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Cardiovascular Research Advance Access originally published online on October 25, 2007
Cardiovascular Research 2008 77(2):432-441; doi:10.1093/cvr/cvm047
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

Bidirectional regulation of Ca2+ sparks by mitochondria-derived reactive oxygen species in cardiac myocytes

Yuan Yan1, Jie Liu2,*, Chaoliang Wei1, Kaitao Li1, Wenjun Xie1, Yanru Wang1 and Heping Cheng1,*

1 Institute of Molecular Medicine, National Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing 100871, China
2 Department of Pathophysiology, Southern Medical University, Guangzhou 510515, China

* Corresponding author. Tel/fax: +86 10 6276 5957. E-mail address: chengp{at}pku.edu.cn (H.C.); jieliu{at}fimmu.com (J.L.)

Received 18 May 2007; revised 13 October 2007; accepted 16 October 2007

Time for primary review: 18 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Result
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: The cardiac ryanodine receptor (RyR) Ca2+ release channel homotetramer harbours ~21 potentially redox-sensitive cysteine residues on each subunit and may act as a sensor for reactive oxygen species (ROS), linking ROS homeostasis to the regulation of Ca2+ signalling. In cardiac myocytes, arrayed RyRs or Ca2+ release units are packed in the close proximity of mitochondria, the primary source of intracellular ROS production. The present study investigated whether and how mitochondria-derived ROS regulate Ca2+ spark activity in intact cardiac myocytes.

Methods and results: Bidirectional manipulation of mitochondrial ROS production in intact rat cardiac myocytes was achieved by photostimulation and pharmacological means. Simultaneous measurement of intracellular ROS and Ca2+ signals was performed using confocal microscopy in conjunction with the indicators 5-(–6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (for ROS) and rhod-2 (for Ca2+). Photoactivated or antimycin A (AA, 5 µg/mL)-induced mitochondrial ROS production elicited a transient increase in Ca2+ spark activity, followed by gradual spark suppression. Intriguingly, photoactivated mitochondrial ROS oscillations subsequent to the initial peaks mirrored phasic depressions of the spark activity, suggesting a switch of ROS modulation from spark-activating to spark-suppressing. Partial deletion of Ca2+ stores in the sarcoplasmic reticulum contributed in part to the gradual, but not the phasic, spark depression. H2O2 at 200 µM elicited a bidirectional effect on sparks and produced sustained spark activation at 50 µM. Lowering basal mitochondrial ROS production, scavenging baseline ROS, and applying the sulphydryl-reducing agent dithiothreitol diminished the incidence of spontaneous Ca2+ sparks and abolished the Ca2+ spark responses to mitochondrial ROS.

Conclusion: Mitochondrial ROS exert bidirectional regulation of Ca2+ sparks in a dose- and time (history)-dependent manner, and basal ROS constitute a hitherto unappreciated determinant for the production of spontaneous Ca2+ sparks. As such, ROS signalling may play an important role in Ca2+ homeostasis as well as Ca2+ dysregulation in oxidative stress-related diseases.

KEYWORDS Reactive oxygen species (ROS); Ca2+ sparks; Oxidative stress; Cardiac myocytes; Mitochondria


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Result
 4. Discussion
 Supplementary material
 Funding
 References
 
The ryanodine receptor (RyR) plays an essential role in cardiac excitation–contraction coupling (EC coupling) and Ca2+ homeostasis1,2 by gating release of Ca2+ from the sarcoplasmic reticulum (SR) via the Ca2+ induced Ca2+ release (CICR) mechanism. Increasing evidence has demonstrated that RyR also acts as a cellular redox sensor due to rich free thiol groups in its structure.3,4 For instance, the cardiac RyR homotetramer harbours 364 cysteine residues, and ~21 of which are free on each subunit.5 Many groups have shown that oxidation of these free thiol groups activates RyRs, whereas their reduction inhibits the channel.5–8 Yano et al.9 have shown that oxidative stress in RyR destabilizes the channel gating and facilitates the channel opening. These implicate that RyR-mediated Ca2+ release may subject to regulation of intracellular reactive oxygen species (ROS) in intact cells.10,11

In cardiac myocytes, mitochondria occupy 30–40% of the cellular volume and constitute the major source of intracellular ROS production. Up to 1–2% of the electrons in the electron transfer chain leaks to molecular oxygen to form superoxide anion and, subsequently, hydrogen peroxide (H2O2).12,13 Mitochondria-related excessive ROS production has been implicated in the pathogenesis of many cardiovascular diseases, including ischaemia–reperfusion injury.10,13 Moreover, electron microscopic studies have shown that mitochondria are in close proximity of Ca2+ releasing units (CRUs) that consist of arrayed RyRs, with the distance varying between 40 and 180 nm in rat myocardium.14 These lines of evidence suggest that mitochondrial ROS might actively modulate local or global RyR-mediated Ca2+ signalling.

