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Evidence for regulation of mitochondrial function by the L-type Ca2+ channel in ventricular myocytes
Journal of Molecular and Cellular Cardiology 46 (2009) 1016–1026
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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y j m c c
Original article
Evidence for regulation of mitochondrial function by the L-type Ca2+ channel in ventricular myocytes Helena M. Viola, Peter G. Arthur, Livia C. Hool ⁎ a b
School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Australia The Western Australian Institute for Medical Research, Australia
a r t i c l e
i n f o
Article history: Received 13 November 2008 Received in revised form 12 December 2008 Accepted 16 December 2008 Available online 7 January 2009 Keywords: Ca2+ channel Mitochondria Calcium Cytoskeleton Mitochondrial membrane potential Mitochondrial NADH Superoxide
a b s t r a c t The L-type Ca2+ channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca2+ channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(−) or voltage-clamp of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calciumdependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltageclamp of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the α1C-subunit of the channel (thereby preventing movement of the β-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca2+ channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton. © 2008 Elsevier Inc. All rights reserved.
1. Introduction In the heart calcium influx through the L-type Ca2+ channel or dihydropyridine receptor (DHPR) is a requirement for calcium release from the ryanodine receptor (RyR) in sarcoplasmic reticulum (SR) stores (a process known as Ca2+-induced Ca2+release). In skeletal muscle, depolarization of the plasma membrane causes SR calcium release and contraction and the DHPR is thought to be the membrane potential sensor that transmits the signal to the RyR to release calcium [1]. Calcium influx is not a requirement. The intracellular loop between domains II and III of the skeletal muscle DHPR is thought to be important in transmitting the signal because peptides directed against the loop can alter RyR gating in skeletal muscle [2,3]. Therefore a direct coupling between DHPR–RyR in skeletal muscle has been proposed. Similarly, in cardiac muscle a coupling between cardiac DHPR and
⁎ Corresponding author. Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway Crawley, WA, 6009, Australia. Tel.: +61 8 6488 3307; fax: +61 8 6488 1025. E-mail address: [email protected] (L.C. Hool). 0022-2828/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.12.015
RyR may exist because peptides directed against the cardiac II–III loop of DHPR also alter RyR gating and the DHPR agonist BayK8644 can rapidly increase resting calcium spark frequency independent of depolarization or calcium entry in ferret ventricular myocytes [4,5]. However it is proposed that a DHPR–RyR coupling plays only a minor role in the heart [4]. In cardiac myocytes mitochondria are responsible for meeting the cellular energy demands required to maintain excitation and contraction on a beat to beat basis. It has been proposed that the mitochondria can rapidly track changes in cytosolic calcium from beat to beat [6,7]. The mitochondria rely on calcium to activate key dehydrogenases in the tricarboxylic acid cycle. This accelerates production of NADH that provides a driving force for increase in proton motive force that maintains ATP production [8]. A rapid uptake mechanism capable of responding to changes in cytosolic calcium has not been identified in the mitochondria and alternative mechanisms for regulating mitochondrial function have been sought. Cardiac myocytes are dynamic cells. L-type Ca2+ channels are anchored to F-actin networks by subsarcolemmal stabilizing proteins that also tightly regulate the function of the channel [9–11]. Cytoskeletal proteins stabilize cell structure but also regulate the subcellular distribution of mitochondria [12]. Therefore L-type Ca2+
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channels may mediate cellular responses through cytoskeletal proteins when conformational changes in the channel occur during activation and inactivation. We examined whether the L-type Ca2+ channel (ICa-L) can regulate mitochondrial function. We examined the effect of activation of ICa-L on a number of mitochondrial parameters including superoxide produc-
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tion, mitochondrial membrane potential, NADH production and metabolic activity. We performed experiments in quiescent guineapig ventricular myocytes with consistent ATP utilisation or where we held ATP concentration constant (in the patch pipette) since this allowed us to more readily explore the effects of channel activation on mitochondrial function. We find that ICa-L can regulate mitochondrial
Fig. 1. Direct activation of ICa-L increases intracellular calcium and superoxide. (A) DHE fluorescence (DHE fluores.) recorded from a myocyte before and after exposure to 2 μM BayK (−) and from a myocyte before and after exposure to 2 μM BayK(+). DHE fluorescence (DHE fluores.) recorded from a myocyte during exposure to 2.5 mM Ca2+ alone, from a myocyte during exposure to 0 mM Ca2+ alone and from a myocyte before and after exposure to BayK(−) in 0 mM Ca2+ are shown at top right inset. Mean ± SEM of the ratio of fluorescence for myocytes exposed to treatments as indicated are shown at bottom right inset. (B) DHE fluorescence (DHE fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) then 45 mM KCl, and from a control myocyte (5 mM KCl). Mean ± SEM of the ratio of fluorescence for myocytes exposed to KCl and/or Nisol as indicated are shown at the right. (C) Fura-2 (340/380 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM BayK(−) and from an untreated myocyte. Arrow indicates when treatments were added. Mean ± SEM of changes in Fura-2 fluorescence (% change in fluores.) for myocytes exposed to BayK(−), 2 μM nisoldipine (Nisol) then BayK(−), KCl ± Nisol as indicated are shown at the right.
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function. The regulation of the mitochondria by the channel requires calcium but is also dependent upon communication through actin filaments. We propose that the structural rearrangement of the
channel during activation is transmitted to the mitochondria via the auxiliary β-subunit of the channel through the cytoskeleton. Preliminary data has been published in abstract form [13].
Fig. 2. Direct activation of ICa-L results in an increase in Ψm. (A) Ratiometric JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 2 μM BayK(−) and from another myocyte before and after exposure to 2 μM BayK(+). Arrow indicates when treatments were added. To establish that the JC-1 signal was indicative of Ψm 20 μM oligomycin (Oligo) and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Individual 535 nm and 580 nm wavelengths are shown above at the upper right. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 2 μM BayK(+) or 2 μM BayK(−) as indicated are shown at the bottom right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) then 45 mM KCl, and from another myocyte before and after exposure to 2 μM Nisol. Arrow indicates when treatments were added. Individual 535 nm and 580 nm wavelengths are shown above at the right. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or Nisol as indicated are shown below at the right. (C) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte patch-clamped and held initially at − 30 mV then voltage-stepped to + 10 mV followed by a voltage-step back to − 30 mV as indicated with arrows (Voltage-stepped) and in another myocyte held at −30 mV and not voltage-stepped to + 10 mV (Not voltage-stepped). Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes as indicated are shown at the right.
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Fig. 3. The increase in Ψm associated with activation of ICa-L does not require calcium. (A) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM Ru360 then 45 mM KCl, and from another myocyte before and after exposure to 2 μM Ru360. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 2 μM Ru360 as indicated are shown at the right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 2 μM BayK(−) and from a myocyte before and after exposure to 2 μM BayK(+) in calcium-free HBS. JC-1 fluorescence recorded from a myocyte patch-clamped with 5 mM BAPTA and 0 mM calcium in the pipette held at − 30 mV and from another myocyte patch-clamped with 5 mM BAPTA and 0 mM calcium in the pipette exposed to 2 μM BayK(−) in calcium-free HBS. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) as indicated are shown at the right. (C) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl in the absence of calcium, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) then 45 mM KCl in the absence of calcium, and from another myocyte before and after exposure to 2 μM Nisol in the absence of calcium. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 2 μM Nisol as indicated are shown at the right. (D) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl in the absence of calcium, from a myocyte before and after exposure to 5 μM amiloride (Amil) and 5 μM tetrodotoxin (TTX) then 45 mM KCl in the absence of calcium, and from another myocyte before and after exposure to 5 μM Amil and 5 μM TTX in the absence of calcium. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl ± 5 μM Amil and 5 μM TTX as indicated are shown at the right.
