Voltage-dependent anion channel 2 modulates resting Ca2+ sparks, but not action potential-induced Ca2+ signaling in cardiac myocytes

Cell Calcium 49 (2011) 136–143

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Voltage-dependent anion channel 2 modulates resting Ca2+ sparks, but not action potential-induced Ca2+ signaling in cardiac myocytes Krishna Prasad Subedi a , Joon-Chul Kim a , Moonkyung Kang b , Min-Jeong Son a , Yeon-Soo Kim b , Sun-Hee Woo a,∗ a b

College of Pharmacy, IDRD, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejeon 305-764, South Korea Indang Institute of Molecular Biology and Department of Medical Laboratory Science, Inje University, Seoul 100-032, South Korea

a r t i c l e

i n f o

Article history: Received 29 July 2010 Received in revised form 12 November 2010 Accepted 20 December 2010 Available online 15 January 2011 Keywords: Voltage-dependent anion channel 2 Knock down Ca2+ spark Cardiac Ca2+ signaling Lentivirus HL-1 cell

a b s t r a c t Voltage-dependent anion channels (VDACs) are pore forming proteins predominantly found in the outer mitochondrial membrane and are thought to transport Ca2+ . In this study, we have investigated the possible role of type 2 VDAC (VDAC2) in cardiac Ca2+ signaling and Ca2+ sparks using a lentiviral knock-down (KD) technique and two-dimensional confocal Ca2+ imaging in immortalized autorhythmic adult atrial cells, HL-1. We confirmed high expression of VDAC2 protein in ventricular, atrial, and HL-1 cells using Western blot analysis. Infection of HL-1 cells with VDAC2-targeting lentivirus reduced the level of VDAC2 protein to ∼10%. Comparisons of autorhythmic Ca2+ transients between wild-type (WT) and VDAC2 KD cells showed no significant change in the magnitude, decay, and beating rate of the Ca2+ transients. Caffeine (10 mM)-induced Ca2+ release, which indicates sarcoplasmic reticulum (SR) Ca2+ content, was not altered by VDAC2 KD. Interestingly, however, the intensity, width, and duration of the individual Ca2+ sparks were significantly increased by VDAC2 KD in resting conditions, with no change in the frequency of sparks. VDAC2 KD significantly delayed mitochondrial Ca2+ uptake during artificial Ca2+ pulses in permeabilized HL-1 cells. These results suggest that VDAC2 may facilitate mitochondrial Ca2+ uptake and restrict Ca2+ spark expansion without regulating activations of sparks under resting conditions, thereby providing evidence on the functional role of VDAC2 in cardiac local Ca2+ signaling. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction The contraction of mammalian cardiac myocytes is controlled by a sequence of events (excitation–contraction coupling, E–C coupling) that includes the Ca2+ current (ICa )-gated opening of Ca2+ release channels (ryanodine receptors, RyRs) and the release of Ca2+ from the sarcoplasmic reticulum (SR) [1–4]. Confocal Ca2+ imaging of cardiac myocytes has revealed the presence of focal Ca2+ transients (Ca2+ sparks) [5] that are activated spontaneously or by ICa [5–7]. The unitary property of Ca2+ sparks, but not their frequency, appears to be independent of ICa and voltage, indicating that Ca2+ sparks may represent the elementary event underlying cardiac E–C coupling [5–8]. The released Ca2+ from the SR in turn binds to the Ca2+ sensing motif on the carboxyl-terminal of the Ltype Ca2+ channel via calmodulin, which prevents the Ca2+ entry through the channel [9]. After having activated the contractile elements, the released Ca2+ is removed from the cytoplasm by the SR

∗ Corresponding author. Tel.: +82 42 821 5924; fax: +82 42 823 6566. E-mail address: [email protected] (S.-H. Woo). 0143-4160/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2010.12.004

Ca2+ ATPase, and by the sarcolemmal Ca2+ ATPase and Na+ –Ca2+ exchanger. The mitochondria, the main role of which is the production of ATP, may also buffer the elevated Ca2+ concentration in cardiac myocytes via carrying Ca2+ into the matrix through its inner membrane [10–13]. Such mechanism may be possible in the intermyofibrillar mitochondria that are positioned closest to the microdomains of the cell membrane–SR junctions [13,14] where Ca2+ sparks occur. Because of their location and function, it is possible that the mitochondria regulate SR Ca2+ cycling and Ca2+ sparks. To control cytosolic Ca2+ concentration, mitochondria have five known pathways for the influx and efflux of Ca2+ across the inner membrane [15,16]. However, Ca2+ transport through the inner mitochondrial membrane requires Ca2+ crossing the mitochondrial outer membrane as well. The most plausible protein entity for Ca2+ transport across the outer mitochondrial membrane is thought to be the voltage-dependent anion channel (VDAC). VDAC, known as porin, is a large channel that transports cations, anions and metabolites, including nucleotides [17–19], and is a component of the mitochondrial permeability transition pore (PTP). A previous report using purified VDAC in liposome has demonstrated that VDAC is

