Recent studies have suggested that mitochondria may play important roles in the Ca2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca2+ flux can regulate the generation of Ca2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca2+ (Cai2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and CaWs were induced in the presence of high (4 mM) external Ca2+ (Cao2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai2+ levels even after depletion of SR Ca2+ in the absence of Cao2+ , suggesting Ca2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψm) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψm) or Ru360 (a mitochondrial Ca2+ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca2+ release and uptake exquisitely control the local Ca2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.
Citation: Zhao Z, Gordan R, Wen H, Fefelova N, Zang W-J, Xie L-H (2013) Modulation of Intracellular Calcium Waves and Triggered Activities by Mitochondrial Ca Flux in Mouse Cardiomyocytes. PLoS ONE 8(11): e80574. https://doi.org/10.1371/journal.pone.0080574
Editor: Vladimir E. Bondarenko, Georgia State University, United States of America
Received: February 26, 2013; Accepted: October 4, 2013; Published: November 7, 2013
Copyright: © 2013 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by National Institutes of Health (NHLBI R01 HL97979 to LHX) , National Natural Science Foundation of China (No. 81170597 to ZZ; No. 81120108002 to WJZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Multiple calcium ion (Ca2+) transporters/channels have been found in the inner membrane of mitochondria [1–3]. For example, cytosolic Ca2+ may be sequestered via the high-capacity mitochondrial Ca2+ uniporter (mCU) [4–6]. The driving force for Ca2+ uptake is created by the mitochondrial membrane potential (ΔΨm, ~ -180 mV more negative than the cytosol), which is generated by the extrusion of protons by the electron transport chain [2,7]. Conversely, mitochondrial Ca2+ efflux predominantly depends on the mitochondrial Na+-Ca2+ exchanger (mNCX). Under certain pathophysiological conditions, mitochondrial depolarization/dissipation causes the opening of mitochondrial permeability transition pores (mPTPs), which also act as an efflux pathway for Ca2+ and other small molecules (<1.5 KDa) . Although controversies still remain [9,10], it has been suggested that mitochondria play an important role in the regulation of intracellular/cytosolic Cai2+ homeostasis in cardiomyocytes under both normal and pathological conditions [11–14].
Previous work has shown that mitochondria and the endoplasmic or sarcoplasmic reticulum (ER or SR) may be physically associated through tethering structures [15,16]. In cardiac ventricular myocytes, the Ca2+ diffusion distance between the RyR clusters at the junctional SR and the ends of the mitochondria is approximately 50-100 nM , which may create a micro-domain for local Ca2+ and generate functional interaction between mitochondria and the SR . While some studies have shown mitochondrial Ca handling may affect the dynamics and magnitudes of Cai2+ oscillations in other cell types [19–22], it is not well understood how the mitochondria and the SR are functionally coupled with relation to the regulation of Cai2+ homeostasis and generation of Cai2+ waves (CaWs) and triggered activities (TAs) in cardiac ventricular myocytes.
In the present study, we aim to test whether mitochondrial Ca2+ fluxes can affect this putative micro-domain [Ca2+] and subsequently alter the Ca2+ handling behavior of the SR. In particular, the regulation of intracellular CaWs by mitochondrial Ca2+ flux under high intracellular Ca2+ conditions was investigated. We found that the protonophore carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) depolarizes Δψm and subsequently causes Ca2+ release from mPTPs, which promotes SR spontaneous Ca2+ release and CaWs. The exacerbation of CaWs by mitochondrial Ca2+ release is able to cause triggered activities, manifesting increased arrhythmogenesis during mitochondrial dysfunction.
Materials and Methods
All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Rutgers-New Jersey Medical School, and in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Mice of 2-4 months old (either sex) were used in this study. All experiments were performed at 35-37°C.
Ventricular myocytes were enzymatically isolated from mouse hearts as previously described . Briefly, the hearts were removed from mice anesthetized with pentobarbital ( 0.07 mg/g, i.v.), and were retrogradely perfused at 37°C in Langendorff fashion with nominally Ca2+-free Tyrode’s solution containing 0.5 mg/ml collagenase (Type II; Worthington) and 0.1 mg/ml protease (type XIV, Sigma) for 10 to 15 minutes. The enzyme solution was then washed out and the hearts were removed from the perfusion apparatus. The left ventricles were placed in petri dishes, and were then gently teased apart with forceps. Finally, the myocytes were filtered through nylon mesh (200 µm). The Ca2+ concentration was gradually increased to 1.0 mM, and the cells were stored at room temperature and used within 8 hours.
Detection of mitochondrial membrane potential, Δψm
Ventricular myocytes were loaded with tetramethylrhodamine methylester (TMRM, 100 nM), a lipophilic cation, for 40 min at room temperature. TMRM fluorescence was monitored with confocal fluorescence microscopy (Ex/Em: 540/580nm) and recorded using an Ixon Charge-Coupled Device (CCD) camera (Andor Technology). FCCP-induced depolarization of Δψm was assessed by the decline in TMRM fluorescence intensity.
Measurement of mPTP opening with calcein
Measurement of the mPTP opening was conducted as previously described [24,25]. Isolated myocytes were co-loaded with 1 μM calcein AM and 1 mM CoCl2 in normal Tyrode's solution at room temperature for 30 min. Loaded myocytes were washed free of calcein (CoCl2 was continuously perfused) just before imagining, and excited at 484 nm. Emitted fluorescence was acquired at 520 nm every 2 min. The percentage of calcein leak, estimated from the fluorescence decrease, was used as the index of mPTP opening.
Measurement of mitochondrial ROS production
Changes in mitochondrial superoxide production were monitored using MitoSOX Red (Invitrogen/Molecular Probes). Isolated cardiac myocytes were loaded with MitoSOX Red (5 μM) for 30 min at 37°C followed by washout. MitoSOX Red fluorescence (EX/EM: 485/585 nm) was monitored using a Nikon Eclipse TE200 inverted microscope and recorded using an Ixon Charge-Coupled Device camera (Andor Technology) operating at ~10 fps with a spatial resolution of 500×400 pixels. MitoSOX Red fluorescence is presented as background-subtracted F/F0 values. The F/F0 value was expressed as 0 during the periods when the cell was not exposed to the excitation light. The average values measured at 3 consecutive 200 ms-exposures were used to evaluate F/F0 values every 2 min. The baseline value (i.e. before perfusion of FCCP) was normalized to 1. The F/F0 level at 6 min after FCCP treatment was compared between different groups.
Estimation of intracellular ATP level ([ATP]i) changes
The changes of intracellular ATP levels were estimated indirectly by using a fluorescent Mg2+ indicator (Mag-fluo-4) , based on the assumption that ATP serves as the dominant buffer of Mg2+. Thus the free intracellular Mg2+ concentration ([Mg2+]i) changes reflect reciprocal changes of [ATP]i. Ventricular myocytes were loaded with Mag-fluo-4 AM (5 µM, Molecular Probes) for 20 min in Tyrode’s solution. Mag-fluo-4 fluorescence (EX/EM: 485/530 nm) was monitored using a Nikon Eclipse TE200 inverted microscope and recorded using an Ixon CCD camera (Andor Technology) operating at ~10 fps with a spatial resolution of 500×400 pixels. The fluorescence images (3 consecutive 200 ms-exposures) were obtained every 1 or 2 min in the absence and presence of 100 nM and 1 µM FCCP, respectively.