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2. Materials and methods 2.1. Measurement of superoxide, intracellular calcium, mitochondrial NADH and mitochondrial membrane potential (Ψm) For all studies adult guinea-pig ventricular myocytes were used. For further detail regarding the isolation and culture of myocytes please see the Supplementary File for Online Publication. Superoxide was measured using the fluorescent indicator dihydroethidium (DHE); intracellular calcium was measured using the indicator Fura-2 and Ψm was measured using JC-1. In some experiments changes in Ψm were assessed while voltage-clamping the cell membrane. For further detail see Supplementary File for Online Publication and [14,15]. 2.2. Measurement of metabolic activity The cleavage of the yellow tetrazolium salt (MTT) to purple formazan crystals by complex I was assessed in myocytes using a modified MTT assay. See Supplementary File for Online Publication for more details. 2.3. Synthesis of AID peptide A peptide against the alpha-interacting domain in the I–II linker of α1C-subunit was synthesized using the following amino acid sequence QQLEEDLKGYLDWITQAE as previously described [16] (Auspep Pty Ltd, Parkville, Australia). An HIV-TAT protein sequence (RKKRRQRRR) was added to the peptide to facilitate plasma membrane transport into the cytoplasm. A scrambled control was also synthesized with a HIV-TAT sequence (QKILGEWDLAQYTDQELE). 3. Results 3.1. Direct activation of ICa-L results in an increase in mitochondrial-derived superoxide Increased cytosolic Ca2+ and matrix Ca2+ overload can lead to enhanced generation of reactive oxygen species [17]. We examined whether calcium influx through ICa-L is sufficient to increase superoxide in myocytes using the fluorescent indicator DHE. Application of the DHPR agonist BayK(−) significantly increased DHE signal compared to controls exposed to the (+) enantiomer of BayK that does not act as an agonist (Fig. 1A). The increase in DHE could be attenuated with application of ICa-L antagonist nisoldipine, when calcium uptake into the mitochondria through the mitochondrial Ca2 + uniporter was blocked with Ru360 or with uncoupling of mitochondrial electron transport with FCCP (Fig. 1A). This is in agreement with data indicating that a predominant source of superoxide in ventricular myocytes is the mitochondria [14,15]. We also used a traditional method for activation of ICa-L through plasma membrane depolarization with a high concentration of KCl [18]. DHE was significantly increased with exposure of cells to 45 mM KCl (Fig. 1B). This was attenuated by nisoldipine suggesting activation of ICa-L was responsible for increased production of superoxide. We measured intracellular calcium by assessing changes in 340/380 fluorescence using the indicator Fura-2. BayK(−) and 45 mM KCl increased 340/380 fluorescence that was significantly attenuated when myocytes were exposed to nisoldipine (Fig. 1C). BayK(−) increased [Ca]i from a resting level of 81.3 ± 19.6 to 92.8 ± 3.2 nM (n = 5) and 45 mM KCl increased [Ca]i to 130.5 ± 15.1 nM (n = 5). We exposed cells to calcium-free HEPES-buffered saline (HBS) for at least 3 h to deplete intracellular calcium stores in the myocytes [19]. In 6 cells the DHE signal was only 48.7 ± 6.1% of DHE signal recorded in myocytes exposed to calcium-containing HBS in the absence of BayK(−) confirming that basal superoxide production is dependent on calcium (Fig. 1A inset at top right). These data suggest that direct activation of ICa-L is sufficient to
alter superoxide production by the mitochondria as a result of increased calcium influx through the channel. 3.2. Direct activation of ICa-L results in an increase in Ψm We examined the effect of activation of ICa-L on Ψm. Exposure of cells to BayK(−) increased JC-1 signal 13.3 ± 1.5% compared to controls exposed to BayK(+) (Fig. 2A). A collapse in JC-1 signal after exposure of cells to oligomycin and FCCP demonstrated that the signal was indicative of Ψm. In addition, consistent with an increase in Ψm the 580 nm signal increased after addition of BayK(−) or 45 mM KCl while the 535 nm signal decreased. The uncoupling of Ψm with oligomycin and FCCP resulted in a reversal of the signals (inset right top Figs. 2A and B). Activation of ICa-L with 45 mM KCl caused a 23.8 ± 3.6% increase in JC-1 signal compared to controls treated with HBS containing 5 mM KCl that was attenuated when myocytes were exposed to nisoldipine (Fig. 2B). We directly activated ICa-L by voltage-stepping the plasma membrane from − 30 mV to + 10 mV using patch-clamp technique in cells that had been loaded with JC-1. Initially cells were patch-clamped with internal pipette solution that contained EGTA and calcium calculated to result in a free calcium concentration of 10 nM [20]. Upon voltagestep to +10 mV the JC-1 signal increased 26.3 ± 3.5% and was reversed when the cell was voltage-stepped to − 30 mV (Fig. 2C). The increase was significantly greater than the signal recorded in cells that were not patch clamped (1.6 ± 0.6%; n = 3) or cells that were patch-clamped and the membrane voltage held at −30 mV for the duration of the experiment (1.2 ± 1.2%, n = 4; Fig. 2C). These data indicate that direct activation of ICa-L can increase Ψm and the response is reversible. 3.3. The increase in Ψm after activation of ICa-L does not appear to require calcium We examined whether an increase in calcium uptake into the mitochondria was necessary for the increase in Ψm after activation of the channel. We exposed myocytes to Ru360 prior to activation of the channel with 45 mM KCl and measured changes in JC-1. Ru360 did not significantly alter the JC-1 signal after activation of ICa-L (Fig. 3A). We examined this further by incubating cells in calcium-free HBS for at least 3 h. In calcium-free HBS, BayK(−) resulted in a 13.0 ± 1.5% increase in JC-1 signal (Fig. 3B) that was not significantly different from the response recorded in calcium-containing HBS (13.3 ± 1.5%; Fig. 2A). In order to buffer calcium as effectively as possible, we exposed cells to calcium-free HBS for 3 h and patch-clamped the cells with pipette solution containing 5 mM BAPTA and 0 mM calcium. Activation of the channel either by voltage-step from −30 mV to +10 mV or application of BayK(−) while the cell was dialysed with BAPTA and 0 mM calcium increased JC-1 signal. The increase in JC-1 signal after application of BayK (−) was similar to the increase in JC-1 signal recorded in cells exposed to calcium-free HBS but not perfused internally with BAPTA and 0 mM calcium (Fig. 3B). Similarly, addition of 45 mM KCl in calcium-free HBS resulted in 31.9 ± 7.3% increase in JC-1 signal (Fig. 3C). This was not significantly different from the increase recorded in the presence of calcium (23.8 ± 3.6%; Fig. 2B). The response was significantly attenuated when myocytes were exposed to nisoldipine (Fig. 3C). Depolarization of the plasma membrane potential with high KCl also activates voltage-dependent fast inward Na+ channels. We examined whether the increase in Ψm was due to alterations in Na+ transport in the myocytes. When cells were exposed to calcium-free HBS containing tetrodotoxin (TTX) to block fast inward Na+ channels and amiloride to block Na+/H+ exchange activity, 45 mM KCl increased JC-1 signal 25.9 ± 4.0% that was similar to the increase in JC-1 recorded in calcium-free HBS alone (31.9 ± 7.3%; Fig. 3D). These results suggest that the increase in Ψm associated with activation of ICa-L is not dependent upon changes in sodium or calcium transport.
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3.4. The increase in Ψm associated with activation of ICa-L appears to be mediated through actin filaments Actin filament (F-actin) networks can regulate the function of the L-type Ca2+ channel [9,21,22]. In addition F-actin associates
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with mitochondria via phalloidin binding sites to stabilize mitochondria within the cell [23]. Since the increase in Ψm after activation of ICa-L did not appear to require calcium, we tested whether the response was mediated as a result of movement through actin networks.
Fig. 4. Depolymerization of actin with latrunculin A attenuates the increase in Ψm without altering mitochondrial superoxide production. (A) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 45 mM KCl, and from another myocyte before and after exposure to 5 μM Latrunc alone. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 5 μM Latrunc as indicated are shown at the right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl in the absence of calcium, from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 45 mM KCl and from another myocyte before and after 5 μM Latrunc alone in the absence of calcium. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 5 μM Latrunc as indicated are shown at the right. (C) DHE fluorescence (DHE fluores.) recorded from a myocyte before and after exposure to 2 μM caffeine (Caff), from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 2 μM Caff and from an untreated myocyte. Mean ± SEM of the ratio of fluorescence for myocytes exposed to 2 μM Caff or 5 μM Latrunc as indicated are shown at the right.
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We exposed myocytes to latrunculin A for 30 min to depolymerize actin and measured changes in Ψm after activation of the channel with 45 mM KCl. Latrunculin A significantly attenuated the increase in Ψm in calcium-containing HBS (Fig. 4A) and in calcium-free HBS (Fig. 4B). Similar results were obtained with BayK(−) in calcium-free HBS (see Supplementary File for Online Publication). We tested whether latrunculin A directly alters mitochondrial function. We exposed myocytes to caffeine that stimulates release of calcium from ryanodine receptors in the sarcoplasmic reticulum, and measured changes in DHE signal. Caffeine increased DHE signal 48.7 ± 6.9% compared to control myocytes not exposed to caffeine. The increase in DHE was not altered when myocytes were also exposed to latrunculin A (Fig. 4C).