Given the diversity of ROS and ROS effector proteins and the clutter of the ROS-active moieties on a given effector protein, the regulation of Ca2+ signals by ROS can be both stimulatory and inhibitory, depending on the dose, history, and cell context. When treated with 100 µM H2O2, cytosolic Ca2+ concentration ([Ca2+]c) of rat cardiac myocytes increases markedly and continues to rise upon washout, whereas 1 µM H2O2 exerts no significant effect on [Ca2+]c.15 At even higher dose, 1 mM H2O2 elicits biphasic response in guinea-pig ventricular myocytes—a transient augmentation followed by a suppression of [Ca2+]c transient after 5 min of exposure.16 Under pathological conditions, such as heart failure,9 hypoxia,17 and ischaemia/reperfusion injury,10 excessive ROS increase mediates, at least in part, the intracellular Ca2+ overload. These previous findings suggest that basal ROS and regulatory ROS increase may play important roles in Ca2+ regulation and excessive ROS may underlie Ca2+ dysregulation in diverse physiological and pathological contexts.

Ca2+ sparks arising from CRU activation constitute elementary events of EC coupling and provide a unique window through which the function and physiological regulation of RyRs can be investigated in intact cells. It has been demonstrated that local Ca2+ release during a spark can be sequestered into mitochondria via Ca2+ uniporter, resulting intramitochondrial Ca2+ transients.18,19 In contrast, direct evidence demonstrating Ca2+ sparks modulation by mitochondrial ROS is still lacking. In the present study, we developed a dual-indicator real-time imaging technique to monitor intracellular ROS and Ca2+ sparks simultaneously. In addition, we adopted a photostimulation method13,20 and various pharmacological means to manipulate local or cell-wide mitochondrial ROS production and investigated the Ca2+ spark responses at high spatial and temporal resolution. We found that mitochondria-derived ROS bidirectionally modulate Ca2+ spark activity in a time- and dose-dependent manner. Furthermore, we demonstrated that basal level of ROS is a major determinant for the occurrence of spontaneous Ca2+ sparks.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Result
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Rat cardiac myocyte isolation
All protocols conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Peking University. Single ventricular cardiac myocytes were isolated from the hearts of adult male Sprague–Dawley rats (200–250 g) by a standard enzymatic technique, as described previous.21 Briefly, following anaesthetized by intraperitoneal injection of trichloroacetaldehyde monohydrate (0.5 g/kg), the heart was removed from the chest, cleaned and flushed with nominally Ca2+-free Tyrode solution consisting of (in mM) 137 NaCl, 5.4 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 10 glucose, and 20 Hepes (pH 7.4), and perfused using a Langendorff apparatus. Perfusion began with nominally Ca2+-free Tyrode solution for 5 min at 37°C, and then switched to the enzyme solution with 0.5 mg/mL collagenase (Worthington, Type II) and 0.06 mg/mL protease (Sigma, Type XIV) for 15 min. Following the perfusion procedure, the heart was minced into small chunks and single cells were shaken loose from the heart tissue and stored in Hepes-buffered solution containing (mM): 137 NaCl, 5.4 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1 CaCl2, 20 glucose and 20 Hepes (pH 7.4, adjusted with NaOH). Cells were used within 8 h after isolation.

2.2 Confocal imaging of intracellular Ca2+ and reactive oxygen species
For confocal Ca2+ imaging, freshly isolated myocytes were loaded with Fluo-4 acetoxymethyl ester (AM, 5 µM) or Rhod-2 AM (5 µM) for 10 min followed by a 20 min washout allowing for deesterification. ROS-sensitive fluorescent probe 5-(–6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-DCF) was used to monitor ROS formation. All fluorescent probes were purchased from Molecular Probes (Eugene, OR, USA). To image the ROS and Ca2+ signals simultaneously (Figure 1A), 3–5 µM CM-DCF and 5 µM Rhod-2 AM were sequentially added to the external solution and incubated the cells for 20 and 10 min, respectively. Under the present loading conditions, rhod-2 was largely retained to the cytosol as judged by the homogenous appearance of the indicator-stained cells (Figure 1A).