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highly Ca2+ permeable, has Ca2+ binding sites, and is involved in PTP regulation [20]. However, while VDAC has been extensively characterized electrophysiologically, its role in intracellular Ca2+ signaling has not been identified. Three mammalian VDAC isoforms (VDAC1, VDAC2, and VDAC3) have been characterized [21–24], and it has been suggested that they each have a distinct physiological function. All three isoforms are abundantly present in mammalian cells [22], but VDAC2 is the only mammalian-specific isoform [25]. VDAC2, the focus of this study, was discovered in the ventricles of mice by Western blotting [26] and detected in mitochondrial membrane fractions prepared from the hearts of rats [27]. Northern blot analysis also detected a VDAC isoform corresponding to human VDAC2 in the hearts of mice [21]. In the present study, we investigated the possible role of VDAC2 in Ca2+ sparks, action potential (AP)-triggered Ca2+ transients, and SR Ca2+ contents using laser scanning two-dimensional (2D) confocal Ca2+ imaging and lentiviral VDAC2 protein knock down (KD) in cardiac cells. We also examined whether lentivirus infection affects those parameters. We adopted immortalized adult cardiac HL-1 cells in this study in order to overcome the limitation of using isolated cardiac myocytes in producing KD cells using lentivirus for the research of Ca2+ sparks. We have previously shown that HL-1 cells have no t-tubule, junctional and nonjunctional Ca2+ sparks, similar to atrial myocytes, and autorhythmic Ca2+ transients at ≥60% confluences [28]. We found that VDAC2 KD significantly increased the duration, intensity, and size of resting Ca2+ sparks.

Reference Reagent Program. In our routine preparation, titers were approximately 106 –107 transduction unit (TU) per ml without further concentration.

2. Methods

2.4. Cytosolic Ca2+ imaging and image analysis

2.1. HL-1 cell culture HL-1 cells were grown and maintained on a gelatin [0.02% (wt/vol)]/fibronectin (12.5 ␮g/ml) matrix containing Claycomb medium (SAFC Biosciences) supplemented with 10% fetal bovine serum (JRH Biosciences), 2 mol/L l-glutamate (Invitrogen), 0.1 mmol/L norepinephrine (Sigma), 100 U/ml penicillin (Invitrogen), and 100 ␮g/ml streptomycin (Invitrogen) [29]. The medium was changed within every 24 h. After trypsinization, the dissociated cells were either plated on standard 60 mm tissue culture dishes for protein expression assays or on 10 cm2 culture dishes containing a glass cover slip for confocal microscopic study. 2.2. Lentivirus production The short hairpin RNA (shRNA) lentiviral vector for targeting VDAC2 mRNA was constructed by inserting synthetic double-stranded oligonucleotides (target sequence 5 -GCTGACAAGGAGTAACTTTGC-3 ) into the shLenti2.4R vector containing a U6 small nuclear RNA promoter upstream of the multicloning sites and a red fluorescence protein (RFP) gene-IRESpuromycin-resistance gene under the control of a human CMV promoter. To create a negative control shRNA vector, the scrambled sequence 5 -AATCGCATAGCGTATGCCGTT-3 was inserted into the vector. The lentivirus was produced by cotransfection of HEK293 T cells with three plasmids: (1) a construct expressing the heterologous envelope protein VSV-G, (2) a packaging defective helper construct expressing the gag-pol gene, and (3) a transfer vector harboring a specific shRNA sequence using Lipofectamine Plus (Invitrogen). At 48 h post-transfection, virus-containing culture supernatants were collected and clarified with a 0.45-mm membrane filter (Nalgene, USA), and immediately stored in a deep freezer at −70 ◦ C. Titers were determined by p24 ELISA (Perkin-Elmer Life Science) or Western blot analysis using a monoclonal anti-p24 antibody obtained through the AIDS Research and

2.3. Protein extraction and Western blotting The cells were scraped in SDS lysis buffer [10 mM Tris–HCl, pH 7.4, 1% (w/v) SDS, 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM Na3 VO4 , and a protease inhibitor cocktail (Roche)]. The cell suspension was then boiled for 5 min, triturated by being passed several times through a 1 ml syringe, and centrifuged at 12,000 × g for 10 min at 4 ◦ C. The resultant supernatant was combined with 2× Laemmli sample buffer, boiled for 5 min, and stored at −80 ◦ C until use. The protein concentrations were determined by the Bradford assay using bovine serum albumin as a standard. Protein samples were separated by SDS-PAGE, and VDAC2 and ␣-actinin were detected using a standard Western blot protocol. Briefly, 15 ␮g each of protein samples were run on a 10% SDS-polyacrylamide gel and subsequently electrotransferred to nitrocellulose membranes. Nitrocellulose membranes were then probed with a VDAC2specific polyclonal antibody and polyclonal antibody to ␣-actinin (Santa Cruz Biotechnology, Inc.) at the recommended dilutions and developed using an enhanced chemiluminescence detection system (Santa Cruz Biotechnology, Inc.). Pre-stained SDS-PAGE molecular weight markers, broad range (Precision Plus Protein Standards, Bio-Rad), were run in parallel to estimate the molecular weight of the immunoreactive bands.