We conclude that latrunculin A does not directly alter mitochondrial function. The increase in Ψm after activation of ICa-L appears to involve actin filaments. Activation of the α1c-subunit of ICa-L is mediated through the auxiliary β-subunit that is also tethered to the cytoskeleton [10]. We examined whether the increase in Ψm in response to activation of ICa-L occurs through the movement of the β-subunit of the channel coupled to actin filaments. We synthesized a peptide directed against the alpha-interacting domain (AID) where the auxiliary β-subunit associates with the α1c-subunit of the channel [16]. Exposure to AID-TAT for 20 min significantly attenuated the increase in JC-1 due to activation of ICa-L (Fig. 5A). For control experiments we exposed cells
Fig. 5. A peptide directed against the alpha interacting domain of the α1C-subunit of ICa-L attenuates the increase in Ψm associated with activation of the channel. (A) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 1 μM scrambled AID-TAT (AID-TAT(S)) then 45 mM KCl and from another myocyte before and after exposure to 1 μM AID-TAT then 45 mM KCl. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 1 μM AID-TAT(S) then KCl or 1 μM AID-TAT then KCl as indicated are shown at the right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte patch-clamped and held initially at − 30 mV then voltage-stepped to +10 mV as indicated with the arrow that had been exposed to 1 μM scrambled AID-TAT peptide (AID-TAT(S)) and in another myocyte patch-clamped and held initially at − 30 mV then voltage-stepped to + 10 mV as indicated with the arrow that had been exposed to 1 μM AID-TAT peptide (AID-TAT). 4 μM FCCP was added at the end of the experiment to collapse Ψm where indicated. Current traces recorded from the patch-clamped cells as indicated are shown inset at the left. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 1 μM AID-TAT(S) or 1 μM AID-TAT are shown at the right.
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Fig. 6. Activation of ICa-L increases mitochondrial NADH. (A) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 2 μM BayK(−) and from another myocyte before and after exposure to 2 μM BayK(+). Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated (see text for explanation). Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes exposed to 2 μM BayK(+) or 2 μM BayK(−) as indicated are shown at the right. (B) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) followed by 45 mM KCl and from an untreated myocyte. Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 2 μM Nisol then 45 mM KCl as indicated are shown at the right. (C) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 0 mM calcium followed by addition of 2 mM external calcium, in a myocyte before and after exposure to 0 mM external calcium only, and in a myocyte before and after exposure to 0 mM external calcium followed by 45 mM KCl. Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes as indicated are shown at the right. (D) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 45 mM KCl, and from an untreated myocyte. Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 5 μM Latrunc then 45 mM KCl as indicated are shown at the right.
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to a scrambled peptide containing a TAT region for 20 min. The scrambled peptide did not attenuate the increase in JC-1 signal after exposure to 45 mM KCl (24.5 ± 1.0%, Fig. 5A). We also patch-clamped myocytes exposed to the AID-TAT peptide and compared the increase in JC-1 after voltage-stepping the cell from −30 mV to +10 mV with patch-clamped cells exposed to the scrambled peptide. The increase in JC-1 after voltage-stepping to +10 mV was significantly attenuated in cells exposed to AID-TAT peptide (1.6±1.6%) but not in cells exposed to scrambled peptide (23.9± 3.5%; Fig. 5B). The attenuation in JC-1 signal after exposure to AID-TAT peptide was greater in cells that were patch-clamped than in non patchclamped cells (Fig. 5A) because during patch-clamp experiments the cells were exposed to the peptide for a longer duration (at least 1 h). The auxiliary β-subunit influences the inactivation rate of the current [24]. Cells exposed to the AID-TAT peptide exhibited a delayed rate of inactivation of currents (Fig. 5B inset left). The time constant (τ) for inactivation of currents obtained from a standard exponential fit (pClamp9, Molecular Devices) in cells exposed to AID-TAT peptide was τ=63.9±5.5 ms (n=16) compared to τ=42.5±0.8 ms in currents from cells exposed to scrambled peptide (n=28). This provided further confirmation that the AID-TAT peptide was binding to AID on the alpha1C subunit of the channel and altering movement of the β-subunit of the channel. These data suggest that the increase in Ψm occurs through activation of the auxiliary β-subunit via the actin cytoskeleton. We propose that the conformational change that occurs upon activation of the channel (including the movement of the auxiliary β-subunit) is transmitted through actin to the mitochondria to increase Ψm. 3.5. Activation of ICa-L increases NADH production We examined whether activation of the channel could alter mitochondrial NADH in myocytes. BayK(−) significantly increased NADH fluorescence compared to myocytes exposed to BayK(+) (Fig. 6A). In all experiments we exposed the myocyte to oligomycin followed by FCCP. An increase in NADH fluorescence was observed after addition of oligomycin consistent with an active TCA cycle, and NADH fluorescence decreased after addition of FCCP consistent with depletion of NADH after collapse of Ψm [25,26]. Activation of ICa-L with 45 mM KCl resulted in a 27.4 ± 5.0% increase in NADH production that was attenuated when myocytes were exposed to nisoldipine (Fig. 6B). In calcium-free HBS, 45 mM KCl was unable to increase NADH signal (Fig. 6C) confirming that NADH production is dependent upon calcium. Consistent with this, addition of 2 mM extracellular calcium caused a further increase in NADH (Fig. 6C). Exposure of myocytes to latrunculin A significantly attenuated the increase in NADH associated with 45 mM KCl (Fig. 6D). In the presence of latrunculin A however oligomycin was still able to increase NADH signal while FCCP decreased NADH signal indicating that latrunculin A did not directly alter mitochondrial function. These data suggest that activation of ICa-L alone is sufficient to increase mitochondrial NADH production. The response requires an increase in influx of calcium through the channel but also appears to involve transmission of channel activation through actin filaments. 3.6. Activation of ICa-L increases mitochondrial metabolic activity in intact myocytes Metabolic activity in myocytes was assessed using a modified MTT assay. BayK(−) significantly increased absorbance at 570 nm by 125.0 ± 16.0% compared to controls treated with BayK(+) (Figs. 7A and B). The increase in absorbance was attenuated when myocytes were exposed to nisoldipine and AID-TAT (Figs. 7A and B). Myocytes were also exposed to the mitochondrial calcium uniporter blocker Ru360 followed by BayK(−). Ru360 significantly attenuated the increase in absorbance in response to BayK(−). The
increase in absorbance associated with BayK(−) was not altered when myocytes were also exposed to dantrolene an inhibitor of the ryanodine receptor (112.0 ± 24.3%). These data suggest that activation of ICa-L increases metabolic activity in myocytes due to increased uptake of calcium by the mitochondria. Preventing movement of the auxiliary β-subunit of the channel with AID-TAT peptide also decreased metabolic activity. 4. Discussion Calcium is integral to mitochondrial function and the mitochondria are capable of tracking changes in intracellular calcium [6,7]. However it is unclear whether calcium influx through ICa-L alone is sufficient to alter mitochondrial function. We report here that calcium influx through the channel increases mitochondrial superoxide, NADH production and metabolic activity in quiescent cardiac myocytes. This occurs as a result of an increase in intracellular calcium and Ru360-sensitive calcium uptake into the mitochondria (Figs. 1 and 7). Exposure of cells to ICa-L antagonist nisoldipine or incubation in calcium-free HBS abolished the increase in superoxide, NADH and metabolic activity (Figs. 1B, 6B, 6C and 7). These results are consistent with previous findings that an increase in ICa-L current and intracellular calcium after exposure to hydrogen peroxide is sufficient to increase mitochondrial superoxide production in quiescent and contracting cardiac myocytes [14]. Our results indicate that activation of ICa-L also increases Ψm. This occurred when the channel was activated directly with the DHPR agonist BayK(−) (Fig. 2A) or with voltage-clamp of the plasma membrane using patch-clamp technique (Fig. 2C). The increase in [Ca]i after addition of BayK(−) or 45 mM KCl was small (Fig. 1C) and insufficient to activate further calcium release from RyR2 [27]. At low intracellular Ca2+ (0–200 nM) Ψm is maintained independent of changes in intracellular Ca2+ [28]. Although we cannot exclude the possibility that calcium mediated the increase in Ψm, calcium uptake into the mitochondria did not appear to be necessary for the increase in JC-1 signal (Fig. 3A). In the absence of calcium (calcium-free HBS and internal calcium buffered with BAPTA to 0 mM) activation of ICa-L alone increased Ψm (Fig. 3B). Furthermore the increase in Ψm after activation of ICa-L in calcium-free HBS was similar in magnitude to the increase in Ψm recorded