Involvement of the mitochondrial calcium uniporter in cardioprotection by ischemic preconditioning

Life Sciences 78 (2006) 738 – 745 www.elsevier.com/locate/lifescie

Involvement of the mitochondrial calcium uniporter in cardioprotection by ischemic preconditioning Shi-zhong Zhang a, Qin Gao a, Chun-mei Cao a, Iain C. Bruce b, Qiang Xia a,* a

Department of Physiology, Zhejiang University School of Medicine, 353 Yan-an Road, Hangzhou 310031, China b Department of Physiology, The University of Hong Kong, Hong Kong, China Received 5 January 2005; accepted 16 May 2005

Abstract The objective of the present study was to determine whether the mitochondrial calcium uniporter plays a role in the cardioprotection induced by ischemic preconditioning (IPC). Isolated rat hearts were subjected to 30 min of regional ischemia by ligation of the left anterior descending artery followed by 120 min of reperfusion. IPC was achieved by two 5-min periods of global ischemia separated by 5 min of reperfusion. IPC reduced the infarct size and lactate dehydrogenase release in coronary effluent, which was associated with improved recovery of left ventricular contractility. Treatment with ruthenium red (RR, 5 AM), an inhibitor of the uniporter, or with Ru360 (10 AM), a highly specific uniporter inhibitor, provided cardioprotective effects like those of IPC. The cardioprotection induced by IPC was abolished by spermine (20 AM), an activator of the uniporter. Cyclosporin A (CsA, 0.2 AM), an inhibitor of the mitochondrial permeability transition pore, reversed the effects caused by spermine. In mitochondria isolated from untreated hearts, both Ru360 (10 AM) and RR (1 AM) decreased pore opening, while spermine (20 AM) increased pore opening which was blocked by CsA (0.2 AM). In mitochondria from preconditioned hearts, the opening of the pore was inhibited, but this inhibition did not occur in the mitochondria from hearts treated with IPC plus spermine. These results indicate that the mitochondrial calcium uniporter is involved in the cardioprotection conferred by ischemic preconditioning. D 2005 Elsevier Inc. All rights reserved. Keywords: Heart; Mitochondrial calcium uniporter; Ischemic preconditioning

Introduction Ischemic preconditioning (IPC), which was originally identified by Murry and co-workers (Murry et al., 1986), provides powerful protection to the ischemic myocardium. Although a variety of intracellular signaling pathways have been implicated in this protection (O’Rourke, 2000), the precise mechanisms remain elusive. Intensive studies showed that prolonged ischemia results in increased intracellular calcium concentration (Allard et al., 1994), and this calcium overload was thought to play a pivotal role in ischemia –reperfusion injury. It has been demonstrated that IPC can lower both intracellular and mitochondrial calcium during reperfusion (Wang et al., 2001). During myocardial performance, mitochondria accumulate significant amounts of

* Corresponding author. Tel.: +86 571 87217146; fax: +86 571 87217147. E-mail address: [email protected] (Q. Xia). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.05.076

calcium from the cytosol through the mitochondrial calcium uniporter (for simplicity, referred to as ‘‘uniporter’’ below), a highly selective calcium ion channel (Kirichok et al., 2004), implying that the uniporter may participate in the cardioprotection conferred by IPC. Opening of the mitochondrial permeability transition pore (referred to below as ‘‘pore’’), which is located in the inner mitochondrial membrane, can lead to mitochondrial swelling and cytochrome C release resulting in cell death (Kroemer et al., 1998). The cardioprotection by IPC may be achieved via inhibiting pore opening during reperfusion (Hausenloy et al., 2002). Therefore, our working hypotheses are that the uniporter may participate in the cardioprotection induced by IPC and that the uniporter may interact with the pore in this process. To test the first hypothesis, we examined the effects of blockade or activation of the uniporter on the cardioprotective effect of IPC as measured by ventricular performance, infarct size and lactate dehydrogenase release in rat hearts

S. Zhang et al. / Life Sciences 78 (2006) 738 – 745

perfused on a Langendorff apparatus. As a preliminary approach to the second hypothesis, the effects of uniporter activity and IPC on pore opening were also assessed. Materials and methods Animals Male Sprague –Dawley rats weighing 210– 240 g were housed in a temperature-controlled (22 –24 -C) room under a 12 h light/12 h dark cycle with water and food freely available. All procedures used in this study were approved by the Ethics Committee for the Use of Experimental Animals in Zhejiang University.