Imaging experiments were carried out by using a laser scanning confocal microscopy system (C1 Eclipse, Nikon, Japan) attached to an inverted microscope (TS2000U, Nikon) fitted with a ×60 oil-immersion objective lens (Plan Apo, Numerical Aperture 1.4, Nikon). To image the Ca2+ fluorescence, HL-1 cells were loaded with 3 ␮M fluo-4 AM for 30 min at 37 ◦ C. The dyes were excited at 488 nm using an argon ion laser and fluorescence emission wavelengths >510 nm was detected. To detect the level of lentivirus infected HL1 cells, the same cells were excited at 543 nm for red fluorescence protein (RFP) fluorescence. The dye-loaded cells were continuously superfused with normal Tyrode solution composed of (in mM) 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2 , 2 CaCl2 , and 10 glucose (pH 7.4, titrated with NaOH). After 10 min of superfusion for washing external fluo-4 AM and for stabilizing cell-beatings, autorhythmic Ca2+ transient signals were two-dimensionally imaged at 60 Hz at 36 ◦ C. A caffeine-containing external solution was shortly applied using a rapid solution exchange system [4] to measure the caffeine-triggered Ca2+ transients. Local averaged Ca2+ signals were measured using a PC program EZ-C1 (Nikon). The average diastolic fluorescence intensity (F0 ) was calculated from several frames measured before Ca2+ upstrokes. Tracings of Ca2+ transients from the region-of-interest (ROI) were shown as the fluorescence of each area normalized relative to the F0 (F/F0 ). In order to measure Ca2+ sparks, autorhythmic Ca2+ changes were suppressed by superfusing a 0.05 mM Ca2+ -containing Tyrode solution. Only cells free of Ca2+ waves were analyzed. Focal Ca2+ releases were automatically identified by a computerized algorithm in the “RealTimeMicroscopy” PC program (upgraded version of “PIC”, own written in C++; [28]). First, this algorithm subtracted the average background signal (F0 ) from the raw image along the x and y directions. If the pixel fluorescence ratio (F/F0 ) was ≤0.3 the signals became zero by low-pass filtering, whereas the signal brighter than the critical value remained intact. The “groups” (“local maxima”), which were defined as connected non-zero signals, were then identified as Ca2+ sparks candidates. If the distance between

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∼ a signal and a group was below a critical distance (=1.2 ␮m), the signal was included in the group. If the number of signals in a group was >5, we then assigned the group as a real spark. To detect local maxima underneath the cell membrane, the extracellular space was excluded by setting up a mark at the cell border in 2D images [30]. The center of a real spark, a position with maximal intensity in the group, was visualized in the image as a mark and exported as a coordinate (Fig. 4A and E in [28]). To calculate the frequency of sparks, whole cell areas were measured using the EZ-C1 (Nikon). To examine the unitary properties of single sparks, Gaussian fitting was performed at the spark center, which was identified by the PC program, in 2D with the same algorithm previously described [30]. Individual Ca2+ sparks in single frames were approximated in a restricted area (30 pixels × 30 pixels) by a Gaussian function at the spark center identified by the RealTimeMicroscopy program in order to give the amplitude (F1 /Fr : F1 is the magnitude of Gaussian curve and Fr means the background fluorescence in the restricted spark area) and the full width at half maximum (FWHM). Full duration at half maximum (FDHM) was evaluated from the signal trace at the centermost part of each spark. 2.5. Mitochondrial Ca2+ measurement After HL-1 cells were loaded with fluo-4 AM (Invitrogen, CA, USA) at 20 ␮M for 1 h in culture media, the cells were washed for 10 min. To remove cytosolic fluo-4, the plasma membrane was permeabilized by perfusion of digitonin (20 ␮M) in a Ca2+ -free internal solution that contained (in mM) 35 KCl, 100 K-glutamate, 6 glucose, 5 NaCl, 20 HEPES, 1 MgCl2 , 3 Mg-ATP, 4 EGTA (pH 7.25 with KOH). After the plasma membrane was permeabilized, the free Ca2+ concentration in the internal solution (extramitochondrial [Ca2+ ], [Ca2+ ]em ) was increased according to the experimental protocol. The [Ca2+ ]em was obtained by mixture of 4 mM EGTA and CaCl2 , and was calculated using the Maxchelator (MAXC) v1.0 (Stanford University) computer program. Confocal Ca2+ imaging was performed using a laser-scanning confocal microscope (A1, Nikon) coupled to an inverted microscope (TS2000U, Nikon) fitted with a ×60 oilimmersion objective lens (Plan Apo, Numerical Aperture 1.4, Nikon) as described above. Mitochondrial Ca2+ change was estimated as fluorescence intensity (F) normalized with fluorescence measured after the exposure to digitonin (F0 ). To localize mitochondria cells were loaded with 200 nM MitoTracker Green for 10 min (excitation wavelength, 488 nm; emission wavelength >510 nm) after Ca2+ imaging. The MitoTracker fluorescence was much brighter than fluo-4 fluorescence, and was detected at the PMT level where fluo-4 signal was not visible. 2.6. Statistical analysis Data are summarized as means ± standard error of mean (SEM). Statistical comparisons were carried out using Student’s t-tests. Differences were considered to be statistically significant to a level of P < 0.05. 3. Results 3.1. Expression of VDAC2 protein in adult rat cardiac myocytes and HL-1 cells We first analyzed the expression of VDAC2 in isolated adult rat cardiomyocytes and autorhythmic atrial cell population HL-1 cells. Our Western blot analysis revealed a high level of VDAC2 expression in isolated atrial and ventricular myocytes, and HL-1 cells (“Atrial” and “HL-1 Con-LV”, Fig. 1). It should be noted that the VDAC2 expression in uninfected HL-1 cells and that in lentivirus