in cells in HBS containing 2.5 mM (Fig. 3B vs Fig. 2A and Fig. 3C and 3D vs Fig. 2B). Therefore we explored an alternative mechanism for the increase in JC-1 signal after activation of the channel. The cardiac L-type Ca2+ channel comprises the poreforming ion conducting α1C-subunit, an auxiliary β-subunit and an auxiliary α2δ-subunit. The β-subunit is anchored to F-actin networks by subsarcolemmal stabilizing proteins such as the 700 kDa protein AHNAK (Fig. 7C) [10]. F-actin networks can regulate the function of cardiac ICa-L and disruption of actin filaments significantly decreases calcium current [9,11]. In conditions where cytoskeletal proteins are destabilized such as in Duchenne Muscular Dystrophy, ICa-L density and inactivation is decreased [22,29]. In addition cytoskeletal proteins associate with mitochondria via docking proteins [12,23,30]. We tested whether the increase in Ψm was mediated as a result of movement of the auxiliary β-subunit of the channel through actin filaments. Exposure of cells to a peptide directed against the alpha interacting domain or exposure to latrunculin A to depolymerize actin attenuated the increase in JC-1 signal (Figs. 4A, 4B and 5). This suggests that activation of the channel may mediate changes in mitochondrial function through the cytoskeletal network. In vivo, calcium influx is a requirement for contraction and for maintenance of metabolic parameters such as NADH and ATP production. Our results suggest that in addition to calcium influx, activation of the channel may also modulate mitochondrial function as a result of transmission of movement from the β-subunit through the cytoskeleton. We have shown that activation of the channel under voltage-clamp conditions can increase Ψm. Inactivation of the channel
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Fig. 7. Activation of ICa-L increases metabolic activity in cardiac myocytes. (A) MTT assay performed in myocytes following exposure to 2 μM BayK(−), 2 μM BayK(+), 2 μM nisoldipine (Nisol) and 2 μM BayK(−), 1 μM scrambled AID-TAT (AID-TAT(S)) and 2 μM BayK(−), and 1 μM AID-TAT and 2 μM BayK(−). Slopes represent change in absorbance at 570 nm (with reference wavelength of 620 nm) measured over 0–2 min. (B) Mean ± SEM of the ratio of slopes measured over 0–2 min for myocytes exposed to 2 μM BayK(+), 2 μM BayK(−), 2 μM nisoldipine (Nisol) and 2 μM BayK(−), 2 μM Ru360 and 2 μM BayK(−), 20 μM dantrolene (Dant) and 2 μM BayK(−), 1 μM scrambled AID-TAT (AID-TAT(S)) and 2 μM BayK(−), and 1 μM AID-TAT and 2 μM BayK(−) as indicated. ★p b 0.05 compared with BayK(−). n: represents number of animals used in each treatment group. (C) Proposed model explaining transmission of movement after activation of the β auxiliary subunit of the L-type Ca2+ channel through cytoskeletal proteins to the mitochondria. For further explanation see text.
by voltage-stepping the cell from +10 mV to − 30 mV reversed the increase in Ψm (Fig. 2C) suggesting that the mitochondria may be capable of responding closely to changes in activity of the channel during the cardiac cycle. We propose that the increase in mitochondrial membrane potential associated with activation of the channel may assist with ATP production to meet the increase in energy demand during each cardiac cycle. Since the L-type Ca2+ channel (ICaL) is the initiator of contraction in cardiac muscle, this may represent a coordinated process by which mitochondrial function is regulated on a beat-to-beat basis. Under conditions where actin is disrupted (e.g.
Duchenne Muscular Dystrophy, cardiomyopathies involving mutations in actin, cardiac failure), we propose this coupling may become compromised and contribute to poor oxygen consumption and energy supply by the mitochondria that are typically observed in these conditions. Acknowledgments This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC). Helena Viola is the
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recipient of a Biomedical Postgraduate Research Scholarship from NHMRC and National Heart Foundation of Australia. Livia Hool is the recipient of a NHMRC Career Development Award. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yjmcc.2008.12.015. References [1] Tanabe T, Beam KG, Adams BA, Niidome T, Numa S. Regions of the skeletal muscle dihydropyridine receptor critical for excitation–contraction coupling. Nature 1990;346:567–9. [2] el-Hayek R, Antoniu B, Wang J, Hamilton SL, Ikemoto N. Identification of calcium release-triggering and blocking regions of the II–III loop of the skeletal muscle dihydropyridine receptor. J Biol Chem 1995;270:22116–8. [3] Zhu X, Gurrola G, Jiang MT, Walker JW, Valdivia HH. Conversion of an inactive cardiac dihydropyridine receptor II–III loop segment into forms that activate skeletal ryanodine receptors. FEBS Lett 1999;450:221–6. [4] Li Y, Bers DM. A cardiac dihydropyridine receptor II–III loop peptide inhibits resting Ca(2+) sparks in ferret ventricular myocytes. J Physiol 2001;537:17–26. [5] Katoh H, Schlotthauer K, Bers DM. Transmission of information from cardiac dihydropyridine receptor to ryanodine receptor: evidence from BayK 8644 effects on resting Ca(2+) sparks. Circ Res 2000;87:106–11. [6] Robert V, Gurlini P, Tosello V, Nagai T, Miyawaki A, Di Lisa F, et al. Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells. EMBO J 2001;20:4998–5007. [7] Maack C, Cortassa S, Aon MA, Ganesan AN, Liu T, O'Rourke B. Elevated cytosolic Na + decreases mitochondrial Ca2+ uptake during excitation–contraction coupling and impairs energetic adaptation in cardiac myocytes. Circ Res 2006;99:172–82. [8] Balaban RS. Cardiac energy metabolism homeostasis: role of cytosolic calcium. J Mol Cell Cardiol 2002;34:1259–71. [9] Lader AS, Kwiatkowski DJ, Cantiello HF. Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels. Am J Physiol 1999;277:C1277–83. [10] Hohaus A, Person V, Behlke J, Schaper J, Morano I, Haase H. The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton. FASEB J 2002;16:1205–16. [11] Rueckschloss U, Isenberg G. Cytochalasin D reduces Ca2+ currents via cofilinactivated depolymerization of F-actin in guinea-pig cardiomyocytes. J Physiol 2001;537:363–70. [12] Varadi A, Johnson-Cadwell LI, Cirulli V, Yoon Y, Allan VJ, Rutter GA. Cytoplasmic dynein regulates the subcellular distribution of mitochondria by controlling the recruitment of the fission factor dynamin-related protein-1. J Cell Sci 2004;117: 4389–400. [13] Viola HM, Arthur PG, Hool LC. Evidence for a functional coupling between the Ltype calcium channel and the mitochondria. Circ Res 2008;103(5):e56.
[14] Viola HM, Arthur PG, Hool LC. A transient exposure to hydrogen peroxide causes an increase in mitochondrial-derived superoxide as a result of sustained alteration in L-type Ca2+ channel function in the absence of apoptosis in ventricular myocytes. Circ Res 2007;100:1036–44. [15] Hool LC, Di Maria CA, Viola HM, Arthur PG. Role of NAD(P)H oxidase in the regulation of cardiac L-type Ca2+ channel function during acute hypoxia. Cardiovasc Res 2005;67:624–35. [16] Hohaus A, Poteser M, Romanin C, Klugbauer N, Hofmann F, Morano I, et al. Modulation of the smooth-muscle L-type Ca2+ channel alpha1 subunit (alpha1Cb) by the beta2a subunit: a peptide which inhibits binding of beta to the I–II linker of alpha1 induces functional uncoupling. Biochem J 2000;348(Pt 3):657–65. [17] Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium, ATP, and ROS: a mitochondrial love–hate triangle. Am J Physiol Cell Physiol 2004;287:C817–33. [18] Saini HK, Dhalla NS. Modification of intracellular calcium concentration in cardiomyocytes by inhibition of sarcolemmal Na+/H+exchanger. Am J Physiol Heart Circ Physiol 2006;291:H2790–800. [19] Damron DS, Darvish A, Murphy L, Sweet W, Moravec CS, Bond M. Arachidonic aciddependent phosphorylation of troponin I and myosin light chain 2 in cardiac myocytes. Circ Res 1995;76:1011–9. [20] Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 1988;157:378–417. [21] Nakamura M, Sunagawa M, Kosugi T, Sperelakis N. Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells. Am J Physiol Cell Physiol 2000;279:C480–7. [22] Sadeghi A, Doyle AD, Johnson BD. Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins alpha-actinin and dystrophin. Am J Physiol Cell Physiol 2002;282:C1502–11. [23] Drubin DG, Jones HD, Wertman KF. Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Mol Biol Cell 1993;4:1277–94. [24] Kobrinsky E, Tiwari S, Maltsev VA, Harry JB, Lakatta E, Abernethy DR, et al. Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions. J Biol Chem 2005;280: 12474–85. [25] Scaduto Jr RC, Grotyohann LW. 2,3-butanedione monoxime unmasks Ca(2+)induced NADH formation and inhibits electron transport in rat hearts. Am J Physiol Heart Circ Physiol 2000;279:H1839–48. [26] Jo H, Noma A, Matsuoka S. Calcium-mediated coupling between mitochondrial substrate dehydrogenation and cardiac workload in single guinea-pig ventricular myocytes. J Mol Cell Cardiol 2006;40:394–404. [27] Bers DM. Cardiac excitation–contraction coupling. Nature 2002;415:198–205. [28] Territo PR, Mootha VK, French SA, Balaban RS. Ca(2+) activation of heart mitochondrial oxidative phosphorylation: role of the F(0)/F(1)-ATPase. Am J Physiol Cell Physiol 2000;278:C423–35. [29] Woolf PJ, Lu S, Cornford-Nairn R, Watson M, Xiao XH, Holroyd SM, et al. Alterations in dihydropyridine receptors in dystrophin-deficient cardiac muscle. Am J Physiol Heart Circ Physiol 2006;290:H2439–45. [30] Morris RL, Hollenbeck PJ. Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons. J Cell Biol 1995;131:1315–26.