Single-cell Cai2+ measurements
Mouse ventricular myocytes were loaded with Fluo-4-AM by incubation in Tyrode’s solution containing 5 μm Fluo-4-AM for 40 min at room temperature. Cai2+ fluorescence (EX/EM: 485/530 nm) was monitored using a Nikon Eclipse TE200 inverted microscope and recorded using an Ixon Charge-Coupled Device camera (Andor Technology) operating at ~50 fps with a spatial resolution of 500×400 pixels, as described in previous studies [27,28]. The fluorescence intensity was measured as the ratio of fluorescence (F) over basal diastolic fluorescence (F0). Our previous report has characterized 3 major kinds of CaWs under Cai2+ overload condition . In the present study, we carried out our experiments in cells exhibiting slow CaWs under the control condition. CaW frequencies were quantified by counting the number of spontaneous CaWs (20% increases in F/F0) and were expressed as the number of waves/min.
Myocyte membrane potential recording using patch clamp
Myocytes were patch-clamped using the whole-cell configuration of the perforated patch-clamp technique (240 μg/ml amphotericin B). Patch pipettes (resistance = 1-3 MΩ) were filled with internal solution containing (in mM): 110 K-aspartate, 30 KCl, 5 NaCl, 10 HEPES, 0.1 EGTA, 5 MgATP, 5 Na2-phosphocreatine, 0.05 cAMP (pH was adjusted to 7.2 with KOH). The cells were superfused with Tyrode’s solution containing (in mM): 136 NaCl, 4.0 KCl, 0.33 Na2PO4, 4.0 CaCl2, 1 MgCl2, 10 glucose and 10 HEPES (pH was adjusted to 7.4 with NaOH). Cell membrane potential signals were measured using a MultiClamp 700A patch-clamp amplifier (Molecular Devices, Sunnyvale, Ca), controlled by a personal computer using a Digidata 1332 acquisition board driven by pCLAMP 10 software (Molecular Devices, Sunnyvale, CA). Acquisitions of cell membrane potential were carried out under the gap-free mode.
Chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, Mo) unless otherwise indicated. FCCP, , IAA, CsA, Ru360, oligomycin, and kaempferol were firstly dissolved in DMSO as stock solutions before diluting into the bath solutions at the final concentrations. Antimycin A stock solution was dissolved in 100% ethanol. The maximum DMSO or ethanol concentration was < 0.2% (vol/vol).
Effect of FCCP on Δψm and mPTP opening
The electron transport chain creates a large negative potential (Δψm: ~ -180 mV) across the mitochondrial inner membrane . By monitoring TMRM fluorescence in intact mouse ventricular myocytes, we confirmed that FCCP strongly depolarized the Δψm in a dose-dependent manner. As shown in Figure 1A, 10 μM FCCP caused maximal dissipation of Δψm with a time constant τ = 68.5 sec, while 1 μM FCCP caused partial depolarization. Next, we evaluated whether the depolarization of Δψm causes the opening of mPTPs. As shown in Figure 1B, FCCP (100 nM, 1, and 10 μM) dose-dependently accelerated the rate of calcein fluorescence decline, which was then attenuated by the mPTP inhibitor CsA (1 µM). These results suggest that the protonophore FCCP depolarizes Δψm and causes opening of mPTP, which may further affect mitochondrial Ca2+ fluxes.
A. TMRM fluorescence was monitored as an indicator of Δψm. FCCP (1 and 10 µM) was perfused as indicated by the horizontal bar. Two snapshots of TMRM fluorescence (control and ~2.5 min after the treatment with 10 µM FCCP) are shown. The fluorescence in the presence of 30 µM FCCP was set as 100% dissipation in each cell. B. Calcein fluorescence intensity was monitored as an indicator of mPTP opening. B-a. Two snapshots of calcein fluorescence at baseline (0 min) and 6 min after the treatment with FCCP (1 and 10 µM) and FCCP (1 µM) + CsA (1 µM). B-b. A representative recording of calcein fluorescence showing the rate of fluorescence decline in the presence of 1 and 10 µM FCCP. B-c. Summary data showing the calcein fluorescence decline in the presence of FCCP (100 nM, 1 and 10 µM) and CsA + FCCP as indicated. The fluorescence in the presence of 30 µM FCCP was set as 0% in each cell. *p < 0.05 vs. Control; #p < 0.05 vs. FCCP (1 µM).
Effect of FCCP on High [Ca2+]o-induced CaWs
We first tested whether opening mPTP by FCCP may alter Cai2+ handling during normal excitation contraction coupling (ECC). The cells were field-paced at 0.5 Hz under a normal Ca2+ concentration (1 mM). Spontaneous CaWs and triggered Ca transients were induced by perfusion with 100 nM FCCP in 3 out of 7 cells (one example is shown in Figure 2A).
A. FCCP (100nM) induced spontaneous CaWs (indicated by arrows) under normal excitation contraction coupling (ECC). The cells were paced by field stimulation at 0.5 Hz in the presence of 1 mM Ca2+ concentration under control (ctl) condition and ~ 5 min after perfusion with 100 nM FCCP. B. A Cai2+ fluorescence trace recorded from a mouse ventricular myocytes. The cell was first perfused with the normal Tyrode's solution (1 mM Ca2+) and then with a high Ca2+ Tyrode's solution (4 mM Ca2+). Cai2+ waves (CaWs) were consistently observed in the presence of high external Ca2+ (Cao2+; 4 mM). Spontaneous Ca2+ CaW were eliminated by Tetracaine (2 mM). C-a. A representative Cai2+ fluorescence trace showing the dose-dependent effects of FCCP on the SCWs. C-b. Effect of FCCP on CaW frequency in a dose-dependent and biphasic manner. C-c. Summary data showing the effect of FCCP on basal Cai2+. ∗p < 0.05,∗∗p < 0.01 vs. control (n = 6). D. Cyclosporin A (CsA), a mPTP inhibitor, significantly counteracted the effects of FCCP (100nM) on SCWs. A representative trace (D-a) and summarized data for CaW frequency (D-b) and basal [Cai2+] level (D-c) are shown. **p < 0.01 vs. Control; #P<0.05, ##p < 0.01 vs. FCCP, n = 8.
To investigate the potential regulatory effect of mitochondrial Ca2+ flux on SR Ca handling behaviors, we established a consistent CaWs model by elevating extracellular Ca2+ concentration ([Ca2+]o) of the Tyrode's solution to 4 mM in mouse ventricular cardiomyocytes. As shown in Figure 2B, consistent CaWs occurred 1-2 min after the perfusion solution was switched from 1 mM to 4 mM Ca2+. Tetracaine (2 mM), an allosteric ryanodine receptor (RyR) blocker, reversibly attenuated the CaWs. This result suggests that these CaWs originate from repetitive spontaneous Ca2+ release from the SR, while high [Ca2+]o functions as a promoting factor. This high [Ca2+]o-induced CaW model was used for the subsequent experiments.