Figure 1
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  		Figure 1 Imaging intracellular Ca2+, reactive oxygen species (ROS) and photochemically elicited ROS waves in rat ventricular myocytes. (A) (a) Overlay of confocal images of a ventricular myocyte stained with Rhod-2 (Ca2+ indicator, red channel) and CM-DCF (ROS indicator, green channel). (b and c) Enlarged view of the boxed region from (A) for ROS (b) and Ca2+ channels (c), respectively. Arrow in (c) marks a typical Ca2+ spark. (B) Sequential xy images of CM-DCF immediately before (a) and after a local laser flash (b–f). A 6.3 x 6.3 µm2 region of the cell (dashed box) was excited with a laser flash between (a) and (b). Except for the flash, an image was acquired every 3 s. Note the propagation of the ROS wave from the site of excitation toward both ends of the cell. (C) A time-line (t-x) image of the leading front of the ROS wave, created by extracting a line along the longitudinal axis of the cell from the series in (B) and displaying the line images from left to right in the order of collection. The dashed line delineates the wave front detected with a computer algorithm. Note that a cell-wide ROS oscillation started at the end of the t-x image. (D) Linear fitting to the leading front of the ROS wave yielded a wave velocity of 4.3 µm/s in this cell. Similar results were obtained in four other cells.

 
Fluorescence images were recorded using a Zeiss LSM 510 inverted confocal microscope with x40 oil immersion lens (numerical aperture = 1.3). Linescan (xt) and frame (xy) imaging modes were used to measure ROS and Ca2+ signals. CM-DCF and rhod-2 were excited alternately by 477 and 543 nm lasers, and fluorescence was measured at 505–530 and >560 nm bands, respectively. Fluo-4 imaging was excited by 488 nm laser and the fluorescence was collected at >505 nm. Linescan rate was 3.07 ms per line; and 512 x 512 pixel, 8 bit xy images of single or dual channels were taken at the speed of 3.0 s per frame. The normal external solution contained (in mM): 137 NaCl, 5.4 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1 CaCl2, 20 glucose, and 20 Hepes (pH 7.4, adjusted with NaOH). All experiments were performed at room temperature (20–25°C).

During imaging data acquisition, the laser intensity used was too low (477, 488 nm at 25% laser power and 1–2% transmission; 543 nm at full laser power and 15% transmission) to excite significant NADH and flavin autofluorescence (data not shown). We therefore made no correction for autofluorescence in the Ca2+ and ROS measurements. However, there was a significant contamination of the rhod-2 channel signal by the CM-DCF channel signal, but not the other way round, during simultaneous Ca2+ and ROS measurement. To correct for this, we determined the ‘bleeding’ coefficient (0.20), and routinely subtracted the contaminating CM-DCF signal before quantification of Ca2+ sparks and other Ca2+ parameters (see Supplementary material online, Figure S1). Further, we found that the resting fluorescence of fluo-4 or rhod-2 stained cells was insensitive to H2O2 (10–200 µM) applied extracellularly (data not shown).

2.3 Photostimulation of mitochondrial reactive oxygen species production
The method of Aon et al.13 was adopted to elicit local mitochondrial ROS production and to trigger whole-cell ROS oscillation. Specifically, laser flash of high intensity (488 nm, 50–100 times greater than that for image acquisition) was applied to region of 6.3 x 6.3 µm2 (xy scan) or 0.4 x 3.3 µm2 (linescan). The laser photostimulation time varied from 0.06 to 1.25 s, depending on the type of experiment. No image data were acquired during the flashes and flash-associated indicator photobleaching appeared to be negligible (see Supplementary material online, Figure S1). The photoactivated mitochondrial ROS may remain local (at low laser dose) or spread over the entire cell in the form of ROS wave (Figure 1B–D), and sometimes trigger oscillatory cell-wide ROS production.13

2.4 Data analysis
Image processing and data analysis were performed using custom-devised algorithms coded in Interactive Data Language (IDL, Research Systems, Inc., Boulder, CO, USA). For visualization of the spatio-temporal properties of CM-DCF response in Figures 1 and  4, a linescan image was reconstructed from the entire time series of xy images in a given experiment.

2.5 Statistical analysis
Data were expressed as means ± SE. The significance of difference between means was determined using ANOVA, Student's t-test or paired t-test, when appropriate. A P < 0.05 was considered statistically significant.