Contents lists available at ScienceDirect
Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y j m c c
Original article
Evidence for regulation of mitochondrial function by the L-type Ca2+ channel in ventricular myocytes Helena M. Viola, Peter G. Arthur, Livia C. Hool ⁎ a b
School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Australia The Western Australian Institute for Medical Research, Australia
a r t i c l e
i n f o
Article history: Received 13 November 2008 Received in revised form 12 December 2008 Accepted 16 December 2008 Available online 7 January 2009 Keywords: Ca2+ channel Mitochondria Calcium Cytoskeleton Mitochondrial membrane potential Mitochondrial NADH Superoxide
a b s t r a c t The L-type Ca2+ channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca2+ channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(−) or voltage-clamp of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calciumdependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltageclamp of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the α1C-subunit of the channel (thereby preventing movement of the β-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca2+ channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton. © 2008 Elsevier Inc. All rights reserved.
1. Introduction In the heart calcium influx through the L-type Ca2+ channel or dihydropyridine receptor (DHPR) is a requirement for calcium release from the ryanodine receptor (RyR) in sarcoplasmic reticulum (SR) stores (a process known as Ca2+-induced Ca2+release). In skeletal muscle, depolarization of the plasma membrane causes SR calcium release and contraction and the DHPR is thought to be the membrane potential sensor that transmits the signal to the RyR to release calcium [1]. Calcium influx is not a requirement. The intracellular loop between domains II and III of the skeletal muscle DHPR is thought to be important in transmitting the signal because peptides directed against the loop can alter RyR gating in skeletal muscle [2,3]. Therefore a direct coupling between DHPR–RyR in skeletal muscle has been proposed. Similarly, in cardiac muscle a coupling between cardiac DHPR and
⁎ Corresponding author. Physiology M311, School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Highway Crawley, WA, 6009, Australia. Tel.: +61 8 6488 3307; fax: +61 8 6488 1025. E-mail address: [email protected] (L.C. Hool). 0022-2828/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.12.015
RyR may exist because peptides directed against the cardiac II–III loop of DHPR also alter RyR gating and the DHPR agonist BayK8644 can rapidly increase resting calcium spark frequency independent of depolarization or calcium entry in ferret ventricular myocytes [4,5]. However it is proposed that a DHPR–RyR coupling plays only a minor role in the heart [4]. In cardiac myocytes mitochondria are responsible for meeting the cellular energy demands required to maintain excitation and contraction on a beat to beat basis. It has been proposed that the mitochondria can rapidly track changes in cytosolic calcium from beat to beat [6,7]. The mitochondria rely on calcium to activate key dehydrogenases in the tricarboxylic acid cycle. This accelerates production of NADH that provides a driving force for increase in proton motive force that maintains ATP production [8]. A rapid uptake mechanism capable of responding to changes in cytosolic calcium has not been identified in the mitochondria and alternative mechanisms for regulating mitochondrial function have been sought. Cardiac myocytes are dynamic cells. L-type Ca2+ channels are anchored to F-actin networks by subsarcolemmal stabilizing proteins that also tightly regulate the function of the channel [9–11]. Cytoskeletal proteins stabilize cell structure but also regulate the subcellular distribution of mitochondria [12]. Therefore L-type Ca2+
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channels may mediate cellular responses through cytoskeletal proteins when conformational changes in the channel occur during activation and inactivation. We examined whether the L-type Ca2+ channel (ICa-L) can regulate mitochondrial function. We examined the effect of activation of ICa-L on a number of mitochondrial parameters including superoxide produc-
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tion, mitochondrial membrane potential, NADH production and metabolic activity. We performed experiments in quiescent guineapig ventricular myocytes with consistent ATP utilisation or where we held ATP concentration constant (in the patch pipette) since this allowed us to more readily explore the effects of channel activation on mitochondrial function. We find that ICa-L can regulate mitochondrial
Fig. 1. Direct activation of ICa-L increases intracellular calcium and superoxide. (A) DHE fluorescence (DHE fluores.) recorded from a myocyte before and after exposure to 2 μM BayK (−) and from a myocyte before and after exposure to 2 μM BayK(+). DHE fluorescence (DHE fluores.) recorded from a myocyte during exposure to 2.5 mM Ca2+ alone, from a myocyte during exposure to 0 mM Ca2+ alone and from a myocyte before and after exposure to BayK(−) in 0 mM Ca2+ are shown at top right inset. Mean ± SEM of the ratio of fluorescence for myocytes exposed to treatments as indicated are shown at bottom right inset. (B) DHE fluorescence (DHE fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) then 45 mM KCl, and from a control myocyte (5 mM KCl). Mean ± SEM of the ratio of fluorescence for myocytes exposed to KCl and/or Nisol as indicated are shown at the right. (C) Fura-2 (340/380 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM BayK(−) and from an untreated myocyte. Arrow indicates when treatments were added. Mean ± SEM of changes in Fura-2 fluorescence (% change in fluores.) for myocytes exposed to BayK(−), 2 μM nisoldipine (Nisol) then BayK(−), KCl ± Nisol as indicated are shown at the right.
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function. The regulation of the mitochondria by the channel requires calcium but is also dependent upon communication through actin filaments. We propose that the structural rearrangement of the
channel during activation is transmitted to the mitochondria via the auxiliary β-subunit of the channel through the cytoskeleton. Preliminary data has been published in abstract form [13].
Fig. 2. Direct activation of ICa-L results in an increase in Ψm. (A) Ratiometric JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 2 μM BayK(−) and from another myocyte before and after exposure to 2 μM BayK(+). Arrow indicates when treatments were added. To establish that the JC-1 signal was indicative of Ψm 20 μM oligomycin (Oligo) and 4 μM FCCP were added at the end of each experiment to collapse Ψm where indicated. Individual 535 nm and 580 nm wavelengths are shown above at the upper right. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 2 μM BayK(+) or 2 μM BayK(−) as indicated are shown at the bottom right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) then 45 mM KCl, and from another myocyte before and after exposure to 2 μM Nisol. Arrow indicates when treatments were added. Individual 535 nm and 580 nm wavelengths are shown above at the right. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or Nisol as indicated are shown below at the right. (C) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte patch-clamped and held initially at − 30 mV then voltage-stepped to + 10 mV followed by a voltage-step back to − 30 mV as indicated with arrows (Voltage-stepped) and in another myocyte held at −30 mV and not voltage-stepped to + 10 mV (Not voltage-stepped). Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes as indicated are shown at the right.
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Fig. 3. The increase in Ψm associated with activation of ICa-L does not require calcium. (A) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM Ru360 then 45 mM KCl, and from another myocyte before and after exposure to 2 μM Ru360. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 2 μM Ru360 as indicated are shown at the right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 2 μM BayK(−) and from a myocyte before and after exposure to 2 μM BayK(+) in calcium-free HBS. JC-1 fluorescence recorded from a myocyte patch-clamped with 5 mM BAPTA and 0 mM calcium in the pipette held at − 30 mV and from another myocyte patch-clamped with 5 mM BAPTA and 0 mM calcium in the pipette exposed to 2 μM BayK(−) in calcium-free HBS. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) as indicated are shown at the right. (C) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl in the absence of calcium, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) then 45 mM KCl in the absence of calcium, and from another myocyte before and after exposure to 2 μM Nisol in the absence of calcium. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 2 μM Nisol as indicated are shown at the right. (D) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl in the absence of calcium, from a myocyte before and after exposure to 5 μM amiloride (Amil) and 5 μM tetrodotoxin (TTX) then 45 mM KCl in the absence of calcium, and from another myocyte before and after exposure to 5 μM Amil and 5 μM TTX in the absence of calcium. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl ± 5 μM Amil and 5 μM TTX as indicated are shown at the right.