739

bicarbonate buffer containing (mM): NaCl 118.0, KCl 4.7, KH2PO4 1.2, NaHCO3 2.5, MgSO4 1.2, CaCl2 1.4, glucose 10.0. The hearts were then mounted to the Langendorff apparatus and perfused retrogradely with K – H buffer gassed with 95% O2/5% CO2 equilibrated at pH 7.3 – 7.4 and maintained at 37 -C. A water-filled latex balloon was inserted into the left ventricle through the left atrium and the pressure was continuously monitored by a transducer connected to the balloon. The initial value of end-diastolic pressure was set to 5– 8 mm Hg by adjusting the volume of the balloon. The parameters measured were left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and maximal rise/fall velocity (TdP/dt max). All hearts were allowed to equilibrate for 20 min before any additional treatment.

Langendorff heart preparation Perfusion protocol of isolated hearts Rats were anesthetized with chloral hydrate (0.4 g/kg, i.p.) and sacrificed by decapitation. The hearts were excised and immediately placed in ice-cold Krebs – Henseleit (K – H)

The experimental protocols for Langendorff perfused hearts are shown in Fig. 1. All isolated hearts received 30 min of

Fig. 1. Perfusion protocols. Perfusion protocols used for hemodynamic measurements in isolated rat hearts. RR: ruthenium red; RR-Pre: RR pretreatment; Ru360Pre: Ru360 pretreatment; Sper: spermine; CsA: cyclosporin A.

740

S. Zhang et al. / Life Sciences 78 (2006) 738 – 745

regional ischemia by ligation of the left anterior descending artery followed by 120 min of reperfusion. IPC was elicited by two cycles of 5-min global ischemia interspersed with 5 min of reperfusion prior to 30 min of regional ischemia. The effects of uniporter blockade on ischemia/reperfusion (I/R) injury were examined by 5 min of pretreatment with ruthenium red (RR) at 5 AM, a concentration known to block the uniporter (Kawahara et al., 2003; Bae et al., 2003), before 30 min of ischemia or during the first 10 min of reperfusion. Ru360, a specific uniporter blocker at 10 AM (Matlib et al., 1998), was also used for 5 min before 30 min of ischemia. The effects of uniporter activation on IPC were examined by perfusion with spermine (20 AM), an activator of the uniporter (Jensen et al., 1989; Lenzen et al., 1986; Rottenberg and Marbach, 1990), during the first 10 min of reperfusion (IPC + Sper). To explore a possible relationship between the uniporter and the pore in cardioprotection by IPC, cyclosporin A (CsA, 0.2 AM), a specific pore inhibitor (Crompton et al., 1988; Davidson and Halestrap, 1990), was applied for 20 min during the last 5 min of the ischemic period and the first 15 min of the reperfusion period (IPC + CsA + Sper).

Mitochondrial enzyme activity

Infarct size measurement

Measurement of mitochondrial calcium content

Infarct size was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) staining method. At the end of 120 min of reperfusion, the coronary artery was re-occluded, and after the heart was perfused with 1% Evans blue, it was frozen at 20 -C for 2 – 3 h. Then the heart was cut into slices and stained with 1% TTC. Infarct and risk areas were measured by planimetry using Image/J software from NIH. Infarct size was expressed as percentage of risk zone.

After mitochondria were isolated, the protein concentration was determined by Bradford assay, with bovine serum albumin as standard. Mitochondrial calcium content was determined by flame atomic absorption spectrophotometry (Panagiotopoulos et al., 1990). Calcium content was expressed as nanomoles calcium per milligram protein (nmol/mg protein).