   3. Result
Top
 Abstract
 1. Introduction
 2. Methods
 3. Result
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Basal level of cytosolic reactive oxygen species was required for spontaneous Ca2+ spark activity
Given that ROS modulate RyR channel activity in vitro,22,23 we first tested the hypothesis that ROS homeostasis plays a role in the regulation of Ca2+ spark activity in intact cells (Figure 2). We showed that Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (TMPyP, 500 µM, 30 min), a membrane-permeant superoxide dismutase (SOD) mimetic, significantly decreased the cytosolic ROS level, evidenced by a 40% reduction of the CM-DCF signal. Concomitantly, TMPyP decreased spontaneous Ca2+ sparks production by 59%, from 15.04 ± 0.59 sparks/6 frames in control group (n = 15 cells) to 6.11 ± 0.74 sparks/6 frames in TMPyP-treated group (n = 5 cells, P < 0.01, Figure 2C). The spark amplitude (F/F0) was also decreased from 1.47 ± 0.01 in control (n = 788 sparks) to 1.39 ± 0.03 in TMPyP-treated group (n = 37 sparks, P < 0.05; Figure 2D), while the spatial width of sparks was unchanged (see Supplementary material online, Figure S2). Since cytosolic ROS production is largely coupled with mitochondrial respiration, we further examined the effect of inhibiting mitochondrial respiratory chain with myxothiazol (Myx), which is thought to suppress ROS generation at Complex III. Similar to the effect of TMPyP, Myx (5 µM, 5 min) lowered the CM-DCF signal by 30% (Figure 2B) and decreased the spark frequency (6.62 ± 0.59 sparks/6 frames, n = 4 cells, P < 0.01 vs. control) and amplitude (1.42 ± 0.03, n = 40 sparks, P < 0.05 vs. control) but not the spark width (see Supplementary material online, Figure S2C). These results indicate that basal level of cytosolic ROS and mitochondrial ROS production are responsible for the majority of spontaneous Ca2+ spark activity. In other words, Ca2+ homeostasis is closely coupled to ROS homeostasis in resting cells.


Figure 2
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  		Figure 2 Effects of lowering cytosolic reactive oxygen species (ROS) concentration on Ca2+ spark incidence and amplitude. (A) Typical Ca2+ spark response to the treatment of myxothiazol (Myx, 5 µM, 5 min), an inhibitor of mitochondrial ROS production at Complex III. (B–D) Cytosolic ROS indexed by CM-DCF fluorescence (B), spark frequency (C) and spark amplitude (D) under various experimental conditions. Myx, 5 µM myxothiazol treated for 5 min; TMPyP: 500 µM TMPyP treated for 30 min. For ratio of F/F0 of rhod-2 (D), F refers to the peak intensity of a spark in the xy confocal image, and F0 the baseline intensity prior to the spark. n = 4–5 cells for each group; *P < 0.05; **P < 0.01 vs. control.

 


Figure 3
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  		Figure 3 Local mitochondrial reactive oxygen species (ROS) bursts enhanced local production of Ca2+ sparks. (A) Representative example showing flash-induced local ROS production and local induction of Ca2+ sparks (ii). Panel (iii) shows the result of subtracting the contaminating CM-DCF signal from the raw data of rhod-2 image (ii). Arrow in (i) denotes the space-time location of the flash (0.06 s, 0.4 x 3.3 µm2). (B) Frequency and unitary properties of Ca2+ sparks near the flash region (CM-DCF {Delta}F/F0 > 1.2 flanking the flash site; n = 60 sparks from six cells) compared with those in regions elsewhere (control, n = 80 sparks). *P < 0.05 vs. control. FWHM, full width at the half maximum; FDHM, full duration at the half maximum.

 


Figure 4
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  		Figure 4 Regulation of Ca2+ sparks by photoactivated cell-wide reactive oxygen species (ROS) oscillation. (A) Time-line plot of CM-DCF fluorescence showing that local photostimulation activated multiple cell-wide ROS oscillations. (B) Ca2+ sparks (denoted by arrowheads) before and after the laser flash. Dashed box marks the flash region, and the bleeding over of CM-DCF signal was not corrected for. (C and D) Time courses of ROS production and Ca2+ spark activity, respectively. Grey arrows (after the flash) in (C) and (D) denote the timing of two subsequent ROS oscillations. (E) Average frequency and amplitude of Ca2+ sparks before the flash (control), during the first ROS oscillation (first reaction), and 10–15 min after the flash. n = 13 cells. *P < 0.05; **P < 0.01 vs. control. (F) Average time course of subsequent ROS oscillations (n = 8 traces) and the corresponding Ca2+ spark activity. Bar graphs to the right show statistics on CM-DCF signal and spark frequency during the ROS oscillations compared to those in time windows of 36 s before and after each oscillation (B&A). *P<0.05 vs. B&A.