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2. Materials and methods 2.1. Measurement of superoxide, intracellular calcium, mitochondrial NADH and mitochondrial membrane potential (Ψm) For all studies adult guinea-pig ventricular myocytes were used. For further detail regarding the isolation and culture of myocytes please see the Supplementary File for Online Publication. Superoxide was measured using the fluorescent indicator dihydroethidium (DHE); intracellular calcium was measured using the indicator Fura-2 and Ψm was measured using JC-1. In some experiments changes in Ψm were assessed while voltage-clamping the cell membrane. For further detail see Supplementary File for Online Publication and [14,15]. 2.2. Measurement of metabolic activity The cleavage of the yellow tetrazolium salt (MTT) to purple formazan crystals by complex I was assessed in myocytes using a modified MTT assay. See Supplementary File for Online Publication for more details. 2.3. Synthesis of AID peptide A peptide against the alpha-interacting domain in the I–II linker of α1C-subunit was synthesized using the following amino acid sequence QQLEEDLKGYLDWITQAE as previously described [16] (Auspep Pty Ltd, Parkville, Australia). An HIV-TAT protein sequence (RKKRRQRRR) was added to the peptide to facilitate plasma membrane transport into the cytoplasm. A scrambled control was also synthesized with a HIV-TAT sequence (QKILGEWDLAQYTDQELE). 3. Results 3.1. Direct activation of ICa-L results in an increase in mitochondrial-derived superoxide Increased cytosolic Ca2+ and matrix Ca2+ overload can lead to enhanced generation of reactive oxygen species [17]. We examined whether calcium influx through ICa-L is sufficient to increase superoxide in myocytes using the fluorescent indicator DHE. Application of the DHPR agonist BayK(−) significantly increased DHE signal compared to controls exposed to the (+) enantiomer of BayK that does not act as an agonist (Fig. 1A). The increase in DHE could be attenuated with application of ICa-L antagonist nisoldipine, when calcium uptake into the mitochondria through the mitochondrial Ca2 + uniporter was blocked with Ru360 or with uncoupling of mitochondrial electron transport with FCCP (Fig. 1A). This is in agreement with data indicating that a predominant source of superoxide in ventricular myocytes is the mitochondria [14,15]. We also used a traditional method for activation of ICa-L through plasma membrane depolarization with a high concentration of KCl [18]. DHE was significantly increased with exposure of cells to 45 mM KCl (Fig. 1B). This was attenuated by nisoldipine suggesting activation of ICa-L was responsible for increased production of superoxide. We measured intracellular calcium by assessing changes in 340/380 fluorescence using the indicator Fura-2. BayK(−) and 45 mM KCl increased 340/380 fluorescence that was significantly attenuated when myocytes were exposed to nisoldipine (Fig. 1C). BayK(−) increased [Ca]i from a resting level of 81.3 ± 19.6 to 92.8 ± 3.2 nM (n = 5) and 45 mM KCl increased [Ca]i to 130.5 ± 15.1 nM (n = 5). We exposed cells to calcium-free HEPES-buffered saline (HBS) for at least 3 h to deplete intracellular calcium stores in the myocytes [19]. In 6 cells the DHE signal was only 48.7 ± 6.1% of DHE signal recorded in myocytes exposed to calcium-containing HBS in the absence of BayK(−) confirming that basal superoxide production is dependent on calcium (Fig. 1A inset at top right). These data suggest that direct activation of ICa-L is sufficient to
alter superoxide production by the mitochondria as a result of increased calcium influx through the channel. 3.2. Direct activation of ICa-L results in an increase in Ψm We examined the effect of activation of ICa-L on Ψm. Exposure of cells to BayK(−) increased JC-1 signal 13.3 ± 1.5% compared to controls exposed to BayK(+) (Fig. 2A). A collapse in JC-1 signal after exposure of cells to oligomycin and FCCP demonstrated that the signal was indicative of Ψm. In addition, consistent with an increase in Ψm the 580 nm signal increased after addition of BayK(−) or 45 mM KCl while the 535 nm signal decreased. The uncoupling of Ψm with oligomycin and FCCP resulted in a reversal of the signals (inset right top Figs. 2A and B). Activation of ICa-L with 45 mM KCl caused a 23.8 ± 3.6% increase in JC-1 signal compared to controls treated with HBS containing 5 mM KCl that was attenuated when myocytes were exposed to nisoldipine (Fig. 2B). We directly activated ICa-L by voltage-stepping the plasma membrane from − 30 mV to + 10 mV using patch-clamp technique in cells that had been loaded with JC-1. Initially cells were patch-clamped with internal pipette solution that contained EGTA and calcium calculated to result in a free calcium concentration of 10 nM [20]. Upon voltagestep to +10 mV the JC-1 signal increased 26.3 ± 3.5% and was reversed when the cell was voltage-stepped to − 30 mV (Fig. 2C). The increase was significantly greater than the signal recorded in cells that were not patch clamped (1.6 ± 0.6%; n = 3) or cells that were patch-clamped and the membrane voltage held at −30 mV for the duration of the experiment (1.2 ± 1.2%, n = 4; Fig. 2C). These data indicate that direct activation of ICa-L can increase Ψm and the response is reversible. 3.3. The increase in Ψm after activation of ICa-L does not appear to require calcium We examined whether an increase in calcium uptake into the mitochondria was necessary for the increase in Ψm after activation of the channel. We exposed myocytes to Ru360 prior to activation of the channel with 45 mM KCl and measured changes in JC-1. Ru360 did not significantly alter the JC-1 signal after activation of ICa-L (Fig. 3A). We examined this further by incubating cells in calcium-free HBS for at least 3 h. In calcium-free HBS, BayK(−) resulted in a 13.0 ± 1.5% increase in JC-1 signal (Fig. 3B) that was not significantly different from the response recorded in calcium-containing HBS (13.3 ± 1.5%; Fig. 2A). In order to buffer calcium as effectively as possible, we exposed cells to calcium-free HBS for 3 h and patch-clamped the cells with pipette solution containing 5 mM BAPTA and 0 mM calcium. Activation of the channel either by voltage-step from −30 mV to +10 mV or application of BayK(−) while the cell was dialysed with BAPTA and 0 mM calcium increased JC-1 signal. The increase in JC-1 signal after application of BayK (−) was similar to the increase in JC-1 signal recorded in cells exposed to calcium-free HBS but not perfused internally with BAPTA and 0 mM calcium (Fig. 3B). Similarly, addition of 45 mM KCl in calcium-free HBS resulted in 31.9 ± 7.3% increase in JC-1 signal (Fig. 3C). This was not significantly different from the increase recorded in the presence of calcium (23.8 ± 3.6%; Fig. 2B). The response was significantly attenuated when myocytes were exposed to nisoldipine (Fig. 3C). Depolarization of the plasma membrane potential with high KCl also activates voltage-dependent fast inward Na+ channels. We examined whether the increase in Ψm was due to alterations in Na+ transport in the myocytes. When cells were exposed to calcium-free HBS containing tetrodotoxin (TTX) to block fast inward Na+ channels and amiloride to block Na+/H+ exchange activity, 45 mM KCl increased JC-1 signal 25.9 ± 4.0% that was similar to the increase in JC-1 recorded in calcium-free HBS alone (31.9 ± 7.3%; Fig. 3D). These results suggest that the increase in Ψm associated with activation of ICa-L is not dependent upon changes in sodium or calcium transport.
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3.4. The increase in Ψm associated with activation of ICa-L appears to be mediated through actin filaments Actin filament (F-actin) networks can regulate the function of the L-type Ca2+ channel [9,21,22]. In addition F-actin associates
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with mitochondria via phalloidin binding sites to stabilize mitochondria within the cell [23]. Since the increase in Ψm after activation of ICa-L did not appear to require calcium, we tested whether the response was mediated as a result of movement through actin networks.
Fig. 4. Depolymerization of actin with latrunculin A attenuates the increase in Ψm without altering mitochondrial superoxide production. (A) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 45 mM KCl, and from another myocyte before and after exposure to 5 μM Latrunc alone. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 5 μM Latrunc as indicated are shown at the right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 45 mM KCl in the absence of calcium, from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 45 mM KCl and from another myocyte before and after 5 μM Latrunc alone in the absence of calcium. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 5 μM Latrunc as indicated are shown at the right. (C) DHE fluorescence (DHE fluores.) recorded from a myocyte before and after exposure to 2 μM caffeine (Caff), from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 2 μM Caff and from an untreated myocyte. Mean ± SEM of the ratio of fluorescence for myocytes exposed to 2 μM Caff or 5 μM Latrunc as indicated are shown at the right.