The enzyme activities of mitochondria were assessed spectrophotometrically using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). We measured the activities of monoamine oxidase as an outer membrane marker enzyme, and succinic dehydrogenase as an inner membrane marker enzyme. Enzyme activity was expressed as specific activity units per h or min per milligram protein. Electron microscopy Electron microscopy was employed before and after calcium loading. Isolated mitochondria were fixed in a solution of 1% osmium tetroxide in 0.1 M cacodylate buffer at 0 – 4 -C for 120 min. Afterwards, the samples were rinsed, dehydrated, and embedded in Epon 812. Sections were cut on an ultramicrotome, stained with uranyl acetate and lead citrate, and examined under a Philips Tecnai-10 electron microscope.

Measurement of mitochondrial permeability transition pore opening

Lactate dehydrogenase measurement Coronary effluent at 5, 10, 15, 20 and 30 min during reperfusion was collected and the activity of lactate dehydrogenase (LDH) was spectrophotometrically assayed and expressed as units per liter. Isolation of mitochondria Mitochondria were isolated from perfused hearts treated with IPC, IPC plus spermine (20 AM, perfused for 10 min after the IPC procedure) and untreated control hearts. After the appropriate perfusion protocol, the extraventricular tissue was removed, then the ventricle was weighed and finely minced in ice-cold buffer (160 mM KCl, 10 mM EGTA, 0.5% fatty acid-free BSA, pH 7.4), and brought to a final concentration of 1 g/10 ml of buffer. This tissue suspension was homogenized and centrifuged at 1000g for 10 min at 2 -C. Then the supernatant was centrifuged at 8000g for 10 min at 2 -C to obtain the original mitochondrial pellet, which was resuspended by use of the homogenizer in suspension buffer (320 mM sucrose, 10 mM Tris –HCl, pH 7.4), and centrifuged again at 8000g for 10 min at 2 -C to obtain the final mitochondrial pellet (Tanonaka et al., 2003).

To measure pore opening, isolated cardiac mitochondria were resuspended in swelling buffer (120 mM KCl, 10 mM Tris – HCl, 20 mM MOPS, 5 mM KH2PO4, pH 7.4), to a final concentration of 0.25 mg/ml. Pore opening was determined by following the absorbance decrease in light scattering at 520 nm that accompanies mitochondrial swelling. Swelling was initiated by addition of CaCl2 (200 AM), and the extent of pore opening was expressed in terms of the maximal rate of mitochondrial swelling (Baines et al., 2003; Halestrap and Davidson, 1990). Chemicals Ruthenium red, spermine and cyclosporin A were purchased from Sigma Co. (USA), and Ru360 was from Merck Co. (Germany). Statistical analysis All values are expressed as mean T S.D. Statistical significance was determined by one-way ANOVA with Newman– Keuls post-hoc test and Student’s t-test. Values of p < 0.05 were considered significant.

S. Zhang et al. / Life Sciences 78 (2006) 738 – 745

Results Role of the mitochondrial calcium uniporter in myocardial injury induced by ischemia/reperfusion Ruthenium red (RR) is a non-competitive inhibitor of the mitochondrial Ca2+ uniporter. In the present study, RR (5 AM) perfused during the first 10 min of reperfusion improved contractile recovery (Fig. 2), which is in accordance with previous reports (Benzi and Lerch, 1992; Miyamae et al., 1996), and decreased infarct size (Fig. 3) and LDH release (Fig. 4). In order to further investigate whether the acceleration of Ca2+ transport by the uniporter during reperfusion can aggravate the injury caused by ischemia/