We then examined the effect of FCCP (10 nM - 1 µM) on CaWs. A representative recording is shown in Figure 2C-a. We found that FCCP increased the frequency (Figure 2C-b) and basal intracellular Ca2+ level ([Cai2+]) ( Figure 2C-c) in a dose-dependent and biphasic manner. The effects of high dose FCCP (1 µM) on CaWs (inhibition) as well as on basal Ca (elevation) were washable. Furthermore, the effects of FCCP on CaWs and basal [Cai2+] were counteracted by the mPTP blocker cyclosporine A (CsA) (Figure 2D). The frequency of CaWs was accelerated from 23.3 ± 2.1 (control) to 57.6 ± 4.28 min-1 by 100 nM FCCP (p < 0.01, n=8), and was subsequently reduced to 27.9 ± 3.06 min-1 by 1 µM CsA (p < 0.01, n=8) (Figure 2 D-b). The basal [Cai2+] levels were elevated from F/F0 =1 to 1.26 ± 0.07 by 100 nM FCCP (p < 0.01), and were subsequently reversed by 1 µM CsA (F/F0:1.05 ± 0.02; p < 0.05, n=8) (Figure 2D-c). In order to exclude any off-target effects of CsA, we tested the effect of CsA by itself on the Ca waves under high [Ca2+]o. CsA alone did not affect CaW frequency (control: 16.4 ± 2.5 vs. CsA (1 µM): 17.3 ± 3.1 min-1 (n = 5, p > 0.05). These results suggest that depolarization of Δψm (by FCCP) may affect CaW behavior via mitochondrial Ca2+ release through mPTP opening.
During perfusion with 100 and 500 nM FCCP (Figure 2C-a & D-a), high-amplitude, spike-like Ca2+ transients were caused, secondarily, by triggered action potentials. This phenomenon is well demonstrated in Figure 3, whereby a line-scan image of Ca2+ fluorescence (Figure 3A-a), whole-cell Ca2+ fluorescence intensity (Figure 3A-b, F/F0), and membrane potential (Figure 3A-c, MP) were displayed simultaneously. Only slow CaWs and correlating sub-threshold depolarizations (SDs) were observed under control conditions. After the cell was perfused with 100 nM FCCP, the amplitudes of CaW-induced SDs were increased. Triggered action potentials (TA) were induced when the SDs reached the threshold level (indicted by the horizontal dashed line in the expanded inset to the right of panel A). Conversely, TAs stimulated synchronized Ca release, which appeared as spike-like Ca transients (indicated by the vertical dashed lines). Notably, the frequency of CaW-triggered APs was profoundly raised from 3.0 ± 2.1 to 62.4 ± 6.2 min−1 (Figure 3B, p < 0.01, n = 4). Since TAs are a major mechanism for arrhythmias, these results strongly imply that mitochondria play a role in arrhythmogenesis via regulation of CaWs. The exacerbation of CaWs by mitochondrial Ca release is sufficient for triggering action potentials and thus increases arrhythmogenicity.
A. A line-scan image along the long axis of the cell (A-a), whole-cell Ca fluorescence intensity (A-b), and membrane potential (A-c), were recorded simultaneously from a ventricular myocyte. CaWs (W) and sub-theshold depolarizations (SD) were exacerbated by FCCP (100 nM) so that triggered APs/activities (TA) and Ca transients (T) were induced. B. Summary data showing that FCCP (100 nM) markedly increased the incidence of TAs (**p < 0.01 vs. control, n = 4).
It should be noted that FCCP at high concentrations (e.g. 1 µM) completely eliminated spontaneous CaWs, while the basal Ca2+ level was dramatically elevated (from F/F0 = 1 to 1.74 ± 0.12; p < 0.01, n = 8, Figure 2C). We determined that the abolition of CaWs by high dose FCCP is attributed to the reduction of SR Ca2+ levels via Ca2+ extrusion by sarcolemmal NCX (sNCX). As shown in Figure 4A, the SR Ca2+ content (as evaluated by a 10 mM caffeine-induced Ca2+ transient) was significantly decreased in the presence of 1 µM FCCP compared to the control. Blocking sNCX by a rapid application of lithium (Li+) raised basal Cai2+ levels extremely high and restored CaWs (Figure 4B), while CaWs could not be restored by Li+ when SERCA2a was blocked simultaneously with 1µM thapsigargin. Likewise, CaWs were also restored by replenishing [Ca2+]i via the elevation of [Ca2+]o from 4 mM to 8 mM (Figure 4C).
A. SR Ca2+ content was assessed by rapid application of 10 mM caffeine in the absence and presence of 1µM FCCP. FCCP significantly reduced SR Ca2+ concentration, presumably via Ca2+ efflux by Na+-Ca2+ exchanger (NCX) (**p < 0.01 vs. control, n = 5). B. Suppressing NCX restored SCWs in the myocytes pretreated with 1µM FCCP, while no SCWs were observed when SERCA2a was inhibited with thapsigargin (TG, 1µM). C. High-dose FCCP-inhibited CaWs were restored by elevating [Ca2+]o from 4 mM to 8 mM. D. Lack of CaW inhibition by high-dose FCCP (1 µM) in the presence of Oligomycin (Oligo 1 µM). The expanded trace in the inset shows a fast Cai2+ oscillation status. E. Mag-fluo-4 fluorescence (evaluation of reciprocal changes of [ATP]i) traces recorded in mouse ventricular myocytes treated with (E-a) 100 nM FCCP or (E-b) 1 µM FCCP. The baseline value (i.e. before perfusion of FCCP) was normalized to 1, as indicated by the leftward-facing arrow in each panel. The F/F0 level at 6 min after FCCP treatment (as indicated by the rightward-facing arrow) was compared between different groups. E-c. Summary data showing the intensity of the fluorescence (F/F0) recorded 6 min after the treatment (**p < 0.01 vs. Ctl).
It has been shown, that in the presence of high concentration of FCCP, a protonophoric uncoupler, there is no driving force for F1F0-ATP synthase, and ATP may be consumed as the ATP synthase operates in the reverse mode. Therefore, one can assume that the decrease of SR Ca2+ content may be caused by the reduction of SERCA2a activity due to the lack of ATP. To test this assumption, we used oligomycin, an inhibitor of F1F0-ATP synthase, to attenuate ATP depletion in the present of FCCP. As shown in Figure 4D, 1 µM alone failed to inhibit CaWs in the presence of oligomycin (1 µM). Instead, the CaW frequency was further enhanced, with the cell entering a fast oscillation status after prolonged exposure to FCCP + oligomycin. These data suggest that the inhibitory effect of high dose FCCP was mediated, at least partially, by ATP hydrolysis through reverse-mode action of F1/F0-ATP synthase. It is likely the decrease of SR Ca2+ content at 1 µM FCCP (as shown in Figure 4A) might be due to ATP depletion and subsequent reduction of SERCA2a activity (which requires ATP). Indeed, using Mg-Fluo-4 as an indicator, we did observe the cellular ATP level was reduced (as indicated by a increase of [Mg2+]i, ) by 1 µM FCCP, but not by 100nM FCCP during the same time course (Fig. 4Ea-c).