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We exposed myocytes to latrunculin A for 30 min to depolymerize actin and measured changes in Ψm after activation of the channel with 45 mM KCl. Latrunculin A significantly attenuated the increase in Ψm in calcium-containing HBS (Fig. 4A) and in calcium-free HBS (Fig. 4B). Similar results were obtained with BayK(−) in calcium-free HBS (see Supplementary File for Online Publication). We tested whether latrunculin A directly alters mitochondrial function. We exposed myocytes to caffeine that stimulates release of calcium from ryanodine receptors in the sarcoplasmic reticulum, and measured changes in DHE signal. Caffeine increased DHE signal 48.7 ± 6.9% compared to control myocytes not exposed to caffeine. The increase in DHE was not altered when myocytes were also exposed to latrunculin A (Fig. 4C).
We conclude that latrunculin A does not directly alter mitochondrial function. The increase in Ψm after activation of ICa-L appears to involve actin filaments. Activation of the α1c-subunit of ICa-L is mediated through the auxiliary β-subunit that is also tethered to the cytoskeleton [10]. We examined whether the increase in Ψm in response to activation of ICa-L occurs through the movement of the β-subunit of the channel coupled to actin filaments. We synthesized a peptide directed against the alpha-interacting domain (AID) where the auxiliary β-subunit associates with the α1c-subunit of the channel [16]. Exposure to AID-TAT for 20 min significantly attenuated the increase in JC-1 due to activation of ICa-L (Fig. 5A). For control experiments we exposed cells
Fig. 5. A peptide directed against the alpha interacting domain of the α1C-subunit of ICa-L attenuates the increase in Ψm associated with activation of the channel. (A) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte before and after exposure to 1 μM scrambled AID-TAT (AID-TAT(S)) then 45 mM KCl and from another myocyte before and after exposure to 1 μM AID-TAT then 45 mM KCl. Arrow indicates when treatments were added. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 1 μM AID-TAT(S) then KCl or 1 μM AID-TAT then KCl as indicated are shown at the right. (B) JC-1 fluorescence (JC-1 fluores.) recorded from a myocyte patch-clamped and held initially at − 30 mV then voltage-stepped to +10 mV as indicated with the arrow that had been exposed to 1 μM scrambled AID-TAT peptide (AID-TAT(S)) and in another myocyte patch-clamped and held initially at − 30 mV then voltage-stepped to + 10 mV as indicated with the arrow that had been exposed to 1 μM AID-TAT peptide (AID-TAT). 4 μM FCCP was added at the end of the experiment to collapse Ψm where indicated. Current traces recorded from the patch-clamped cells as indicated are shown inset at the left. Mean ± SEM of changes in JC-1 fluorescence (% change in fluores.) for myocytes exposed to 1 μM AID-TAT(S) or 1 μM AID-TAT are shown at the right.
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Fig. 6. Activation of ICa-L increases mitochondrial NADH. (A) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 2 μM BayK(−) and from another myocyte before and after exposure to 2 μM BayK(+). Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated (see text for explanation). Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes exposed to 2 μM BayK(+) or 2 μM BayK(−) as indicated are shown at the right. (B) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 2 μM nisoldipine (Nisol) followed by 45 mM KCl and from an untreated myocyte. Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 2 μM Nisol then 45 mM KCl as indicated are shown at the right. (C) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 0 mM calcium followed by addition of 2 mM external calcium, in a myocyte before and after exposure to 0 mM external calcium only, and in a myocyte before and after exposure to 0 mM external calcium followed by 45 mM KCl. Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes as indicated are shown at the right. (D) NADH fluorescence (NADH fluores.) recorded from a myocyte before and after exposure to 45 mM KCl, from a myocyte before and after exposure to 5 μM latrunculin A (Latrunc) then 45 mM KCl, and from an untreated myocyte. Arrow indicates when treatments were added. 20 μM oligomycin (Oligo) and 4 μM FCCP were added where indicated. Mean ± SEM of changes in NADH fluorescence (% change in fluores.) for myocytes exposed to 45 mM KCl or 5 μM Latrunc then 45 mM KCl as indicated are shown at the right.
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to a scrambled peptide containing a TAT region for 20 min. The scrambled peptide did not attenuate the increase in JC-1 signal after exposure to 45 mM KCl (24.5 ± 1.0%, Fig. 5A). We also patch-clamped myocytes exposed to the AID-TAT peptide and compared the increase in JC-1 after voltage-stepping the cell from −30 mV to +10 mV with patch-clamped cells exposed to the scrambled peptide. The increase in JC-1 after voltage-stepping to +10 mV was significantly attenuated in cells exposed to AID-TAT peptide (1.6±1.6%) but not in cells exposed to scrambled peptide (23.9± 3.5%; Fig. 5B). The attenuation in JC-1 signal after exposure to AID-TAT peptide was greater in cells that were patch-clamped than in non patchclamped cells (Fig. 5A) because during patch-clamp experiments the cells were exposed to the peptide for a longer duration (at least 1 h). The auxiliary β-subunit influences the inactivation rate of the current [24]. Cells exposed to the AID-TAT peptide exhibited a delayed rate of inactivation of currents (Fig. 5B inset left). The time constant (τ) for inactivation of currents obtained from a standard exponential fit (pClamp9, Molecular Devices) in cells exposed to AID-TAT peptide was τ=63.9±5.5 ms (n=16) compared to τ=42.5±0.8 ms in currents from cells exposed to scrambled peptide (n=28). This provided further confirmation that the AID-TAT peptide was binding to AID on the alpha1C subunit of the channel and altering movement of the β-subunit of the channel. These data suggest that the increase in Ψm occurs through activation of the auxiliary β-subunit via the actin cytoskeleton. We propose that the conformational change that occurs upon activation of the channel (including the movement of the auxiliary β-subunit) is transmitted through actin to the mitochondria to increase Ψm. 3.5. Activation of ICa-L increases NADH production We examined whether activation of the channel could alter mitochondrial NADH in myocytes. BayK(−) significantly increased NADH fluorescence compared to myocytes exposed to BayK(+) (Fig. 6A). In all experiments we exposed the myocyte to oligomycin followed by FCCP. An increase in NADH fluorescence was observed after addition of oligomycin consistent with an active TCA cycle, and NADH fluorescence decreased after addition of FCCP consistent with depletion of NADH after collapse of Ψm [25,26]. Activation of ICa-L with 45 mM KCl resulted in a 27.4 ± 5.0% increase in NADH production that was attenuated when myocytes were exposed to nisoldipine (Fig. 6B). In calcium-free HBS, 45 mM KCl was unable to increase NADH signal (Fig. 6C) confirming that NADH production is dependent upon calcium. Consistent with this, addition of 2 mM extracellular calcium caused a further increase in NADH (Fig. 6C). Exposure of myocytes to latrunculin A significantly attenuated the increase in NADH associated with 45 mM KCl (Fig. 6D). In the presence of latrunculin A however oligomycin was still able to increase NADH signal while FCCP decreased NADH signal indicating that latrunculin A did not directly alter mitochondrial function. These data suggest that activation of ICa-L alone is sufficient to increase mitochondrial NADH production. The response requires an increase in influx of calcium through the channel but also appears to involve transmission of channel activation through actin filaments. 3.6. Activation of ICa-L increases mitochondrial metabolic activity in intact myocytes Metabolic activity in myocytes was assessed using a modified MTT assay. BayK(−) significantly increased absorbance at 570 nm by 125.0 ± 16.0% compared to controls treated with BayK(+) (Figs. 7A and B). The increase in absorbance was attenuated when myocytes were exposed to nisoldipine and AID-TAT (Figs. 7A and B). Myocytes were also exposed to the mitochondrial calcium uniporter blocker Ru360 followed by BayK(−). Ru360 significantly attenuated the increase in absorbance in response to BayK(−). The