741

reperfusion, administration of spermine at 20 AM, a concentration that can activate Ca2+ transport by the uniporter (Deryabina and Zvyagilskaya, 2000; Knox et al., 2004; Nicchitta and Williamson, 1984), during the first 10 min of reperfusion increased the LDH release (Fig. 4), but did not induce significant changes in infarct size (Fig. 3), LVDP, TdP/dt max or LVEDP (Fig. 2) compared to the hearts that underwent ischemia/reperfusion only. Role of the mitochondrial calcium uniporter in ischemic preconditioning From the above results, since blockade of the uniporter during reperfusion can protect hearts from ischemia/reperfu-

Fig. 2. Effects of ischemia/reperfusion (I/R), ruthenium red (RR), Ru360, spermine (Sper), cyclosporin A (CsA) and ischemic preconditioning (IPC) on LVDP (A), TdP/dt max (B and C) and LVEDP (D) at 30 min of reperfusion following 30 min of ischemia in isolated rat hearts. I/R was achieved by ligation of the left anterior coronary artery for 30 min as ischemia, then release of the ligation as reperfusion for 120 min. RR (5 AM) was administered 5 min before the onset of 30 min of ischemia (RR-Pre) or during the first 10 min of reperfusion. Ru360 (10 AM) was added 5 min before the onset of 30 min of ischemia (Ru360-Pre). Sper (20 AM) was given during the first 10 min of reperfusion. CsA (0.2 AM) was perfused for 20 min during the last 5 min of the ischemic period and the first 15 min of the reperfusion period. IPC was achieved by two episodes of 5-min global ischemia separated by 5 min of reperfusion. IPC + Sper: IPC + Sper (20 AM) during the first 10 min of reperfusion. **P < 0.01 vs. I/R; ##P < 0.01 vs. IPC; ++P < 0.01 vs. IPC + Sper (mean T S.D., n = 8).

742

S. Zhang et al. / Life Sciences 78 (2006) 738 – 745

A

90

I/R

80

Sper RR RR-Pre Ru360-Pre

LDH (U/L)

70 60 50 40 30 20 10 0 0

5

10

15

20

25

30

35

Reperfusion time (min)

B 90

IPC IPC+Sper IPC+CsA+Sper

80 70

LDH (U/L)

Fig. 3. Effects of ischemia/reperfusion (I/R), ruthenium red (RR, 5 AM), Ru360 (10 AM), spermine (Sper, 20 AM), cyclosporin A (CsA, 0.2 AM) and ischemic preconditioning (IPC) on infarct size in isolated rat hearts after 120 min of reperfusion following 30 min of ischemia. The protocols were those of Fig. 1. **P < 0.01 vs. I/R; ##P < 0.01 vs. IPC; ++P < 0.01 vs. IPC + Sper (mean T S.D., n = 8).

60 50 40 30 20

sion injury, we hypothesized that blockade of the uniporter before ischemia can also provide protection. We found that pretreatment with RR (5 AM) for 5 min before the onset of 30 min of ischemia mimicked the protective effect of IPC, including decreased infarct size (Fig. 3) and LDH release during reperfusion (Fig. 4), which was associated with augmented LVDP, T dP/dt max and LVEDP recovery (Fig. 2). To further confirm that the RR-induced protection was through inhibition of Ca2+ uptake by mitochondria, we perfused hearts with Ru360 (10 AM), a more specific uniporter inhibitor than RR (Matlib et al., 1998), for 5 min before the onset of 30 min of ischemia and found a protective effect similar to that of RR (Figs. 2 – 4). To further investigate the role of the uniporter in myocardial protection by IPC, we perfused the heart with spermine (20 AM) during the first 10 min of reperfusion and the myocardial protective effect of IPC was abolished (Figs. 2 –4). To further investigate cardiac effect of spermine, an inhibitor of the pore, cyclosporin A (CsA, 0.2 AM) was perfused for 20 min during the last 5 min of the ischemic period and the first 15 min of the reperfusion period; this attenuated the injury induced by spermine described above (Figs. 2– 4). Enzyme activities and morphology of isolated mitochondria Monoamine oxidase and succinate dehydrogenase activities were higher in isolated mitochondria than in the cytosol, and the induction of swelling with high Ca2+ reduced these values (Table 1). Under electron microscopy, mitochondria showed intact membranes and dense matrix space before swelling (Fig. 5A), but disruption of the outer membrane and disappearance of the cristae was evident after Ca2+-induced swelling (Fig. 5B).