Fig. 1. Expression of VDAC2 in cardiac myocytes and knock down (KD) of VDAC2 using lentivirus. (A) Representative immunoblots showing expression level of VDAC2 in atrial myocytes (Atrial), HL-1 cells infected with control lentivirus (HL-1 Con-LV) and lentivirus targeting VDAC2 mRNA (HL-1 VDAC2-LV), and ventricular myocytes (Ventricular). ␣-Actinin was used as a loading control. The cells were lysed in a SDS containing buffer, and 15 ␮g of total protein was run on 10% SDSpolyacrylamide gel. Blots were sequentially probed with VDAC2 (upper panel) and ␣-actinin (lower panel) antibodies. (B) Summarized densitometric data to show relative intensity. Mean ratio of the densities of VDAC2 vs. ␣-actinin was expressed in arbitrary unit (a.u.). Four experiments were done in duplicate. VDAC2 was highly expressed in atrial myocytes and HL-1 cells, and infection of HL-1 cells with lentivirus targeting VDAC2 mRNA resulted in significantly reduced level (∼90% reduction) of VDAC2 protein expression as compared to uninfected and control lentivirus infected cells. **** P < 0.0001, HL-1 Con-LV vs. HL-1 VDAC2-LV. ## P < 0.01, Atrial vs. Ventricular; P > 0.05, Atrial vs. HL-1 Con-LV.

infected cells were similar (data not shown). VDAC2 protein was also highly expressed in isolated ventricular myocytes (Fig. 1, “Ventricular”), which is consistent with a previous report [27]. The level of VDAC2 protein in atrial cells was significantly higher than that in ventricular myocytes (Fig. 1B, P < 0.01). The results support the physiological significance of VDAC2 in the heart. 3.2. Down regulation of VDAC2 expression using lentivirus in HL-1 cells In order to examine the role of VDAC2 in cardiac Ca2+ signaling, we knocked down VDAC2 expression using lentivirus in HL-1 cells. For this purpose, we generated lentivirus targeting VDAC2 mRNA as described in ‘Section 2’. Two consecutive infections of HL1 cells with the lentivirus resulted in 70–80% of the cells expressing RFP, indicating that the majority of the cells were infected with lentivirus. Consistent with this, our Western blot data showed very low levels (approximately 10% of the control) of VDAC2 protein expression in HL-1 cells infected with VDAC2-targeting lentivirus

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(“HL-1 VDAC2-LV”, Fig. 1) compared to cells infected with control lentivirus (“HL-1 Con-LV”, Fig. 1). This result indicates that the lentivirus, we generated, is able to target and significantly downregulate VDAC2 expression in HL-1 cells. 3.3. Lentivirus infection and VDAC2 KD do not alter Ca2+ transients and SR Ca2+ contents To examine whether VDAC2 regulates intracellular Ca2+ signaling on depolarization, we compared autorhythmic Ca2+ transients in uninfected, wild type virus infected (WT), and VDAC2 KD HL-1 cells using confocal Ca2+ imaging. HL-1 cells with 70–80% confluences normally showed autorhythmic Ca2+ transients, as shown in Fig. 2B. The Ca2+ transients are known to be triggered by action potentials and the autorhythmic nature is caused by a small portion of nodal cells in the HL-1 cell population [31]. The infected cells were distinguished from uninfected cells by visualizing the expression of RFP and superimposing that with fluo-4-Ca2+ fluorescence (green) (Figs. 2A and 3A). Fig. 2B shows representative Ca2+ signals measured from uninfected and virus infected HL-1 cells, illustrated in Fig. 2A (see dotted lines). Both uninfected and infected HL-1 cells displayed autorhythmic Ca2+ transients. The magnitudes (F/F0 ) and decay time (half decay time, T1/2 ) of the autorhythmic Ca2+ transients were similar in the uninfected (0.35 ± 0.03 and 0.30 ± 0.01 s, respectively, n = 50) and WT (0.35 ± 0.03 and 0.32 ± 0.02 s, respectively, n = 40) HL-1 cells (Fig. 2B and C, P > 0.05). The SR Ca2+ contents, measured as the magnitudes (F/F0 ) of caffeine (10 mM)-induced Ca2+ transients, were not significantly different between the uninfected (1.39 ± 0.06, n = 57) and WT cells (1.40 ± 0.07, n = 41, P > 0.05; Fig. 2B and D). In addition, the decay of caffeine-induced Ca2+ transients was not altered by virus infection (P > 0.05; Fig. 2B and D). The beating rates measured in the infected (1.24 ± 0.02, n = 37, 6 batches of cells) and uninfected (1.18 ± 0.02, n = 37, 6 batches of cells) cells were not significantly different (P > 0.05; Fig. 2B and E). This indicates that lentivirus infection may not affect depolarizationinduced Ca2+ transients or the amount of Ca2+ in the SR of HL-1 cells. In Fig. 3, the characteristics of autorhythmic Ca2+ transients and caffeine-induced Ca2+ transients in WT (n = 40) and VDAC2 KD cells (n = 21) were compared. The magnitude (F/F0 ) and decay time (half decay time, T1/2 ) of autorhythmic Ca2+ transient were not significantly different between the VDAC2 KD cells (0.39 ± 0.08 and 0.34 ± 0.03 s, respectively) and WT cells (0.36 ± 0.03 and 0.32 ± 0.02 s, respectively) (P > 0.05; Fig. 3B and C). The magnitude (F/F0 ) and decay time (half decay time, T1/2 ) of caffeine-induced Ca2+ transients were also similar between the WT (1.40 ± 0.07 and 1.29 ± 0.06 s, respectively) and VDAC2 KD (1.44 ± 0.09 and 1.35 ± 0.08 s, respectively) cells (P > 0.05; Fig. 3B and D). The beating rate was not changed by VDAC2 KD (P > 0.05; Fig. 3B and E). These results suggest that VDAC2 does not play a role in regulating global Ca2+ transients or SR Ca2+ loading in beating atrial cells.