Effect of FCCP on mitochondrial Ca2+ efflux
We have observed FCCP-induced elevation of basal Cai2+ levels (Figure 2). This elevation may represent three components: 1) mitochondrial Ca2+ release, 2) the entry of Ca2+ through the sarcolemmal membrane, and 3) SR Ca2+ release. In the following experiments, we dissected the component of the Cai2+ elevation caused by mitochondrial Ca2+ efflux in the presence of 1 µM FCCP (Figure 5). The total basal [Cai2+] was elevated from 1 to 1.62 ± 0.04 (n = 11; Figure 5 A and D) by 1 µM FCCP in Tyrode’s solution containing 4 mM Ca2+. To eliminate the component formed by the entry through the sarcolemmal membrane, we perfused the cells with a Ca2+-free Tyrode’s solution before adding FCCP. As shown in Figure 5B, the CaWs ceased in the Ca2+-free Tyrode’s solution. However, the basal [Cai2+] was still raised by treatment with FCCP (1 μM; from 1 to 1.34 ± 0.02, n = 11), suggesting the elevated basal [Cai2+] in the absence of extracellular Ca2+ can be attributed to intracellular sources, i.e. the mitochondria and SR. To further analyze the contribution from mitochondrial release, we depleted the SR Ca2+ by fast exposure to caffeine (10mM) before treatment with FCCP in Ca2+-free Tyrode’s solution. A representative recording is shown in Figure 5C. After confirming the SR Ca was completely depleted by repetitive caffeine doses, the cell was then treated with FCCP (1µM). The basal [Cai2+] was still elevated (from 1 to 1.13 ± 0.01, n = 11) by FCCP when both extracellular and SR sources were eliminated, suggesting that mitochondrial Ca2+ release contributes partially to the basal [Cai2+]. We postulate that this elevation of [Cai2+] may locally regulate RyR Ca release function at cytosolic face of RyRs and effectively facilitate CaW and TA generation.
A. Effects of FCCP (1 µM) on CaWs and basal Cai2+ level under control condition (4 mM Cao2+). B. Effects of FCCP on basal [Cai2+] level in a Cao2+-free condition. C. Effects of FCCP on basal Cai2+ level in Cao2+-free, and SR-Ca depleted condition. D. Summary data showing the effect of 1 µM FCCP on basal Cai2+ (∗∗p < 0.01 vs. control, n =11).
Regulation of CaWs by other mitochondrial Ca transporters
If local [Cai2+] plays an important role in influencing the RyRs, and subsequently modulating CaWs, Ca2+ flux via other mitochondrial Ca2+ transporters should also be effective . This assumption was tested by modifying the function of mCUs, which mediate Ca2+ uptake into mitochondria [4–6]. As shown in Fig. 6Aa & b, kaempferol (10 µM), a mCU opener, significantly attenuated CaWs induced by 100 nM FCCP (p < 0.01 vs. FCCP, n=7). The frequency of CaWs was increased from 19.2 ± 1.9 (control) to 50.5 ± 3.9 min-1 by 100 nM FCCP (p < 0.01, n = 5), and was subsequently reduced to 23.2 ± 3.1 min-1 by 10 µM kaempferol (p < 0.05, n = 5) (Figure 6A-b). The basal [Cai2+] level was elevated from F/F0 = 1 to 1.38 ± 0.09 by 100 nM FCCP (p < 0.01, n = 5), and was subsequently attenuated by 10 µM kaempferol (F/F0: 1.18 ± 0.06; p < 0.05, n = 5) (Figure 6A-a & c).
A. Kaempferol, a mitochondrial uniporter (mCU) opener, significantly attenuated CaWs induced by 100nM FCCP. A representative trace (A-a) and summarized data for CaW frequency (A-b) and basal [Cai2+] level (c) are shown (**p < 0.01 vs. control; #p < 0.05 vs. FCCP, n = 5). B. The effect of Ru360, a mCU blocker, on CaW frequency and basal [Cai2+] level in the presence of 50 nM FCCP. A representative trace (B-a) and summarized data (B-b & c) are shown. #p < 0.05 vs. FCCP (n = 5). C. The effect of Ru360 on CaW in the absence of FCCP. Note a persistent and fast oscillating status after Ru360 treatment.
In addition, we also tested the effects of the mCU blocker Ru360 on CaWs. As shown in Figure 6B, the application of 50 nM FCCP alone produced a weak effect on CaWs and basal [Cai2+], however its effect was strongly potentiated by the addition of 2 µM Ru360. A direct potentiating effect of Ru360 on CaWs was also observed in the absence of FCCP (Figure 6C). Based on the results shown in Figure 1, we believe that only a certain percentage of total mitochondria were dissipated by 100 nM FCCP, and the voltage-dependent mCUs should still remain functional in the residual mitochondria. Thus the mCU opener keampferol or inhibitor Ru360 can be still effective in controlling mitochondrial Ca2+ uptake, although we cannot completely exclude the possibility they may also have other off-target effects. However, we did not observe any effect of CGP37157 (a mNCX blocker, 10 µM) on CaWs (data not shown).
Less direct effect of metabolic inhibitors on CaWs
FCCP can also uncouple mitochondrial oxidative phosphorylation and reduce ATP production . To determine whether the metabolic status is involved in the CaW regulation in our experimental conditions, we tested the effects other metabolic inhibitors have on CaW behaviors. The effect of FCCP on CaWs and basal [Cai2+] were mimicked by antimycin A (10 µM), an electron transport chain (complex III) inhibitor, which also depolarizes Δψm (Figure 7A). The frequency of CaWs was increased from 20.9 ± 2.9 to 45.4 ± 3.5 min-1 (p < 0.01, n= 6, Figure 7A-b) before stopping, once the basal [Cai2+] reached a high level (F/F0 from 1 to 1.44 ± 0.05, p <0.01, n = 6) (Figure 7A-c).
A. The effect of antimycin A (an electron transport chain inhibitor disrupting ΔΨm) on CaW frequency and basal [Cai2+] level. Representative traces (5 and 10 µM antimycin A) (A-a) and summarized data (A-b & c) are shown. ∗∗p < 0.01 vs. control (n=6). B. The effects of oligomycin alone (B-a) and in the presence (B-b) of high-dose antimycine A (10 µM) on CaWs. B-b & c. Summary data showing the CaW frequencies under each condition. (n = 5 for each, ∗∗p < 0.01 vs. control). C. the same as A, except for Iodoacetic acid (IAA, a glycolytic inhibitor, n = 7) was tested. IAA exerted no significant effects on CaWs or basal [Cai2+] (p > 0.05).
On the contrary, neither oligomycin (Fig. 7Bb), an ATP synthase (F0F1-ATPase) inhibitor (n=5) nor iodoacetic acid (Figure 7C), a glycolytic inhibitor (n = 7), affected CaW frequency and basal [Cai2+], excluding the direct contribution of intracellular metabolic condition (or ATP levels) in CaW activation under our present experimental conditions.
Similar to the result shown in Figure 4D, the inhibitory effect of high dose of antimycin A on CaWs was also attenuated by oligomycin (1 µM) (Figure 7B a & c), suggesting ATP depletion by reverse-mode action of F1/F0-ATP synthase may also be involved in CaW inhibition.