 
3.2 Photoactivation of mitochondrial reactive oxygen species waves and oscillations
In order to augment mitochondrial ROS for investigation of Ca2+ spark response, we resorted to photostimulation to activate local and global mitochondrial ROS production, as suggested by previous studies.13,20 With a laser flash delivered at precise time and location, punctuated microstructures of bright CM-DCF signals were observed in the laser-radiated area, which resembled individual mitochondria (Figure 1B), consistent with that photostimulation induces local burst production of mitochondrial ROS.20 If the laser flash was brief (≤0.2 s), these mitochondria-derived ROS signal remained localized (Figure 3A). At increasing duration of the flash, photoactivated mitochondrial ROS bursts sometimes spread over the entire cytoplasm in the form of propagating ROS waves (velocity = 3–10 µm/s, n = 5 cells) (Figure 1C and D), perhaps via the ROS-induced ROS release mechanism.20 Moreover, in a subpopulation of cells (~70%), such ROS waves triggered self-sustaining cell-wide ROS oscillations that displayed a duration of 51.85 ± 6.07 s (measured as the full duration at half maximum) and a periodicity of 100.51 ± 16.01 s (n = 32) (Figure 4A and C). Though the exact mechanism underlying mitochondrial ROS bursts and oscillations is not fully understood,13 we exploited them as novel means to elicit local and global mitochondrial ROS signals to probe Ca2+ spark response (see below).

3.3 Ca2+ sparks evoked by local mitochondrial reactive oxygen species bursts
To test the possibility of local regulation of Ca2+ spark activity by mitochondrial ROS, we applied brief photostimulation to a small area (≤0.2 s, 0.4 x 3.3 µm2) and monitored local ROS and spark production simultaneously (Figure 3), using the dual-indicator imaging technique developed in this study. We found that photostimulated mitochondrial ROS burst (Figure 3A, top panel) rapidly evoked 2.4-fold greater Ca2+ spark activity in the proximity of the flash-radiated area (5.01 ± 1.08 sparks/100 µm s) as compared with other region of the cytosol (2.11 ± 0.93 sparks/100 µm s, n = 6 cells, P < 0.05) (Figure 3). The morphology of Ca2+ sparks, indexed by the amplitude, spatial width, and duration, however, was largely unchanged (Figure 3B). That is, mitochondrial ROS locally increased the propensity of spark activation without affecting properties of individual events.

3.4 Bidirectional regulation of Ca2+ spark frequency by global mitochondrial reactive oxygen species oscillation
Photoactivated oscillatory cell-wide mitochondrial ROS production afforded a novel experimental setting which permitted us to further probe the kinetic response of Ca2+ sparks (Figure 4). In 270 s control period at normal imaging laser intensity, the incidence of Ca2+ sparks was at a near constant rate of 15.09 ± 1.34 sparks/6 frames (n = 13 cells), despite a gradual rise of CM-DCF signal; the latter likely reflects ROS accumulation stimulated by the low-level laser of imaging acquisition.13 After a >0.8 s laser flash and step increase of cytosolic ROS level, Ca2+ spark frequency was immediately increased. On average, Ca2+ spark frequency during the period of first ROS oscillation was 21.72 ± 2.90 sparks/6 frames (averaged over 0–54 s after the flash, n = 13 cells), which was 44% higher than that in the control period (P < 0.05). However, the mitochondrial ROS activation of Ca2+ sparks appeared to be only transient, as the sparks frequency declined gradually, opposite to the continuous ROS accumulation. At 10 min after the laser flash, the spark frequency was reduced to a level that was even 27% lower than control (11.05 ± 1.29 sparks/6 frames, n = 13 cells, P < 0.01 vs. control) (Figure 4E). It is noteworthy that the flash and the ROS accumulation did not appreciably alter the average cytosolic Ca2+ (see Supplementary material online, Figure S1).

In addition to the biphasic time course of spark response, a careful examination revealed that subsequent ROS oscillation was mirrored by phasic depression of the spark activity (Figure 4D and F). The average rate of Ca2+ spark occurrence during subsequent ROS oscillation was 10.25 ± 0.95 sparks/6 frames (8 oscillations from 7 cells), whereas the average spark frequency 36 s before and after the oscillation was significantly higher (12.85 ± 0.88 sparks/6 frames, P < 0.05) (Figure 4F). Thus, kinetically resolved measurement unmasked a mitochondrial ROS-induced spark depression following the initial mitochondrial ROS-induced spark activation. This indicates that mitochondrial ROS modulation of Ca2+ spark activity is bimodal and history-dependent.