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peripheral junctions of the SR domain and cell membrane since HL1 cells lack a t-tubule structure [28]. We found that there was no significant difference in the frequencies of Ca2+ sparks among the uninfected (1.29 ± 0.22, n = 8), WT (1.33 ± 0.72, n = 5), and VDAC2 KD (1.49 ± 0.24, n = 5) HL-1 cells (Fig. 4B; P > 0.05, uninfected vs. WT; P > 0.05, WT vs. KD). Next, we examined whether lentivirus infection or KD of VDAC2 alters the unitary properties of individual Ca2+ sparks. Twodimensional confocal images of single sparks were fitted with the Gaussian function in order to measure amplitude (F1 /Fr ) and FWHM (see Section 2). In addition, the time courses of fluorescence intensity at the centermost part of sparks were measured to evaluate the FDHM. The distribution histogram shown in Fig. 5A–C compares the peak amplitude, FWHM, measured at peak spark area, and FDHM of the sparks recorded in the uninfected, WT, and VDAC2 KD HL1 cells. There was no difference in the spark intensity (F1 /Fr ) and duration (FDHM) of Ca2+ sparks between the uninfected and WT cells (Fig. 5A and C, upper two graphs). Compared with the uninfected cells, the sparks recorded in the WT cells were significantly narrower in space (Fig. 5B, upper two graphs). Interestingly, the spark amplitude, FWHM, and FDHM were significantly larger in the VDAC2 KD cells than in the WT cells (Fig. 5A–C, lower two graphs), indicating larger and more prolonged Ca2+ sparks in the absence of VDAC2. These data suggest that VDAC2 may restrict Ca2+ spark expansion, either directly or indirectly. 3.5. The effect of VDAC2 knock down on mitochondrial Ca2+ responses Since no significant difference between VDAC2 KD and WT HL-1 cells was measured in the SR Ca2+ loading and action potential induced Ca2+ transients, we next examined whether VDAC2 increases the efficiency of mitochondria in accumulating Ca2+ that it directly stimulates Ca2+ uptake. We monitored intramitochondrial Ca2+ , using higher concentrations of fluo-4 AM, which is known to compartmentalize into mitochondria. Cytoplasmic fluo-4 was removed by membrane permeabilization with digitonin (see Section 2). This method has been previously described and successfully used to estimate mitochondrial free Ca2+ in cardiac myocytes and other cell type [12]. Fig. 6A and B shows representative traces of mitochondrial Ca2+ accumulation in permeabilized WT and VDAC2 KD HL-1 cells. The two sets of traces correspond to [Ca2+ ] in the perfusion solution of 300 nM and 10 ␮M. There was significant difference in the rate of mitochondrial Ca2+ uptake at 300 nM and 10 ␮M [Ca2+ ]em used (Fig. 6C, right). However, total amounts of mitochondrial Ca2+ uptake at both [Ca2+ ]em used were similar between the WT and VDAC2 KD cells (Fig. 6C, left). These results indicate that VDAC2 may contribute to facilitating Ca2+ uptake through the inner mitochondrial membrane. 4. Discussion

3.4. Modulation of Ca2+ sparks by VDAC2 in HL-1 cells In the next series of experiments, we examined whether VDAC2 is involved in the regulation of Ca2+ sparks in resting conditions. We have previously characterized the spatio-temporal properties of resting Ca2+ sparks, which are sensitive to ryanodine and tetracaine, in HL-1 cells [28]. In order to record distinct Ca2+ sparks, the bulk Ca2+ transients in HL-1 cells were stopped by superfusing a 0.05 mM Ca2+ -containing external solution. Fig. 4A illustrates the sequential 2D confocal Ca2+ images recorded in uninfected, WT, and VDAC2 KD HL-1 cells under this condition, showing the developments and dissipations of individual Ca2+ sparks. Spontaneous Ca2+ sparks were mostly observed in the cell periphery, where there are

The present experimental approach was developed to determine whether the VDAC2 mitochondrial protein could regulate rhythmic Ca2+ signaling and Ca2+ sparks in cardiac myocytes. We were able to knock down VDAC2 expression to approximately 10% of the control expression level using lentivirus in adult atrial cell line HL-1 (Fig. 1), and found that the KD of the VDAC2 significantly increased spark intensity, width, and duration without affecting spark occurrences (Figs. 4 and 5). When cells lacked VDAC2, mitochondrial Ca2+ uptake was significantly delayed (Fig. 6). Interestingly, however, VDAC2 KD did not alter the rate and magnitude of autorhythmic global Ca2+ transients and the status of SR Ca2+ loading (Fig. 3), which suggests that VDAC2 plays a minor role in