10 0 0

5

10

15

20

25

30

35

Reperfusion time (min) Fig. 4. Effects of ischemia/reperfusion (I/R), ruthenium red (RR, 5 AM), Ru360 (10 AM), spermine (Sper, 20 AM), cyclosporin A (CsA, 0.2 AM), ischemic preconditioning (IPC) and IPC + Sper (20 AM) on lactate dehydrogenase (LDH) release during reperfusion in isolated rat hearts. The protocols were those of Fig. 1. **P < 0.01 vs. I/R; #P < 0.05, ##P < 0.01 vs. IPC + Sper (mean T S.D., n = 8).

Effects of mitochondrial calcium uniporter activity and ischemic preconditioning on calcium content in isolated mitochondria In order to observe whether the mitochondrial Ca2+ content can be changed by activation of the uniporter or ischemic preconditioning, we measured the Ca2+ content of isolated mitochondria using flame atomic absorption spectrophotometry. This revealed that the Ca2+ content in mitochondria isolated

Table 1 Activities of monoamine oxidase and succinic dehydronesa in cytosol and mitochondria (specific activity) Mitochondrial enzymes

Cytosol

Mitochondria

Succinate dehydrogenase (U/min/mg protein) Monoamine oxidase (U/h/mg protein)

15.23 T 2.09

62.98 T 9.02*

38.66 T 6.54**

2.34 T 0.59

23.68 T 3.37*

17.86 T 2.72***

Before swelling

* P < 0.01 vs. Cytosol. ** P < 0.01 vs. Before swelling (mean T S.D., n = 5). *** P < 0.05 vs. Before swelling (mean T S.D., n = 5).

After swelling

S. Zhang et al. / Life Sciences 78 (2006) 738 – 745

743

treatment inhibited the pore opening induced by Ca2+(200 AM). Spermine (20 AM) added together with Ca2+(200 AM) accelerated the opening of the pore, and this was blocked by CsA (0.2 AM), a specific pore inhibitor (Fig. 7). Inhibitory effect of ischemic preconditioning on mitochondrial permeability transition pore opening In order to observe the effect of IPC on pore opening, mitochondria prepared from hearts that had been treated with global preconditioning were examined. We found that the pore opening caused by addition of Ca2+ (200 AM) was inhibited. To further explore whether the uniporter is involved in this process, mitochondria were isolated from hearts treated with IPC plus spermine. In this case, the inhibitory effect of IPC on pore opening was abolished (Fig. 7). Discussion Prolonged ischemia produces an increased Ca2+ concentration in the cytosol, which results in mitochondrial accumulation during reperfusion, leading to mitochondrial Ca2+ overload, myocardial damage and cell death (Allard et al., 1994). Therefore, preventing or reducing mitochondrial accumulation by inhibiting the mitochondrial Ca2+ uniporter might be expected to exert a beneficial effect on the heart during ischemia and reperfusion. The uniporter is one of the mechanisms by which mitochondria exchange Ca2+ with the cytosol. The uniporter transports Ca2+ from the cytosol into the intramitochondrial compartment and increases the Ca2+ level in the matrix. We hypothesized that blockade of the uniporter during reperfusion may provide protection against ischemia/reperfusion injury. In the present study, we found that the I/R-induced

Fig. 5. Electron photomicrographs of mitochondria. Fixation was performed following isolation (A), or following exposure to 200 AM CaCl2 (B).