increase in absorbance associated with BayK(−) was not altered when myocytes were also exposed to dantrolene an inhibitor of the ryanodine receptor (112.0 ± 24.3%). These data suggest that activation of ICa-L increases metabolic activity in myocytes due to increased uptake of calcium by the mitochondria. Preventing movement of the auxiliary β-subunit of the channel with AID-TAT peptide also decreased metabolic activity. 4. Discussion Calcium is integral to mitochondrial function and the mitochondria are capable of tracking changes in intracellular calcium [6,7]. However it is unclear whether calcium influx through ICa-L alone is sufficient to alter mitochondrial function. We report here that calcium influx through the channel increases mitochondrial superoxide, NADH production and metabolic activity in quiescent cardiac myocytes. This occurs as a result of an increase in intracellular calcium and Ru360-sensitive calcium uptake into the mitochondria (Figs. 1 and 7). Exposure of cells to ICa-L antagonist nisoldipine or incubation in calcium-free HBS abolished the increase in superoxide, NADH and metabolic activity (Figs. 1B, 6B, 6C and 7). These results are consistent with previous findings that an increase in ICa-L current and intracellular calcium after exposure to hydrogen peroxide is sufficient to increase mitochondrial superoxide production in quiescent and contracting cardiac myocytes [14]. Our results indicate that activation of ICa-L also increases Ψm. This occurred when the channel was activated directly with the DHPR agonist BayK(−) (Fig. 2A) or with voltage-clamp of the plasma membrane using patch-clamp technique (Fig. 2C). The increase in [Ca]i after addition of BayK(−) or 45 mM KCl was small (Fig. 1C) and insufficient to activate further calcium release from RyR2 [27]. At low intracellular Ca2+ (0–200 nM) Ψm is maintained independent of changes in intracellular Ca2+ [28]. Although we cannot exclude the possibility that calcium mediated the increase in Ψm, calcium uptake into the mitochondria did not appear to be necessary for the increase in JC-1 signal (Fig. 3A). In the absence of calcium (calcium-free HBS and internal calcium buffered with BAPTA to 0 mM) activation of ICa-L alone increased Ψm (Fig. 3B). Furthermore the increase in Ψm after activation of ICa-L in calcium-free HBS was similar in magnitude to the increase in Ψm recorded in cells in HBS containing 2.5 mM (Fig. 3B vs Fig. 2A and Fig. 3C and 3D vs Fig. 2B). Therefore we explored an alternative mechanism for the increase in JC-1 signal after activation of the channel. The cardiac L-type Ca2+ channel comprises the poreforming ion conducting α1C-subunit, an auxiliary β-subunit and an auxiliary α2δ-subunit. The β-subunit is anchored to F-actin networks by subsarcolemmal stabilizing proteins such as the 700 kDa protein AHNAK (Fig. 7C) [10]. F-actin networks can regulate the function of cardiac ICa-L and disruption of actin filaments significantly decreases calcium current [9,11]. In conditions where cytoskeletal proteins are destabilized such as in Duchenne Muscular Dystrophy, ICa-L density and inactivation is decreased [22,29]. In addition cytoskeletal proteins associate with mitochondria via docking proteins [12,23,30]. We tested whether the increase in Ψm was mediated as a result of movement of the auxiliary β-subunit of the channel through actin filaments. Exposure of cells to a peptide directed against the alpha interacting domain or exposure to latrunculin A to depolymerize actin attenuated the increase in JC-1 signal (Figs. 4A, 4B and 5). This suggests that activation of the channel may mediate changes in mitochondrial function through the cytoskeletal network. In vivo, calcium influx is a requirement for contraction and for maintenance of metabolic parameters such as NADH and ATP production. Our results suggest that in addition to calcium influx, activation of the channel may also modulate mitochondrial function as a result of transmission of movement from the β-subunit through the cytoskeleton. We have shown that activation of the channel under voltage-clamp conditions can increase Ψm. Inactivation of the channel
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Fig. 7. Activation of ICa-L increases metabolic activity in cardiac myocytes. (A) MTT assay performed in myocytes following exposure to 2 μM BayK(−), 2 μM BayK(+), 2 μM nisoldipine (Nisol) and 2 μM BayK(−), 1 μM scrambled AID-TAT (AID-TAT(S)) and 2 μM BayK(−), and 1 μM AID-TAT and 2 μM BayK(−). Slopes represent change in absorbance at 570 nm (with reference wavelength of 620 nm) measured over 0–2 min. (B) Mean ± SEM of the ratio of slopes measured over 0–2 min for myocytes exposed to 2 μM BayK(+), 2 μM BayK(−), 2 μM nisoldipine (Nisol) and 2 μM BayK(−), 2 μM Ru360 and 2 μM BayK(−), 20 μM dantrolene (Dant) and 2 μM BayK(−), 1 μM scrambled AID-TAT (AID-TAT(S)) and 2 μM BayK(−), and 1 μM AID-TAT and 2 μM BayK(−) as indicated. ★p b 0.05 compared with BayK(−). n: represents number of animals used in each treatment group. (C) Proposed model explaining transmission of movement after activation of the β auxiliary subunit of the L-type Ca2+ channel through cytoskeletal proteins to the mitochondria. For further explanation see text.
by voltage-stepping the cell from +10 mV to − 30 mV reversed the increase in Ψm (Fig. 2C) suggesting that the mitochondria may be capable of responding closely to changes in activity of the channel during the cardiac cycle. We propose that the increase in mitochondrial membrane potential associated with activation of the channel may assist with ATP production to meet the increase in energy demand during each cardiac cycle. Since the L-type Ca2+ channel (ICaL) is the initiator of contraction in cardiac muscle, this may represent a coordinated process by which mitochondrial function is regulated on a beat-to-beat basis. Under conditions where actin is disrupted (e.g.
Duchenne Muscular Dystrophy, cardiomyopathies involving mutations in actin, cardiac failure), we propose this coupling may become compromised and contribute to poor oxygen consumption and energy supply by the mitochondria that are typically observed in these conditions. Acknowledgments This study was supported by grants from the National Health and Medical Research Council of Australia (NHMRC). Helena Viola is the
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recipient of a Biomedical Postgraduate Research Scholarship from NHMRC and National Heart Foundation of Australia. Livia Hool is the recipient of a NHMRC Career Development Award. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yjmcc.2008.12.015. References [1] Tanabe T, Beam KG, Adams BA, Niidome T, Numa S. Regions of the skeletal muscle dihydropyridine receptor critical for excitation–contraction coupling. Nature 1990;346:567–9. [2] el-Hayek R, Antoniu B, Wang J, Hamilton SL, Ikemoto N. Identification of calcium release-triggering and blocking regions of the II–III loop of the skeletal muscle dihydropyridine receptor. J Biol Chem 1995;270:22116–8. [3] Zhu X, Gurrola G, Jiang MT, Walker JW, Valdivia HH. Conversion of an inactive cardiac dihydropyridine receptor II–III loop segment into forms that activate skeletal ryanodine receptors. FEBS Lett 1999;450:221–6. [4] Li Y, Bers DM. A cardiac dihydropyridine receptor II–III loop peptide inhibits resting Ca(2+) sparks in ferret ventricular myocytes. J Physiol 2001;537:17–26. [5] Katoh H, Schlotthauer K, Bers DM. Transmission of information from cardiac dihydropyridine receptor to ryanodine receptor: evidence from BayK 8644 effects on resting Ca(2+) sparks. Circ Res 2000;87:106–11. [6] Robert V, Gurlini P, Tosello V, Nagai T, Miyawaki A, Di Lisa F, et al. Beat-to-beat oscillations of mitochondrial [Ca2+] in cardiac cells. EMBO J 2001;20:4998–5007. [7] Maack C, Cortassa S, Aon MA, Ganesan AN, Liu T, O'Rourke B. Elevated cytosolic Na + decreases mitochondrial Ca2+ uptake during excitation–contraction coupling and impairs energetic adaptation in cardiac myocytes. Circ Res 2006;99:172–82. [8] Balaban RS. Cardiac energy metabolism homeostasis: role of cytosolic calcium. J Mol Cell Cardiol 2002;34:1259–71. [9] Lader AS, Kwiatkowski DJ, Cantiello HF. Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels. Am J Physiol 1999;277:C1277–83. [10] Hohaus A, Person V, Behlke J, Schaper J, Morano I, Haase H. The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton. FASEB J 2002;16:1205–16. [11] Rueckschloss U, Isenberg G. Cytochalasin D reduces Ca2+ currents via cofilinactivated depolymerization of F-actin in guinea-pig cardiomyocytes. J Physiol 2001;537:363–70. [12] Varadi A, Johnson-Cadwell LI, Cirulli V, Yoon Y, Allan VJ, Rutter GA. Cytoplasmic dynein regulates the subcellular distribution of mitochondria by controlling the recruitment of the fission factor dynamin-related protein-1. J Cell Sci 2004;117: 4389–400. [13] Viola HM, Arthur PG, Hool LC. Evidence for a functional coupling between the Ltype calcium channel and the mitochondria. Circ Res 2008;103(5):e56.
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