Parametric analysis of Ca2+ spark morphology suggested that, during the first ROS oscillation, the spark amplitude (Figure 4E) and spatial size (see Supplementary material online, Figure S2A) were unaltered compared with control, whereas spark amplitude between 10 and 15 min after the flash was reduced from 1.47 ± 0.01 (n = 788 sparks in control) to 1.37 ± 0.02 (n = 266 sparks, P < 0.01 vs. control, Figure 4E). Importantly, no significant change in spark morphology was observed during ROS oscillation-induced phasic spark depression (see Supplementary material online, Figure S2B).

3.5 Biphasic response of Ca2+ spark activity to antimycin A-induced mitochondrial reactive oxygen species production
Inhibiting Complex III of the mitochondrial respiratory chain with antimycin A (AA) has been shown to stimulate mitochondrial superoxide anion and consequential H2O2 production,13 affording means for augmenting mitochondrial ROS independently of photostimulation. We found that 5 µg/mL AA altered the Ca2+ spark frequency in a biphasic, time-dependent manner while monotonically enhanced the CM-DCF signal (Figure 5A–C). An initial 2.8-fold increase in spark activity peaked at 4 min, and then a progressive decline resulted in a significant 53% suppression at 10 min. Characteristics of individual spark displayed no significant changes except for a small decline in amplitude at 10 min after AA application (see Supplementary material online, Figure S3). Taken together, both photochemical and pharmacological enhancements of mitochondrial ROS production exert bidirectional, time-dependent effects on Ca2+ spark activity.


Figure 5
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  		Figure 5 Effects of antimycin A (AA) and H2O2 on Ca2+ spark production. (A) Time-dependent effect of AA (5 µg/mL) on Ca2+ spark activity. (B and C) Time courses of CM-DCF signal (F/F0) (B) and spark frequency (C) after AA treatment. n = 6; *P < 0.05; **P < 0.01 vs. control at the corresponding time points. (D) Pretreatment of cells with TMPyP (250 µM, 30 min, n = 9 cells) decreased the incidence of spontaneous Ca2+ sparks and prevented AA from altering the spark activity. (E) Biphasic effect of 200 µM H2O2 on Ca2+ spark frequency (n = 12 cells); *P < 0.05; **P < 0.01 vs. control.

 


Figure 6
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  		Figure 6 Possible mechanism underlying mitochondrial reactive oxygen species modification of Ca2+ sparks. (A) The SR Ca2+ contents during photostimulation (left panel, n = 9 cells) or AA treatment (right panel, n = 6 cells). Data were expressed as percentage of {Delta}F/F0 under control conditions. * P< 0.05 vs. control. (B) Effect of DTT on flash-induced Ca2+ spark response. (Insert) Cell-wide ROS accumulation in the presence or absence of DTT. n = 6 cells for each group.

 
In attempt to define which specific ROS were responsible for the spark modulatory effects described above, we showed that the SOD mimetic TMPyP, while decreasing spontaneous spark activity, effectively abolished the AA-induced spark response (Figure 5D). This result suggests the involvement of superoxide anions—the primal ROS from the mitochondria. Furthermore, we found that application of 200 µM H2O2, a more stable and membrane permeable derivative of superoxide anion, reproduced to a large extent the AA-induced spark response (Figure 5E). Notably, H2O2 at 50 µM H2O2 increased Ca2+ spark activity over the entire 10 min observation period and was ineffective at 10 µM in eliciting any significant changes in spark production (see Supplementary material online, Figure S4). These results suggest that H2O2 may also contribute to mitochondrial ROS regulation of Ca2+ spark activity, if it is presented at sufficiently high local concentrations.

3.6 Possible molecular mechanisms underlying mitochondrial reactive oxygen species modulation of Ca2+ sparks
The Ca2+ content inside the SR is thought to be an important determinant of spark frequency and characteristics.24,25 We thus examined the effect of mitochondrial ROS on the caffeine-sensitive Ca2+ store under the present experimental conditions and the results are summarized in Figure 6A. During either photostimulation or AA treatment, there was a trend of progressive SR store depletion; a significant effect was first detected at about 10 min after the flash or AA application. This result is consistent with the corresponding data on mitochondrial ROS modulation of spark amplitude, suggesting that partial store depletion may contribute to the time-dependent depression of Ca2+ sparks (see Section 4).