Less involvement of reactive oxygen species (ROS) in CaW facilitation by FCCP
It has been suggested that production of reactive oxygen species (ROS) in mitochondria may lead to aberrant calcium homeostasis [31–33]. In order to assess whether ROS was involved in the FCCP-induced regulation of CaWs in the setting of our present study, we measured mitochondrial superoxide production (by using MitoSOX Red) in the presence of FCCP at 100 nM and 1 µM, respectively. While 1 µM FCCP induced significant mitochondrial ROS generation, a lower concentration of FCCP (100 nM) did not promote mitochondrial ROS production (Figure 8A). Next, we tested the effects of antioxidants (MnTMPyP) on the frequency of spontaneous CaWs in the presence of 100 nM FCCP. Oligomycin was applied to exclude potential influences of cellular metabolic status (i.e. ATP consumption). As shown in Figure 8B, we still observed the significant activation of CaWs by FCCP in the presence of MnTMPyP, consistent with our assumption that Ca efflux via mitochondria (but neither ROS nor ATP depletion) plays a major role in the activation of CaWs by FCCP at low doses.
A. Mitosox Red fluorescence (evaluation of mitochondrial superoxide level) traces recorded in mouse ventricular myocytes treated with (A-a) 100 nM FCCP or (A-b) 1 µM FCCP. The baseline value (i.e. before perfusion of FCCP) was normalized to 1, as indicated by the leftward-facing arrow in each panel. The F/F0 level at 6 min after FCCP treatment (as indicated by the rightward-facing arrow) was compared between different groups. (A-c) Summary data showing the intensity of the fluorescence (F/F0) recorded 6 min after the treatment (**p < 0.01 vs. Ctl). B. Effect of MnTMPyP (10 µM) on spontaneous CaWs. Oligomycin was applied to exclude potential influences of cellular metabolic status. FCCP (100 nM) promoted CaWs either in the absence (B-a) or the presence (B-b) of MnTMPyP. (A-c). Summary data showing the CaW frequencies under differential treatments. The cell number for measurement in each group is indicated (** p < 0.01).
We have investigated the roles of mitochondrial Ca2+ flux in modulation of CaWs and TAs in mouse ventricular myocytes. The major findings of this present study include: 1) The protonophore FCCP, which can depolarize Δψm and open mPTPs, increase basal [Cai2+] and potentiate CaWs and TAs in a dose-dependent manner in mouse ventricular myocytes; 2) the effect of FCCP on CaWs can be antagonized by CsA, an mPTP blocker; 3) Ca2+ flux through mCU also affects CaW behaviors; 4) the effect of FCCP on SCWs can be mimicked by antimycin A (an electron transport chain inhibitor which can also depolarize Δψm), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor). We postulate that mitochondrial Ca2+ flux controls the local Ca2+ homeostasis in the micro-domain at the cytosolic face of RyR. It is well known that the majority of RyRs are located in the dyadic regions of the junctional SR membrane and face the transverse tubule in ventricular myocytes . However, at least some regions of the mitochondria (i.e. the Z-dist ends of an intermyofibrillar mitochondrion) are very close to the junctional SR and RyRs . Additionally, in their recent study Lu et al revealed the presence of an intra-mitochondrial Ca gradient suggesting that there does exist the functional interaction between mitochondria and SR in terms of Ca crosstalk . We postulate that mitochondrial Ca flux controls the local Ca homeostasis in the microdomain within the region close to the junctional SR and RyRs. Our study provides further evidence showing the functional coupling that occurs between the mitochondria and the SR under pathological conditions. These data implicate mitochondrial dysfunction as a potential cellular mechanism of arrhythmogenesis under stress/pathological conditions, such as ischemia-reperfusion and heart failure. A recent study by Yaniv Y, et al  revealed that mitochondrial, cytosolic, and SR Ca2+ crosstalk occurs in single rabbit sinoatrial-node cells, and regulates the automaticity of sinoatrial-node cells under physiological condition. Our current study provides insights into how a similar mechanism accounts for the arrhythmogenesis in ventricular myocytes under Ca2+-overload conditions.
Regulation of CaWs by both cytosolic and SR luminal Ca2+
Recent data have also demonstrated that SR Ca2+ leaks (Ca sparks) and CaWs are regulated by Ca2+ at both the SR luminal face and cytosolic face of RyRs [36,37]. Thus, elevation of cytosolic Ca2+ is likely to increase SR Ca2+ leak and facilitates CaWs by the direct activation of RyRs. Our present study provides evidence showing inter-organelle regulation between the mitochondria and SR via cytosolic Ca2+ and its subsequent effect on CaW regulation and generation.
We first established a CaW model by elevating [Cao2+] (4 mM). This model indicates that Ca2+ influx from sarcolemma can elevate cytosolic levels and trigger CaWs. This sarcolemmal Ca2+ influx may be caused by non-selective cationic channels, such as TRPCs [29,38,39]. The central finding of our study is that Ca2+ fluxes via mitochondrial Ca2+ transporters dynamically change cytosolic Ca2+ levels and consequently regulate CaW behaviors. Although only a mild (but significant) elevation of basal [Cai2+] induced by mitochondrial efflux was detected by measuring global Ca2+ (Figure 5 C and D), it is conceivable that the local [Cai2+] at the mitochondria-SR micro-domain could be much higher.
It should be noted that FCCP has biphasic effects on CaWs (Figure 2C). FCCP promotes CaWs only at moderate concentrations (50 -500nM), while at high concentrations it suppresses CaWs (1 µM), and elevates basal [Cai2+] (Figure 2B). Early studies on biophysical properties of RyRs reconstituted in lipid bilayers have proposed a negative feedback regulation mechanism for the regulation of RyR activities by cytosolic Ca2+ [40–43]. However, this negative feedback regulation cannot explain the data obtained from our experiments, since fast CaWs were restored by blocking sNCX, (substituting Li+ for Na + in the perfusion solution) when basal [Cai2+] was elevated. Based on the results shown in Figure 4, we postulate that the cessation of CaWs was most likely due to the decrease of SR Ca2+ content below the CaW threshold level.
We have also shown that FCCP facilitated CaWs at both low (100nM) and high concentrations (1 µM) while in the presence of oligomycin. It is known that FCCP treatment may result in ATP depletion, which is attenuated by the F1F0-ATP synthase inhibitor oligomycin. Therefore, it seems likely the biphasic effects of FCCP on CaW frequency are mediated via different mechanisms. The stimulation of CaWs by FCCP at lower concentrations could arise from increased mitochondrial Ca2+ release (via mPTP) and/or reduced mitochondrial Ca2+ uptake (via mCU) followed by increased diastolic [Ca2+]i and enhanced SR Ca2+ release. On the contrary, the inhibition of CaWs at higher concentrations (e.g. 1 µM) could be caused by reduced SR Ca2+ content due to consumption of ATP and consequent reduction of SERCA activity, as well as excessive extrusion of Ca2+ through the sarcolemma. In addition, we cannot exclude the possibility of reduced RyR activity since ATP may act as a direct activator of SR RyR . A recent study by Zima et al investigated how mitochondrial Ca2+ signaling and ATP production affect SR Ca2+ release and E-C coupling in cat atrial myocytes. They used a high dose of FCCP (2 µM) and found similar dual effects of FCCP based on Ca and ATP, respectively .