from hearts treated with IPC and ischemia/reperfusion was lower than that in hearts with ischemia/reperfusion only. Pretreatment with spermine (20 AM) after IPC increased the Ca2+ content. Treatment with RR (5 AM) in the I/R heart reduced the mitochondrial Ca2+, while application of spermine (20 AM) in the normally perfused heart increased the Ca2+ gain in mitochondria (Fig. 6). Effects of mitochondrial calcium uniporter activity on mitochondrial permeability transition pore opening in isolated mitochondria In order to explore the possibility that pore opening may be modulated by activity of the uniporter, mitochondria isolated from control hearts were preincubated (3 min) with the uniporter inhibitors Ru360 (10 AM) or RR (1 AM). This

Fig. 6. Effects of ischemia/reperfusion (I/R), ischemic preconditioning (IPC), spermine (20 AM), IPC + Sper (20 AM) and ruthenium red (RR, 5 AM) on Ca2+ content in mitochondria. After 20 min of equilibration, I/R was achieved by 30 min of global ischemia and 60 min of reperfusion. Protocols for IPC and IPC + Sper groups followed those in Fig. 1, but the reperfusion time was 60 min. Sper and RR were given during the first 10 min of reperfusion. **P < 0.01 vs. I/R; ##P < 0.01 vs. IPC; ++P < 0.01 vs. control (mean T S.D., n = 5).

744

S. Zhang et al. / Life Sciences 78 (2006) 738 – 745

Fig. 7. The effects of ruthenium red (RR), Ru360, spermine (Sper), cyclosporin A plus spermine (CsA + Sper), ischemic preconditioning (IPC) and IPC plus spermine (IPC + Sper) treatment, on pore opening. (A) Pore opening in mitochondria from either control hearts or from hearts that had been subjected to IPC or IPC plus spermine (20 AM) treatment was measured by spectrophotometric monitoring of the decrease in absorbance at 520 nm (A 520) after addition of CaCl2 (200 AM). RR (1 AM), Ru360 (10 AM) or CsA (0.2 AM) was added 3 min before Ca2+ addition. Spermine (20 AM) and Ca2+ were added together; (B) Maximal swelling rates of mitochondria. **P < 0.01 vs. control; ##P < 0.01 vs. IPC; ++P < 0.01 vs. Sper (mean T S.D., n = 5).

myocardial injury was attenuated by RR or Ru360, blockers of the uniporter, but was aggravated by spermine, an activator of the uniporter, suggesting that it plays a role in I/R injury. It has been demonstrated that IPC lowers the mitochondrial Ca2+ level during reperfusion (Wang et al., 2001), so we extended these findings by testing the hypothesis that the uniporter may also be involved in the cardioprotection conferred by ischemic preconditioning. The present study showed that activation of the uniporter with spermine (20 AM) blocked the protective effect of IPC, and pretreatment with the uniporter inhibitors Ru360 or RR mimicked the protective effect induced by IPC, suggesting a possible inhibition of the uniporter in myocardial protection by IPC.