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Fig. 2. Autorhythmic and caffeine-induced Ca2+ transients in uninfected and control lentivirus infected (WT) HL-1 cells. Panel (A) shows confocal fluorescence images of fluo-4 loaded HL-1 cells (green), lentivirus infected HL-1 cells (red, WT; see red arrows in the middle images) and their merged images (lower). Uninfected cells were marked by white arrows. (B) Time courses of autorhythmic Ca2+ transients followed by caffeine (10 mM)-induced Ca2+ transients measured from the indicated regions-of-interests (ROIs) shown in panel (A). (C) Comparison of mean magnitude of Ca2+ transients and half decay time (T1/2 ) between uninfected (50 cells, 6 bathes) and control lentivirus infected (40 cells, 6 batches) HL-1 cells. (D) Comparison of mean magnitude of caffeine-induced Ca2+ transients and half decay time (T1/2 ) in uninfected (57 cells, 6 bathes) and control lentivirus infected (41 cells, 6 bathes) HL-1 cells (P > 0.05, uninfected vs. infected cells). (E) Comparison of mean beating rate of uninfected (37 cells, 6 batches) and WT (37 cells, 6 batches) HL-1 cells (P > 0.05, uninfected vs. infected cells). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3. Autorhythmic and caffeine-induced Ca2+ transients in WT and VDAC2 KD HL-1 cells. Panel (A) shows confocal fluorescence images of fluo-4 loaded HL-1 cells (green), WT (left image, RFP stained) and VDAC2 KD (right image, RFP stained) HL-1 cells and their merged images (lower). (B) Time courses of autorhythmic Ca2+ transients followed by caffeine (10 mM)-induced Ca2+ transients measured from the indicated regions-of-interests (ROIs) shown in panel (A). (C) Comparison of mean magnitude of Ca2+ transients and half decay time (T1/2 ) between WT (40 cells, 6 batches) and VDAC2 KD (21 cells, 6 batches) HL-1 cells. (D) Comparison of mean magnitude of caffeine-induced Ca2+ transients and half decay time (T1/2 ) in WT (40 cells, 6 batches) and KD (21 cells, 6 batches) HL-1 cells. (E) Comparison of mean beating rate of WT (37 cells, 6 batches) and KD (21 cells, 6 batches) HL-1 cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Fig. 4. Comparison of the frequency of Ca2+ sparks among uninfected, WT, and VDAC2 KD HL-1 cells. (A) Sequential 2D confocal Ca2+ images, measured in uninfected, WT, and VDAC2 KD HL-1 cells at 60 Hz, show Ca2+ sparks (see red arrowheads). Beating of HL-1 cells was stopped by superfusing 0.05 mM Ca2+ -containing Tyrode solution in order to record individual Ca2+ sparks. Focal Ca2+ transients measured from the centermost part of each sparks (arrowheads) were displayed above the images. (B) Average frequency of Ca2+ sparks measured in uninfected (8 cells, 4 batches), WT (5 cells, 3 batches), and VDAC2 KD (5 cells, 3 batches) HL-1 cells. There was no difference in the spark frequency among the three groups. There was no significant difference among three groups of cells (P > 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

B

40

No. of sparks

Uninfected

A 50 3.30 ± 0.35 (n = 166)

30

C

60 2.52 ± 0.17 (n = 172)

40

20

20

20

0

2

4

6

8

10

0

0

2

4

6

8

10

25

WT

20

No. of sparks

0.062 ± 0.002 (n = 173)

40

10 0

0 0.0

20

5

5

10

4

6

8

10

No. of sparks

20

0

0

2

4

6

8

10

0 0.0

0.1

0.5

0.2

0.3

0.4

0.5

††††

†††

3.39 ± 0.16 (n = 63)

0.4

15

15 ††

15

0.3

0.045 ± 0.000 (n = 112)

30

10

2

0.2

40

2.19 ± 0.12** n = 68

15

10

0

0.1

50

20

2.82 ± 0.32 (n = 65)

15

0

VDAC2 KD

60

3.39 ± 0.37 (n = 72)

10

0.126 ± 0.009 (n = 96)

10

10 5

5 0

0

2 4 6 Amplitude (F1/Fr)

8

10

0

5

0

2

4 6 FWHM (µm)

8

10

0 0.0

0.1

0.2 0.3 FDHM (s)

0.4

0.5

Fig. 5. Unitary properties of Ca2+ sparks in uninfected, WT, and VDAC2 KD HL-1 cells. Distribution histograms of peak amplitude (F1 /Fr ; A), full width at half maximal amplitude (FWHM, measured at peak area; B), and full duration at half maximal amplitude (FDHM; C). The amplitude, width, and duration of sparks were larger in VDAC2 KD cells than in WT cells. Data were represented as mean ± SEM. n, number of sparks. ** P < 0.01, uninfected (8 cells from 4 batches) vs. WT (5 cells from 3 batches). †† P < 0.01, ††† P < 0.001, †††† P < 0.0001, WT (5 cells from 3 batches) vs. KD (5 cells from 3 batches).