Effect of FCCP on mitochondrial Ca2+ flux: Ca2+ efflux via mPTP and Ca2+ influx via mCU
As shown in the schematic illustration (Figure 9), mitochondrial Ca2+ flux is regulated by several mechanisms: while mCU is responsible for Ca2+ influx, mNCX and mPTP mediate Ca2+ efflux. The uptake of Ca2+ through mCUs is driven by the large electrochemical gradient (ΔΨm = −180 mV) across the inner mitochondrial membrane. It has been reported that FCCP depolarizes ΔΨm in RBL-2H3 cells , which is also confirmed in the setting of our experiments (Figure 1). Accordingly, FCCP-induced ΔΨm depolarization should reduce the driving force for Ca2+ uptake by mCU. Additionally, ΔΨm depolarization also leads to the opening of mPTP . mPTP is a voltage-dependent, high conductance (~1200 pS) complex comprised of several components including the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase (ANT), and cyclophilin D . The opening of mPTPs leads to the release of mitochondrial factors less than 1.5 kDa, including Ca2+ and cytochrome c . Therefore, the exacerbation of CaWs by FCCP may be mediated by reduced Ca uptake via mCU and/or increased Ca release via mPTP. This notion was supported by the results that either activation of mCU by kaempferol (Figure 6A) or inhibition of mPTP by CsA (Figure 2D) suppressed CaWs. We did not observe any apparent effect of the mNCX inhibitor CGP37157, suggesting mNCX contributes less to this CaW regulation under our experimental condition.
Mitochondrial Ca2+ is released into the cytoplasm via the permeability transition pore (mPTP) and up-taken by the mitochondrial calcium uniporter (mCU). An increase in local cytosolic [Ca2+] prompts additional uptake of Ca2+ by the SERCA pump, and triggers a greater release of Ca2+ by the ryanodine receptor (RyR), thus generating/enhancing CaWs. Either inhibition of mPTP by CsA or activation of mCU by kaempferol suppresses CaWs.
Regulation of CaWs by local Cai2+ versus metabolic inhibition
It is known that FCCP is an uncoupler of oxidative phosphorylation inhibiting the coupling between the electron transport and phosphorylation reactions and thus reducing ATP production. Since ATP is required for normal function of ionic pumps, including sarcoendoplasmic reticulum calcium ATPase (SERCA2), which mediates Cai2+ uptake into the SR, it is possible that metabolic inhibition may cause abnormalities in Ca handling and arrhythmias. For example, inhibition of glycolysis with IAA causes elevation of diastolic Cai2+ and a decrease of the amplitude of Cai2+ transients in cat atrial myocytes . Our previous studies have reported that inhibition of either glycolysis or oxidative phosphorylation affects Ca handling and arrhythmogenesis in embryonic mouse hearts . Oligomycin (1 or 5 µM) or IAA (100 µM) by themselves did not affect CaWs, respectively (Figure 7B & 7C), suggesting that inhibition of mitochondrial ATP production (via F1F0-ATP synthase or glycolysis) alone was not involved in the CaW activation in the setting of our present experiments. Although we cannot exclude potential effects of long-term metabolic inhibition, our present data shows that the regulatory effects of FCCP and antimycin A are most likely due to their acute, direct effect on Ca efflux through mPTP (and reduced Ca uptake by mCU), rather than any secondary effects mediated by metabolic inhibition or ROS production. This notion is consistent with previous observations made in other cell types . It should be noted that the ATP depletion induced by reverse-mode action of F1/F0-ATP synthase may account for, at least partially, the inhibition of CaWs in the presence of high doses of FCCP (1 µM) or antimycin A (10 µM), which are thought to cause either reduction of either SERCA2a or RyR activities, or both.
Pathophysiological and clinical relevance
It has been shown that mPTP opening can be induced by direct treatment with ROS [49,50] or myocardial ischemia-reperfusion (I/R) . Thus, mitochondrial uncouplers, such as FCCP, have been used previously as a tool to produce cellular models of ischemia or hypoxia . Our present study demonstrates that mPTP opening may cause the disruption of mitochondrial Ca2+ handling and therefore promote CaW generation via mitochondria-SR functional crosstalk/communication. CaWs can activate sNCX current (INCX, or inward transient current Iti), which causes subthreshold depolarizations (SDs). The SDs trigger action potentials when they reach the threshold and predispose arrhythmias.
In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure [8,53–57], suggesting the mPTP as a promising therapeutic target in human cardiovascular disease. It has also been suggested that the mitochondria may contribute to arrhythmogenesis by introducing electrical heterogeneity into the heart tissue . Inhibition of mPTP has been shown to reduce mortality following acute myocardial infarction in mice . It remains unknown whether inhibition of mPTP opening may prevent arrhythmias in intact animal models or in clinical settings.
Our present study exclusively investigates the role of mitochondria in the regulation of cardiac myocyte function by buffering cytosolic Ca2+. It should be noted that mitochondrial metabolic products, such as ATP and ROS can affect SR Ca homeostasis via regulation of SERCA and RyR activity [31,60,61]. On the other hand, SR Ca2+ release and mitochondrial Ca2+ can reversely affects ATP and ROS productions through activation of the Krebs Cycle [62,63]. Thus the interaction between SR and mitochondria is complicated and can be linked via either intracellular Ca2+ (as shown in our present study) or intracellular metabolic products.
In order to induce CaWs consistently and study the regulatory effects of mitochondrial dysfunction, we have exclusively used a high (4 mM) [Ca2+]o-induced CaW model in this study. We believe that this model is still relevant since Ca overload may occur under various pathological conditions, such as ischemia-reperfusion, hypertrophy, and heart failure.
Conceived and designed the experiments: LHX ZZ. Performed the experiments: ZZ HW RG NF. Analyzed the data: ZZ HW RG NF WJZ LHX. Wrote the manuscript: ZZ RG LHX.
- 1. O'Rourke B, Cortassa S, Aon MA (2005) Mitochondrial ion channels: gatekeepers of life and death. Physiology (Bethesda) 20: 303-315. doi:https://doi.org/10.1152/physiol.00020.2005. PubMed: 16174870.
- 2. Griffiths EJ (2009) Mitochondrial calcium transport in the heart: physiological and pathological roles. J Mol Cell Cardiol 46: 789-803. doi:https://doi.org/10.1016/j.yjmcc.2009.03.001. PubMed: 19285504.
- 3. Griffiths EJ, Balaska D, Cheng WH (2010) The ups and downs of mitochondrial calcium signalling in the heart. Biochim Biophys Acta 1797: 856-864. doi:https://doi.org/10.1016/j.bbabio.2010.02.022. PubMed: 20188059.
- 4. Bernardi P (1999) Mitochondria in muscle cell death. Ital J Neurol Sci 20: 395-400. doi:https://doi.org/10.1007/s100720050057. PubMed: 10937859.
- 5. Gunter TE, Buntinas L, Sparagna G, Eliseev R, Gunter K (2000) Mitochondrial calcium transport: mechanisms and functions. Cell Calcium 28: 285-296. doi:https://doi.org/10.1054/ceca.2000.0168. PubMed: 11115368.
- 6. Kirichok Y, Krapivinsky G, Clapham DE (2004) The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427: 360-364. doi:https://doi.org/10.1038/nature02246. PubMed: 14737170.
- 7. Hoppe UC (2010) Mitochondrial calcium channels. FEBS Lett 584: 1975-1981. doi:https://doi.org/10.1016/j.febslet.2010.04.017. PubMed: 20388514.
- 8. Halestrap AP, Pasdois P (2009) The role of the mitochondrial permeability transition pore in heart disease. Biochim Biophys Acta 1787: 1402-1415. doi:https://doi.org/10.1016/j.bbabio.2008.12.017. PubMed: 19168026.