To test the hypothesis that oxidative modification of RyR underlies the spark response to mitochondrial ROS, we examined the effect of dithiothreitol (DTT),5 an SH-reducing agent, on flash-induced Ca2+ spark response. Administration of 20 mM DTT decreased the resting CM-DCF signal by 30%. Concomitantly, the rate of spontaneous Ca2+ sparks was markedly decreased by 72%, from 14.81 ± 0.41 to 4.09 ± 0.12 sparks/6 frames, n = 6 cells (P < 0.01 vs. control). In the presence of DTT, flash-induced step increase of ROS failed to elicit any significant changes in Ca2+ spark activity; subsequent time-dependent rise of the global ROS did not suppress the spark activity, either (Figure 6B). That DTT abolished flash-induced spark activation and depression are consistent with the idea that mitochondrial ROS modulation of sparks is mediated, at least in part, by altering the redox status of RyRs in intact cells.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Result
 4. Discussion
Supplementary material
 Funding
 References
 
4.1 Regulation of Ca2+ sparks by basal mitochondrial reactive oxygen species production
Spontaneous sparks occur in quiescent unstimulated ventricular myocytes and these sparks have been thought to simply reflect the probabilistic opening of RyRs by the CICR mechanism operating at resting [Ca2+]c of about 100 nM with a normal (i.e. not overloaded) SR.21,26 The present study has demonstrated that baseline mitochondrial ROS production plays an important role in maintaining such spontaneous Ca2+ spark activity. Inhibition of mitochondrial ROS production decreased the basal cytosolic ROS level and diminished spontaneous Ca2+ spark production by as much as 60%. That basal mitochondrial ROS are spark activators underscores a physiological role of ROS in Ca2+ homeostasis, in contrast to the prevalent view that mitochondrial ROS are inevitable, potentially harmful by-products of oxidative respiration. The above finding also provides supporting evidence for the notion on RyR as a cellular redox senor.3,4 Indeed, lowering the resting ROS level with TMPyP or inhibiting mitochondrial ROS production with Myx decimated the spontaneous Ca2+ spark activity. Similar result was obtained with the reducing reagent DTT presented at millimolar concentrations. The latter is in general agreement with the report that NADH, a reducing reagent as well as an energy metabolite, inhibits RyR channel activity in lipid bilayers and spontaneous Ca2+ sparks in saponin-permeablized cardiac myocytes.27

However, caution should be excised in translating the present findings to physiological situations. Our experiments used glucose as the only substrate for energy production and were performed at the room temperature in freshly isolated adult cardiac myocytes, as opposed to physiological temperature (37°C) and the use of fatty acid as the major metabolic substrate in vivo. Limitations in the present study also include the presence of a variety of fluorescent probes which might affect the metabolic state of the mitochondria under study, and the presence of oxygen tension (in ambient air) that is higher than is usually seen in myocardium.

4.2 Regulation of Ca2+ sparks by stimulated mitochondrial reactive oxygen species production
Another key finding of this study, we have shown that photochemically and pharmacologically stimulated excessive mitochondrial ROS production exerts a bidirectional regulation of Ca2+ spark activity in a dose- and time (history)-dependent fashion. This finding was made possible by two technical improvements. First, we adopted the photostimulation method developed by Aon et al.13 to create local mitochondrial ROS bursts, ROS waves, or self-sustaining cell-wide ROS oscillations. Second, we developed a dual-indicator imaging method for simultaneous visualization of ROS and Ca2+ signals, permitting investigation of Ca2+ spark response to local and global mitochondrial ROS at high temporal and spatial resolution. Several lines of evidence have been provided to support our conclusion. After an intense laser flash that evoked global ROS oscillation, a transient spark activation was found to accompany a step increase of cell-wide ROS level. However, subsequent gradual rise in global ROS mirrored a progressive decline in Ca2+ spark frequency, suggesting bidirectional regulation of spark activity by mitochondrial ROS. Intriguingly, ROS oscillations that succeeded the initial spark activation were associated with phasic depression of Ca2+ spark activity, indicative of a modality switch of spark regulation by mitochondrial ROS from excitatory to inhibitory. Similar time-dependent bidirectional regulation of spark activity was observed when cells were treated with AA at 5 µg/mL, which increased cytosolic ROS level presumably by stimulating mitochondrial ROS production at Complex III.