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WT

VDAC2 KD

ΔF/F0 = 2

A

10 0

[Ca2+]em (nM)

C

5 min 0

300

0

4

5 Rate of Ca2+ uptake (ΔF/F0)/min

B

10 0

ΔF/F0 = 2

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2 min

[Ca2+]m (ΔF/F0)

4 3 2 1 0

300 nM

10 μM

300

WT KD

3

**

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10 μM

Fig. 6. Mitochondrial Ca2+ uptake in permeabilized WT and VDAC2 KD HL-1 cells. Panel (A) and (B) show changes in mitochondrial matrix Ca2+ signal (F/F0 ) during pulses with 10 ␮M (A) and 300 nM (B) free Ca2+ -containing internal solutions in WT and VDAC2 KD HL-1 cells, permeabilized with digitonin (see Section 2). The [Ca2+ ]em indicates extramitochondrial Ca2+ concentration. (C) Comparisons of changes in mitochondrial matrix Ca2+ signal ([Ca2+ ]m : F/F0 ) and the rate of Ca2+ uptake during pulses with 300 nM or 10 ␮M free Ca2+ -containing internal solutions between WT (45 cells, 6 batches) and VDAC2 KD (34 cells, 5 bathes) HL-1 cells. * P < 0.05, ** P < 0.01 vs. WT.

the regulation of global SR Ca2+ handling. Our findings are the first to demonstrate that VDAC2 may regulate the statio-temporal properties of spontaneous Ca2+ sparks in cardiac myocytes under resting conditions, providing evidence about the functional role of VDAC2 in cardiac local Ca2+ signaling. We found that autorhythmic cardiac cell line HL-1 and isolated adult rat atrial and ventricular myocytes express significantly high levels of VDAC2 protein (Fig. 1). This finding is consistent with earlier reports that expression of VDAC2 was detected by Western blot analysis in whole hearts [27,32] as well as in the ventricular preparations of rats and mice [26]. In cardiac tissue, types 1–3 VDAC isoforms are known to be expressed [21,26,27,32], and it appears they are localized to the mitochondrial outer membrane [27,33], which is different from the skeletal muscles, where VDACs are also present in the SR membrane [34]. The presence of plasma membrane channels with physiological properties similar to VDAC1 has been reported [35,36]. In fact, a monoclonal antibody, raised against VDAC1, completely blocks a high-conductance anion channel found in the plasma membrane of bovine astrocytes [36]. However, there is no evidence on the presence of plasma membrane VDAC in cardiac cells. Since there is no specific inhibitor for VDAC isoforms, a genetic knock down technique is useful for finding the physiological functions of the VDAC isoforms. The functional difference of VDAC isoforms has been reported. For example, VDAC isoforms display different properties with respect to the permeability of the outer mitochondrial membrane to solutes [24]. VDAC1-deficient mice are viable and have mitochondrial functions that are slightly affected. While VDAC2 gene deletion is lethal [25], mice lacking VDAC3 are

healthy; however, males are infertile [37]. We adopted lentivirus and adult cardiac cell line HL-1 to produce a stable cell line, with null expression of VDAC2, in order to keep a stable KD level. This method overcame the limitations that stable genetic knock down was technically difficult using isolated cardiac myocytes and adenovirus. It takes 4–5 days of infection in order to achieve altered protein expressions using lentiviral gene delivery in cultured adult ventricular myocytes [38]. After infection with WT lentivirus, the morphology and viability significantly changed and Ca2+ sparks were no longer observed in the cultured adult rat ventricular myocytes (data not shown). We found that spontaneous Ca2+ sparks in VDAC2 KD HL-1 cells were brighter, wider, and more prolonged, and occurred with the same frequency when compared with those in the WT cells (Figs. 4 and 5). The brighter and more prolonged Ca2+ sparks, with no change in their occurrences, indicate that the amounts of SR Ca2+ releases from single Ca2+ release units are larger, and that the activations of Ca2+ release sites are not affected. Considering that there were no differences in the SR Ca2+ content (Fig. 3) and spark frequency, larger focal Ca2+ releases, under resting conditions, may be explained by the increased mean open time of RyRs (slower inactivation) and/or opening of more numbers of RyRs for single spark. A larger spark width may also result from increased Ca2+ releases. Although the mechanism for limitation of Ca2+ spark expansion by VDAC2 is not clear, one possible mechanism may be the facilitation of mitochondrial Ca2+ uptake by VDAC2, because a lack of VDAC2 resulted in a significantly delayed mitochondrial Ca2+ uptake (Fig. 6). Inhibition of Ca2+ diffusion, through the outer mitochondrial membrane by VDAC2 KD, may increase local [Ca2+ ] and enhance Ca2+ movement to other directions, which may increase the chances of activation of adjacent RyRs. Such regulation of Ca2+ sparks by VDAC2 may be possible because VDAC2 is known to be partially co-localized with RyR2 in the subsarcolemmal region of HL-1 cells [39,40]. The possibility on direct modulation of RyR2 by VDAC2 may not be excluded, since in vitro biochemical assays (GST-pull down and co-immunoprecipitation) recently revealed physical interactions between the two proteins [40]. Interestingly, it has been reported that Ca2+ sparks are sufficient for activating the low affinity Ca2+ uptake in adjacent mitochondria [41]. This study simultaneously measured mitochondrial matrix Ca2+ and cytosolic Ca2+ , using rhod-2 AM and fluo-3 in permeabilized cardiac H9c2 cells, and found that Ca2+ sparks could elicit focal mitochondrial matrix Ca2+ transients (“Ca2+ marks”) [41]. This study also showed that blockade of inner mitochondrial Ca2+ uptake increased the size and duration of Ca2+ sparks with only a small rise in global cytosolic Ca2+ [41]. In permeabilized HL-1 cells, mitochondrial Ca2+ uptake, during artificial Ca2+ pulses, was significantly slowed in VDAC2 KD cells compared with WT cells (Fig. 6). However, there was no difference in overall magnitude of Ca2+ uptake between the WT and the VDAC2 KD cells (Fig. 6). The mitochondrial Ca2+ uptake was almost completely inhibited by FCCP, a mitochondrial uncoupler, which disrupts inner mitochondrial potential (data not shown). The slower rate of Ca2+ accumulation in the mitochondrial matrix in VDAC2 KD HL-1 cells provides the functional role of VDAC2 as a Ca2+ channel, which allows Ca2+ uptake through the inner mitochondrial membrane in cardiac cells. Although the lack of VDAC2 decreased the rate of Ca2+ uptake, other VDAC isoforms (i.e. VDAC1 and VDAC3; [21,26,27,32]) and the remaining VDAC2 (∼10% of total VDAC2), might have carried Ca2+ through the outer membrane. It is thought that mitochondria may uptake cytosolic Ca2+ and regulate Ca2+ transients in cardiac cells. However, there is controversy with regard to the kinetics of mitochondrial Ca2+ uptake during action potential and on the percentage of mitochondrial contribution to cytosolic Ca2+ signaling. This controversy appears to be dependent on the experimental methods, species, and prepara-