- 9. Bers DM (2002) Cardiac excitation-contraction coupling. Nature 415: 198-205. doi:https://doi.org/10.1038/415198a. PubMed: 11805843.
- 10. Dedkova EN, Blatter LA (2008) Mitochondrial Ca2+ and the heart. Cell Calcium 44: 77-91. doi:https://doi.org/10.1016/j.ceca.2007.11.002. PubMed: 18178248.
- 11. Ruiz-Meana M, Fernandez-Sanz C, Garcia-Dorado D (2010) The SR-mitochondria interaction: a new player in cardiac pathophysiology. Cardiovasc Res 88: 30-39. doi:https://doi.org/10.1093/cvr/cvq225. PubMed: 20615915.
- 12. García-Pérez C, Hajnóczky G, Csordás G (2008) Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle. J Biol Chem 283: 32771-32780. doi:https://doi.org/10.1074/jbc.M803385200. PubMed: 18790739.
- 13. Andrienko TN, Picht E, Bers DM (2009) Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes. J Mol Cell Cardiol 46: 1027-1036. doi:https://doi.org/10.1016/j.yjmcc.2009.03.015. PubMed: 19345225.
- 14. Szabadkai G, Duchen MR (2008) Mitochondria: the hub of cellular Ca2+ signaling. Physiology (Bethesda) 23: 84-94. doi:https://doi.org/10.1152/physiol.00046.2007. PubMed: 18400691.
- 15. Csordás G, Hajnóczky G (2009) SR/ER-mitochondrial local communication: calcium and ROS. Biochim Biophys Acta 1787: 1352-1362. doi:https://doi.org/10.1016/j.bbabio.2009.06.004. PubMed: 19527680.
- 16. Csordás G, Renken C, Várnai P, Walter L, Weaver D et al. (2006) Structural and functional features and significance of the physical linkage between ER and mitochondria. J Cell Biol 174: 915-921. doi:https://doi.org/10.1083/jcb.200604016. PubMed: 16982799.
- 17. Williams GS, Boyman L, Chikando AC, Khairallah RJ, Lederer WJ (2013) Mitochondrial calcium uptake. Proc Natl Acad Sci U S A.
- 18. Lu X, Ginsburg KS, Kettlewell S, Bossuyt J, Smith GL et al. (2013) Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release. Circ Res 112: 424-431. doi:https://doi.org/10.1161/CIRCRESAHA.111.300501. PubMed: 23243207.
- 19. Werth JL, Thayer SA (1994) Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons. J Neurosci 14: 348-356. PubMed: 8283242.
- 20. Friel DD, Tsien RW (1994) An FCCP-sensitive Ca2+ store in bullfrog sympathetic neurons and its participation in stimulus-evoked changes in [Ca2+]i. J Neurosci: 14: 4007-4024.
- 21. Fluegge D, Moeller LM, Cichy A, Gorin M, Weth A et al. (2012) Mitochondrial Ca(2+) mobilization is a key element in olfactory signaling. Nat Neurosci 15: 754-762. doi:https://doi.org/10.1038/nn.3074. PubMed: 22446879.
- 22. Sergeant GP, Bradley E, Thornbury KD, McHale NG, Hollywood MA (2008) Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra. J Physiol 586: 4631-4642. doi:https://doi.org/10.1113/jphysiol.2008.159194. PubMed: 18703577.
- 23. Shanmugam M, Gao S, Hong C, Fefelova N, Nowycky MC et al. (2011) Ablation of phospholamban and sarcolipin results in cardiac hypertrophy and decreased cardiac contractility. Cardiovasc Res 89: 353-361. doi:https://doi.org/10.1093/cvr/cvq294. PubMed: 20833651.
- 24. Petronilli V, Miotto G, Canton M, Brini M, Colonna R et al. (1999) Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 76: 725-734. doi:https://doi.org/10.1016/S0006-3495(99)77239-5. PubMed: 9929477.
- 25. Fraysse B, Nagi SM, Boher B, Ragot H, Lainé J et al. (2010) Ca2+ overload and mitochondrial permeability transition pore activation in living delta-sarcoglycan-deficient cardiomyocytes. Am J Physiol Cell Physiol 299: C706-C713. doi:https://doi.org/10.1152/ajpcell.00545.2009. PubMed: 20592245.
- 26. Zima AV, Pabbidi MR, Lipsius SL, Blatter LA (2013) Effects of mitochondrial uncoupling on Ca(2+) signaling during excitation-contraction coupling in atrial myocytes. Am J Physiol Heart Circ Physiol 304: H983-H993. doi:https://doi.org/10.1152/ajpheart.00932.2012. PubMed: 23376829.
- 27. Xie LH, Chen F, Karagueuzian HS, Weiss JN (2009) Oxidative-stress-induced afterdepolarizations and calmodulin kinase II signaling. Circ Res 104: 79-86. PubMed: 19038865.
- 28. Xie LH, Sato D, Garfinkel A, Qu Z, Weiss JN (2008) Intracellular Ca alternans: coordinated regulation by sarcoplasmic reticulum release, uptake, and leak. Biophys J 95: 3100-3110. doi:https://doi.org/10.1529/biophysj.108.130955. PubMed: 18539635.
- 29. Xie LH, Wen H, Zhao Z, Fefelova N, Allen CA (2011) Unique “Cellular Fibrillation” Status In Mouse Ventricular Myocytes. Circulation the AHA Scientific Sessions. November. Orlando, Florida.
- 30. Terada H (1990) Uncouplers of oxidative phosphorylation. Environ Health Perspect 87: 213-218. doi:https://doi.org/10.1289/ehp.9087213. PubMed: 2176586.
- 31. Yan Y, Liu J, Wei C, Li K, Xie W et al. (2008) Bidirectional regulation of Ca2+ sparks by mitochondria-derived reactive oxygen species in cardiac myocytes. Cardiovasc Res 77: 432-441. PubMed: 18006452.
- 32. Bovo E, Lipsius SL, Zima AV (2012) Reactive oxygen species contribute to the development of arrhythmogenic Ca²⁺ waves during β-adrenergic receptor stimulation in rabbit cardiomyocytes. J Physiol 590: 3291-3304. doi:https://doi.org/10.1113/jphysiol.2012.230748. PubMed: 22586224.
- 33. Ho HT, Stevens SC, Terentyeva R, Carnes CA, Terentyev D et al. (2011) Arrhythmogenic adverse effects of cardiac glycosides are mediated by redox modification of ryanodine receptors. J Physiol 589: 4697-4708. PubMed: 21807619.
- 34. Franzini-Armstrong C (2007) ER-mitochondria communication. How privileged? Physiol (Bethesda) 22: 261-268. doi:https://doi.org/10.1152/physiol.00017.2007.
- 35. Yaniv Y, Spurgeon HA, Lyashkov AE, Yang D, Ziman BD et al. (2012) Crosstalk between mitochondrial and sarcoplasmic reticulum Ca2+ cycling modulates cardiac pacemaker cell automaticity. PLOS ONE 7: e37582. doi:https://doi.org/10.1371/journal.pone.0037582. PubMed: 22666369.
- 36. Venetucci LA, Trafford AW, O'Neill SC, Eisner DA (2008) The sarcoplasmic reticulum and arrhythmogenic calcium release. Cardiovasc Res 77: 285-292. PubMed: 18006483.
