alterations in membrane potential and Ca2+i dynamics reminiscent of myocardial stunning.

• Computer modeling provided further insights on Ca-voltage coupling mediated by TRPC activation.

Nonstandard Abbreviations and Acronyms

8-HQ

8-hydroxyquinoline

AP

action potential

APD

action potential duration

BPD

2,2'-bipyridyl

Ca²⁺_i

intracellular calcium ion

CaT

Ca²⁺_i transient

CaW

Ca²⁺_i wave

[Ca²⁺]_i

intracellular calcium ion concentration

CRU

Ca²⁺ release unit

DAD

delayed afterdepolarization

EAD

early afterdepolarization

Fe

iron

Gd³⁺

gadolinium

iPSC-CM

induced pluripotent stem cell-derived cardiomyocyte

I_Ca,L

L-type Ca²⁺ current

I_K

potassium current

I_Na

sodium current

I_TRPC

TRPC channel current

LTCC

L-type Ca²⁺ channel

NCX

Na⁺-Ca²⁺ exchanger

RMP

resting membrane potential

ROS

reactive oxygen species

SERCA

sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase

SOCE

store-operated Ca²⁺ entry

SR

sarcoplasmic reticulum

TRPC

transient receptor potential canonical