K.P. Subedi et al. / Cell Calcium 49 (2011) 136–143

tions [42]. Although we found that VDAC2 KD affected spontaneous individual Ca2+ sparks in resting conditions, we observed no changes in the action potential-triggered Ca2+ transients and in the caffeine-induced Ca2+ transients (Fig. 3). The reason why the effects of VDAC2 KD on focal and global Ca2+ signals are different is not clear. It may be partly related to the previous finding that VDAC2 proteins are co-localized with RyR2 only in the subsarcolemma [40]. Consistent with our finding overexpression of VDAC in HeLa cells did not show any changes in the global SR Ca2+ handling [43]. The precise mechanisms for the regulation of Ca2+ sparks by VDAC2 need further investigation. Acknowledgements We thank Dr. W. Claycomb for HL-1 cells. This work was supported by National Research Foundation (NRF) of Korea grants funded by the Ministry of Education, Science and Technology (2009-0053266 and 2009-0065568). This work was also in part supported by Priority Research Centers Program through the NRF funded by the Ministry of Education, Science and Technology (2009-0093815). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ceca.2010.12.004. References [1] D.J. Beuckelmann, W.G. Wier, Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells, J. Physiol. 405 (1988) 233–255. [2] M. Näbauer, G. Callewaert, L. Cleemann, M. Morad, Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes, Science 244 (1989) 800–803. [3] E Niggli, W.J. Lederer, Voltage-independent calcium release in heart muscle, Science 250 (1990) 565–568. [4] L. Cleemann, M. Morad, Role of Ca2+ channel in cardiac excitation–contraction coupling in the rat: evidence from Ca2+ transients and contraction, J. Physiol. 432 (1991) 283–312. [5] H. Cheng, W.J. Lederer, M.B. Cannell, Calcium sparks: elementary events underlying excitation–contraction coupling in heart muscle, Science 262 (1993) 740–744. [6] M.B. Cannell, H. Cheng, W.J. Lederer, Spatial non-uniformities in [Ca2+ ]i during excitation–contraction coupling in cardiac myocytes, Biophys. J. 67 (1994) 1942–1956. [7] L. Cleemann, W. Wang, M. Morad, Two-dimensional confocal images of organization, density, and gating of focal Ca2+ release sites in rat cardiac myocytes, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 10984–10989. [8] P.S. Shacklock, W.G. Wier, C.W. Balke, Local Ca2+ transients (Ca2+ sparks) originate at transverse tubules in rat heart cells, J. Physiol. 487 (1995) 601–608. [9] N.M. Soldatov, Ca2+ channel moving tail: link between Ca2+ -induced inactivation and Ca2+ signal transduction, Trends Pharmacol. Sci. 24 (2003) 167–171. [10] J.A. Sánchez, M.C. García, V.K. Sharma, K.C. Young, M.A. Matlib, S.S. Sheu, Mitochondria regulate inactivation of L-type Ca2+ channels in rat heart, J. Physiol. 536 (2001) 387–396. [11] V. Robert, P. Gurlini, V. Tosello, T. Nagal, A. Miyawaki, F. Di Lisa, T. Pozzan, Beatto-beat oscillations of mitochondrial [Ca2+ ] in cardiac cells, EMBO J. 20 (2001) 4998–5007. [12] M. Sedova, E.N. Dedkova, L.A. Blatter, Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes, Am. J. Physiol. Cell Physiol. 291 (2006) C840–C850. [13] V. Lukyanenko, A. Chikando, W.J. Lederer, Mitochondria in cardiomyocyte Ca2+ signaling, Int. J. Biochem. Cell Biol. 41 (2009) 1957–1971. [14] A.S. Parfenov, V. Salnikov, W.J. Lederer, V. Lukyánenko, Aqueous diffusion pathways as a part of the ventricular cell ultrastructure, Biophys. J. 90 (2006) 1107–1119. [15] T.E. Gunter, K.K. Gunter, S.S. Sheu, C.E. Gavin, Mitochondrial calcium transport: physiological and pathological relevance, Am. J. Physiol. 267 (1994) C313–C339. [16] T.E. Gunter, L. Buntinas, G.C. Sparagna, K.K. Gunter, The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients, Biochim. Biophys. Acta 1366 (1998) 5–15. [17] M. Colombini, A candidate for the permeability pathway of the outer mitochondrial membrane, Nature 279 (1979) 643–645.

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