- 37. Bovo E, Mazurek SR, Blatter LA, Zima AV (2011) Regulation of sarcoplasmic reticulum Ca²⁺ leak by cytosolic Ca²⁺ in rabbit ventricular myocytes. J Physiol 589: 6039-6050. PubMed: 21986204.
- 38. Kojima A, Kitagawa H, Omatsu-Kanbe M, Matsuura H, Nosaka S (2010) Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes. Br J Pharmacol 161: 1734-1750. doi:https://doi.org/10.1111/j.1476-5381.2010.00986.x. PubMed: 20718730.
- 39. Wen H, Zhao Z, Fefelova N, Allen C, Xie LH (2011) Arrhythmogenic Effect of Store-operated Calcium Entry in Mouse Ventricular Myocytes. Circulation 124: A15884.
- 40. Fabiato A (1985) Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol 85: 291-320. doi:https://doi.org/10.1085/jgp.85.2.291. PubMed: 2580044.
- 41. Meissner G, Rios E, Tripathy A, Pasek DA (1997) Regulation of skeletal muscle Ca2+ release channel (ryanodine receptor) by Ca2+ and monovalent cations and anions. J Biol Chem 272: 1628-1638. doi:https://doi.org/10.1074/jbc.272.3.1628. PubMed: 8999838.
- 42. Sham JS, Song LS, Chen Y, Deng LH, Stern MD et al. (1998) Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes. Proc Natl Acad Sci U S A 95: 15096-15101. doi:https://doi.org/10.1073/pnas.95.25.15096. PubMed: 9844021.
- 43. Laver DR, Lamb GD (1998) Inactivation of Ca2+ release channels (ryanodine receptors RyR1 and RyR2) with rapid steps in [Ca2+] and voltage. Biophys J 74: 2352-2364. doi:https://doi.org/10.1016/S0006-3495(98)77944-5. PubMed: 9591662.
- 44. Meissner G, Henderson JS (1987) Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. J Biol Chem 262: 3065-3073. PubMed: 2434495.
- 45. Inoue T, Suzuki Y, Ra C (2010) Epigallocatechin-3-gallate inhibits mast cell degranulation, leukotriene C4 secretion, and calcium influx via mitochondrial calcium dysfunction. Free Radic Biol Med 49: 632-640. doi:https://doi.org/10.1016/j.freeradbiomed.2010.05.015. PubMed: 20510351.
- 46. Duchen MR (2000) Mitochondria and calcium: from cell signalling to cell death. J Physiol 529 1: 57-68. doi:https://doi.org/10.1111/j.1469-7793.2000.00057.x.
- 47. Kockskämper J, Zima AV, Blatter LA (2005) Modulation of sarcoplasmic reticulum Ca2+ release by glycolysis in cat atrial myocytes. J Physiol 564: 697-714. doi:https://doi.org/10.1113/jphysiol.2004.078782. PubMed: 15695247.
- 48. Chen F, De Diego C, Xie LH, Yang JH, Klitzner TS et al. (2007) Effects of metabolic inhibition on conduction, Ca transients, and arrhythmia vulnerability in embryonic mouse hearts. Am J Physiol Heart Circ Physiol 293: H2472-H2478. doi:https://doi.org/10.1152/ajpheart.00359.2007. PubMed: 17660398.
- 49. Sheu SS, Wang W, Cheng H, Dirksen RT (2008) Superoxide flashes: illuminating new insights into cardiac ischemia/reperfusion injury. Future Cardiol 4: 551-554. doi:https://doi.org/10.2217/14796618.104.22.1681. PubMed: 19649173.
- 50. Saotome M, Katoh H, Yaguchi Y, Tanaka T, Urushida T et al. (2009) Transient opening of mitochondrial permeability transition pore by reactive oxygen species protects myocardium from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 296: H1125-H1132. doi:https://doi.org/10.1152/ajpheart.00436.2008. PubMed: 19202002.
- 51. Halestrap AP, Clarke SJ, Javadov SA (2004) Mitochondrial permeability transition pore opening during myocardial reperfusion - a target for cardioprotection. Cardiovasc Res 61: 372-385. doi:https://doi.org/10.1016/S0008-6363(03)00533-9. PubMed: 14962470.
- 52. Suh SH, Droogmans G, Nilius B (2000) Effects of cyanide and deoxyglucose on Ca2+ signalling in macrovascular endothelial cells. Endothelium 7: 155-168. PubMed: 10912910.
- 53. Hausenloy DJ, Lim SY, Ong SG, Davidson SM, Yellon DM (2010) Mitochondrial cyclophilin-D as a critical mediator of ischaemic preconditioning. Cardiovasc Res 88: 67-74. doi:https://doi.org/10.1093/cvr/cvq113. PubMed: 20400621.
- 54. Baines CP (2009) The mitochondrial permeability transition pore and ischemia-reperfusion injury. Basic Res Cardiol 104: 181-188. doi:https://doi.org/10.1016/j.amjcard.2009.08.522. PubMed: 19242640.
- 55. Elrod JW, Wong R, Mishra S, Vagnozzi RJ, Sakthievel B et al. (2010) Cyclophilin D controls mitochondrial pore-dependent Ca(2+) exchange, metabolic flexibility, and propensity for heart failure in mice. J Clin Invest 120: 3680-3687. doi:https://doi.org/10.1172/JCI43171. PubMed: 20890047.
- 56. Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H et al. (2005) Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434: 658-662. doi:https://doi.org/10.1038/nature03434. PubMed: 15800627.
- 57. Weiss JN, Korge P, Honda HM, Ping P (2003) Role of the mitochondrial permeability transition in myocardial disease. Circ Res 93: 292-301. doi:https://doi.org/10.1161/01.RES.0000087542.26971.D4. PubMed: 12933700.
- 58. Brown DA, O'Rourke B (2010) Cardiac mitochondria and arrhythmias. Cardiovasc Res 88: 241-249. doi:https://doi.org/10.1093/cvr/cvq231. PubMed: 20621924.
- 59. Gomez L, Thibault H, Gharib A, Dumont JM, Vuagniaux G et al. (2007) Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice. Am J Physiol Heart Circ Physiol 293: H1654-H1661. doi:https://doi.org/10.1152/ajpheart.01378.2006. PubMed: 17557911.
- 60. Lanner JT (2012) Ryanodine receptor physiology and its role in disease. Adv Exp Med Biol 740: 217-234. doi:https://doi.org/10.1007/978-94-007-2888-2_9. PubMed: 22453944.
- 61. Fill M, Copello JA (2002) Ryanodine receptor calcium release channels. Physiol Rev 82: 893-922. PubMed: 12270947.
- 62. Kohlhaas M, Liu T, Knopp A, Zeller T, Ong MF et al. (2010) Elevated cytosolic Na+ increases mitochondrial formation of reactive oxygen species in failing cardiac myocytes. Circulation 121: 1606-1613. doi:https://doi.org/10.1161/CIRCULATIONAHA.109.914911. PubMed: 20351235.
- 63. De Giusti VC, Caldiz CI, Ennis IE, Perez NG, Cingolani HE et al. (2013) Mitochondrial reactive oxygen species (ROS) as signaling molecules of intracellular pathways triggered by the cardiac renin-angiotensin II-aldosterone system (RAAS). Front Physiol 4: 126.