A spark-activating component of ROS action has recently been reported in cardiac, skeletal, and smooth muscle cells.20,28,29 Zorov et al.20 demonstrated that photoactviated burst production of ROS from individual mitochondria enhanced local spark incidence. Exogenous ROS generated from xanthine/xanthine oxidase reaction stimulate single cardiac RyR channel activity in planar bilayers.27 Elevating ROS production with diazoxide, which is thought to be an opener of putative mitochondrial KATP channel, results in an increase of Ca2+ spark frequency in arterial smooth muscle cells.29 Isaeva et al.28 reported that mitochondrial redox potential determines the appearance of Ca2+ sparks in permeabilized skeletal muscle fibre.

The apparent discrepancy between the present and previous studies might be explained by the level of ROS and the time window of observation involved. In this regard, our results showed that bidirectional spark regulation is usually associated with relatively high ROS levels, while basal ROS appear to be excitatory in spark regulation. In response to a flash subthreshold to activating ROS oscillation, local mitochondrial ROS bursts evoked a flurry of regional Ca2+ spark activity with no spark-inhibiting component (Figure 3), consistent with previous report.20 Furthermore, we showed that, while extracellular application of 200 µM H2O2 reproduced the bidirectional spark response to AA and photostimulation, lower concentration of H2O2 (50 µM) elevated spark activity that did not show sign of depression over the entire period of experiment (10 min) (see Supplementary material online, Figure S4). In permeabilized cardiac cells, superoxide anions generated in situ from xanthine/xanthine oxidase reaction cause a slight transient increase followed by slowly developing decrease of Ca2+ spark frequency.28 Previous report has also shown that H2O2 at 1 mM concentration elicits a transient augmentation followed by a suppression of action potential-evoked [Ca2+]c transient after 5 min of exposure.16 In planar lipid bilayer experiments, only steady-state behaviour of ROS modulation of RyR activity has thus far been explored. Favero et al.23 demonstrated that the open probability of skeletal RyRs incorporated into lipid bilayer was bidirectionally regulated by H2O2: 100 µM H2O2 increases RyR open probability while inhibiting RyR open probability at very high concentrations (1–10 mM). This result is in general agreement with the present findings in intact cells.

4.3 Possible role of SR Ca2+ store depletion
It has been shown that SR lumenal Ca2+ affects RyR open probability30 through interactions with the Ca2+ binding protein and both junctin and triadin.31,32 To determine possible contribution of the SR Ca2+ to the bidirectional spark regulation by mitochondrial ROS, we have shown that the initial increase in spark activity in photostimulation or AA treatment is associated with no change in SR Ca2+ store, whereas the SR Ca2+ content decreased to ~70% of control level at the later experimental time point (5–10 min after photostimulation or 10 min after addition of AA). This partial store depletion is due perhaps to initial ROS increase in spark-mediated SR leak and ROS inhibition of the SR Ca2+ ATPase.11

The above results suggest that the time-dependent decrease in the SR content contribute at least in part to the gradual decrease in spark frequency. However, it appears that partial SR depletion alone cannot fully account for diminishing spark production. In previous study by Song et al.,24 decreasing the SR Ca2+ content to the same extent (i.e. 70% of control) caused merely 20% decrease in the apparent Ca2+ spark frequency. In the present study, however, the spark frequency declined by 49% and 90% relative to the peak activity in photostimulation and AA experiments, respectively. This suggests that additional time-dependent negative regulatory mechanism underlies the biphasic response of spark activity. This view is supported by the finding that, during subsequent ROS oscillations, phasic depression of spark frequency occurred without changing the spark amplitude (and perhaps, the SR Ca2+ load).

In summary, our data indicate that the mitochondrial ROS regulation of Ca2+ signalling can be both excitatory and inhibitory, depending on ROS levels and recent history of ROS modification. Mitochondrial ROS level in the physiological regime is predominantly excitatory in regulating the Ca2+ spark activity. At excess, mitochondrial ROS appear to be inhibitory. Regulatory or pathophysiological transient increase of ROS can either produce phasic activation (starting from an excitatory regime) or phasic depression (starting from an inhibitory regime) of SR Ca2+ release. In addition, local coupling of mitochondrial ROS to Ca2+ sparks would enrich spatio-temporal dynamics of the interplay between ROS and Ca2+ signalling. Thus, the present findings may bear important implications in understanding role of mitochondrial ROS in Ca2+ homeostasis as well as Ca2+ dysregulation in oxidative stress-related diseases.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Result
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Result
 4. Discussion
 Supplementary material
 Funding
References
 
This study was supported in part by grants National Natural Science Foundation of China (No. 30630021, No. 30628009) and the Major State Basic Research Development Program of China (973 Program, No. 2007CB512100).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Result
 4. Discussion
 Supplementary material
 Funding
 References
 

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