The mitochondrial permeability transition pore is located in the inner mitochondrial membrane and can mediate cell death by apoptosis or necrosis when opened. Previous studies indicated that inhibition of pore opening during reperfusion may mediate the cardioprotection induced by IPC. In the present study, the cardioprotective effect of IPC was abolished by administration of spermine during reperfusion, which was reversed by cyclosporin A, a specific inhibitor of the pore, raising the possibility of a relationship between the pore and the uniporter in cardioprotection induced by IPC. In experiments with isolated mitochondria, we found that inhibition of the uniporter with RR or Ru360 diminished the opening of the pore; further, activating the uniporter with spermine increased pore opening, indicating that the activity of the pore may be related to uniporter activity. The exact relationship between the uniporter and the pore is not yet clear. In the study of isolated mitochondria, inhibition of the uniporter with RR reduced the Ca2+ content in the mitochondria like IPC; however, activation of the uniporter with spermine caused an increase of Ca2+ content in mitochondria, which may influence pore opening. To observe the relationship between IPC and pore opening, we measured the opening induced by Ca2+ (200 AM) and found it was markedly inhibited in mitochondria isolated from preconditioned rat hearts. Furthermore, the inhibitory effect of IPC on pore opening was blocked in mitochondria isolated from hearts treated with IPC plus spermine. These results raise the possibility that the inhibitory effect of IPC on pore opening may occur via inhibition of the uniporter. The present study demonstrated the possible involvement of the uniporter in cardioprotection induced by IPC. However, the question remains open as to what mechanisms are responsible for the inhibition of the uniporter. A recent study showed that the uniporter is a highly Ca2+-selective and inwardly rectifying ion channel, and the open probability of the channel is about 99% at 200 mV, declining to approximately 11% at 80 mV (Kirichok et al., 2004). So, depolarization of the mitochondrial membrane may inhibit the activity of the uniporter. Opening of mitochondrial ATPsensitive potassium channels (mitoKATP) has been shown to depolarize the mitochondrial membrane potential and thus protect the heart during ischemia and reperfusion (Holmuhamedov et al., 1998). We speculate that in the case of cardioprotection, the inhibition of uniporter activity may be induced by depolarization of the mitochondrial membrane via mitoKATP activation, and then pore opening is inhibited. Further studies are needed to confirm or refute this speculation. Conclusion In conclusion, this study shows that inhibition of the mitochondrial calcium uniporter mimics the cardioprotective effect of IPC, and cancellation of the protective effect of IPC induced by activating the uniporter reveals its potential role in this process. Experiments on isolated perfused rat hearts and isolated mitochondria suggested that the protective effect induced by inhibition of the uniporter may be related to inhibition of mitochondrial permeability transition pore opening.

S. Zhang et al. / Life Sciences 78 (2006) 738 – 745

References Allard, M.F., Flint, J.D., English, J.C., Henning, S.L., Salamanca, M.C., Kamimura, C.T., English, D.R., 1994. Calcium overload during reperfusion is accelerated in isolated hypertrophied rat hearts. Journal of Molecular and Cellular Cardiology 26 (12), 1551 – 1563. Bae, J.H., Park, J.W., Kwon, T.K., 2003. Ruthenium red, inhibitor of mitochondrial Ca2+ uniporter, inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2+ depletion and cytochrome c release. Biochemical and Biophysical Research Communications 303 (4), 1073 – 1079. Baines, C.P., Song, C.X., Zheng, Y.T., Wang, G.W., Zhang, J., Wang, O.L., Guo, Y., Bolli, R., Cardwell, E.M., Ping, P., 2003. Protein kinase Cepsilon interacts with and inhibits the permeability transition pore in cardiac mitochondria. Circulation Research 92 (8), 873 – 880. Benzi, R.H., Lerch, R., 1992. Dissociation between contractile function and oxidative metabolism in postischemic myocardium. Attenuation by ruthenium red administered during reperfusion. Circulation Research 71 (3), 567 – 576. Crompton, M., Ellinger, H., Costi, A., 1988. Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochemical Journal 255 (1), 357 – 360. Davidson, A.M., Halestrap, A.P., 1990. Partial inhibition by cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by submicromolar [Ca2+], but not by butyrate. Evidence for two distinct swelling mechanisms. Biochemical Journal 268 (1), 147 – 152. Deryabina, Y.I., Zvyagilskaya, R.A., 2000. The Ca2+-transport system of yeast (Endomyces magnusii) mitochondria: independent pathways for Ca2+ uptake and release. Biochemistry (Moscow) 65 (12), 1352 – 1356. Halestrap, A.P., Davidson, A.M., 1990. Inhibition of Ca2+-induced largeamplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidylprolyl cis – trans isomerase and preventing it interacting with the adenine nucleotide translocase. Biochemical Journal 268 (1), 153 – 160. Hausenloy, D.J., Maddock, H.L., Baxter, G.F., Yellon, D.M., 2002. Inhibiting mitochondrial permeability transition pore opening: a new paradigm for myocardial preconditioning? Cardiovascular Research 55 (3), 534 – 543. Holmuhamedov, E.L., Jovanovic, S., Dzeja, P.P., Jovanovic, A., Terzic, A., 1998. Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function. American Journal of Physiology. Heart and Circulatory Physiology 275 (5 Pt 2), H1567 – H1576. Jensen, J.R., Lynch, G., Baudry, M., 1989. Allosteric activation of brain mitochondrial Ca2+ uptake by spermine and by Ca2+: brain regional differences. Journal of Neurochemistry 53 (4), 1182 – 1187. Kawahara, K., Takase, M., Yamauchi, Y., 2003. Ruthenium red-induced transition from ventricular fibrillation to tachycardia in isolated rat hearts:

745

possible involvement of changes in mitochondrial calcium uptake. Cardiovascular Pathology 12 (6), 311 – 321. Kirichok, Y., Krapivinsky, G., Clapham, D.E., 2004. The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427 (6972), 360 – 364. Knox, C.D., Belous, A.E., Pierce, J.M., Wakata, A., Nicoud, I.B., Anderson, C.D., Pinson, C.W., Chari, R.S., 2004. Novel role of phospholipase Cdelta1: regulation of liver mitochondrial Ca2+ uptake. American Journal of Physiology: Gastrointestinal and Liver Physiology 287 (3), G533 – G540. Kroemer, G., Dallaporta, B., Resche-Rigon, M., 1998. The mitochondrial death/life regulator in apoptosis and necrosis. Annual Review of Physiology 60, 619 – 642. Lenzen, S., Hickethier, R., Panten, U., 1986. Interactions between spermine and Mg2+ on mitochondrial Ca2+ transport. Journal of Biological Chemistry 261 (35), 16478 – 16483. Matlib, M.A., Zhou, Z., Knight, S., Ahmed, S., Choi, K.M., Krause-Bauer, J., Phillips, R., Altschuld, R., Katsube, Y., Sperelakis, N., Bers, D.M., 1998. Oxygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2+ uptake into mitochondria in vitro and in situ in single cardiac myocytes. Journal of Biological Chemistry 273 (17), 10223 – 10231. Miyamae, M., Camacho, S.A., Weiner, M.W., Figueredo, V.M., 1996. Attenuation of postischemic reperfusion injury is related to prevention of [Ca2+]m overload in rat hearts. American Journal of Physiology. Heart and Circulatory Physiology 271 (5 Pt 2), H2145 – H2153. Murry, C.E., Jennings, R.B., Reimer, K.A., 1986. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74 (5), 1124 – 1136. Nicchitta, C.V., Williamson, J.R., 1984. Spermine. A regulator of mitochondrial calcium cycling. Journal of Biological Chemistry 259 (21), 12978 – 12983. O’Rourke, B., 2000. Myocardial KATP channels in preconditioning. Circulation Research 87 (10), 845 – 855. Panagiotopoulos, S., Daly, M.J., Nayler, W.G., 1990. Effect of acidosis and alkalosis on postischemic Ca gain in isolated rat heart. American Journal of Physiology. Heart and Circulatory Physiology 258 (3 Pt 2), H821 – H828. Rottenberg, H., Marbach, M., 1990. Regulation of Ca2+ transport in brain mitochondria: I. The mechanism of spermine enhancement of Ca2+ uptake and retention. Biochimica et Biophysica Acta 1016 (1), 77 – 86. Tanonaka, K., Iwai, T., Motegi, K., Takeo, S., 2003. Effects of N-(2mercaptopropionyl)-glycine on mitochondrial function in ischemic – reperfused heart. Cardiovascular Research 57 (2), 416 – 425. Wang, L., Cherednichenko, G., Hernandez, L., Halow, J., Camacho, S.A., Figueredo, V., Schaefer, S., 2001. Preconditioning limits mitochondrial Ca2+ during ischemia in rat hearts: role of KATP channels. American Journal of Physiology. Heart and Circulatory Physiology 280 (5), H2321 – H2328.