Skip Navigation


Cardiovascular Research Advance Access first published online on October 18, 2007
This version [Corrected Proof] published online on November 23, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm039
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
77/2/416    most recent
cvm039v3
cvm039v2
cvm039v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Javadov, S.
Right arrow Articles by Karmazyn, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Javadov, S.
Right arrow Articles by Karmazyn, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

NHE-1 inhibition-induced cardioprotection against ischaemia/reperfusion is associated with attenuation of the mitochondrial permeability transition

Sabzali Javadov1, Angel Choi1, Venkatesh Rajapurohitam1, Asad Zeidan1, Alexei G. Basnakian2 and Morris Karmazyn1,*

1 Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, Medical Sciences Building, London, Ontario, Canada N6A 5C1
2 Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

* Corresponding author. Tel: +1 519 661 3872; fax: +1 519 661 3827. E-mail address: morris.karmazyn{at}schulich.uwo.ca

Time for primary review: 23 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: The possible contribution of the cardiac mitochondrial permeability transition pore (PTP) towards the cardioprotective effects of Na+–H+ exchanger-1 (NHE-1) inhibition was studied in hearts subjected to ischaemia/reperfusion (IR).

Methods and results: Langendorff-perfused rat hearts were subjected to 40 min of global ischaemia and 60 min of reperfusion in the presence or absence of the NHE-1 specific inhibitor AVE-4890 (AVE, 5 µM). Mitochondrial PTP opening was determined in the intact heart using 2-deoxy-[3H]-glucose entrapment and in isolated mitochondria by monitoring the decrease of the calcium-induced light scattering. Mitochondrial respiration was measured with a Clark-type oxygen electrode whereas release of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) and levels of cleaved poly-(ADP-ribose) polymerase (PARP) were analysed by western blotting. IR induced mitochondrial PTP opening, which was inhibited by 28% (P < 0.05) with AVE treatment. Mitochondria isolated from AVE-treated hearts demonstrated significantly less calcium-induced swelling and higher substrate oxidation at complex I and II as well as cytochrome c oxidase and citrate synthase activity. AVE treatment also suppressed IR-induced release of AIF and EndoG from mitochondria, prevented the IR-induced rise in cleaved PARP levels, and was associated with significantly enhanced postischemic recovery of left ventricular developed pressure and a significant decrease in lactate dehydrogenase release. AVE did not affect PTP opening directly in isolated mitochondria.

Conclusion: The beneficial effect of NHE-1 inhibition in hearts subjected to IR is associated with attenuation of mitochondrial PTP opening and apoptosis and the resultant mitochondrial dysfunction. The effect of AVE on PTP opening most likely is indirect, as pore opening was not affected by direct administration of AVE to mitochondrial suspensions.

KEYWORDS Heart; Ischaemia; Reperfusion; Mitochondria; Sodium–hydrogen exchanger; Apoptosis

Received May 1, 2007; revised October 12, 2007; accepted October 16, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Na+–H+ exchanger-1 (NHE-1) represents a major mechanism for H+ extrusion and pHi regulation under both normal physiological and pathological conditions, especially during ischaemia/reperfusion (IR).1 Activation of the NHE-1 due to intracellular acidosis during ischaemia leads to [Na+] elevation in the cytoplasm that, in turn, causes the Na+/Ca2+ exchanger (NCE) to act in reverse mode leading to Ca2+ accumulation.1 Therefore, attenuation of NHE-1 activity by specific inhibitors could represent one of the potential pharmacological approaches available to protect the heart from IR injury. Indeed, after the initial report showing NHE inhibition as an effective approach to improve heart function,2 diverse NHE-1 specific inhibitors have been demonstrated to elicit cardioprotective effects in various models of cardiac IR.1,3 NHE-1 inhibitors have been shown to attenuate IR-induced heart dysfunction by reducing infarct size, reduction of arrhythmias and improvement of functional recovery, and that was associated with normalization of intracellular Na+ and Ca2+ level and reduction of apoptosis.1 Furthermore, the cardioprotection produced by NHE-1 inhibitors appears to be more robust than other cardioprotective strategies.4,5 Despite the extensive evidence identifying cardioprotective effect of NHE-1 inhibitors against IR, the molecular and cellular mechanisms underlying the beneficial effect have not been completely elucidated.

In addition to ATP synthesis, mitochondria also have been recognized as key players in regulation of cell death in cardiac injury such as that produced by IR. Cellular dysfunctions induced by IR converge on mitochondria and induce a sudden increase in the permeability of the inner mitochondrial membrane by the opening of permeability transition pores (PTP) in the inner mitochondrial membrane with subsequent loss of ionic homeostasis, matrix swelling, and outer membrane rupture.6,7 During mild stress, transient (reversible) opening of the PTP can occur in highly Ca2+-sensitive mitochondrial populations leading to the release of apoptotic proteins such as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) from the intermembrane space and induce caspase-dependent and/or caspase-independent apoptosis.8,9 AIF and EndoG have been shown to induce apoptosis without caspase-3 activation by translocation into nuclei following an apoptotic signal.8,10 However, the presence of functionally competent mitochondria is very much essential to support ATP-dependent processes including apoptosis. It can be speculated that during severe IR there is massive swelling and membrane depolarization of mitochondria leading to induction of more ROS production, ATP hydrolysis, and caspase activation resulting in cell death. Molecular mechanisms underlying the PTP-induced cellular dysfunction during cardiac IR remain to be elucidated. However, a growing body of evidence supports the concept that pharmacological inhibition of the PTP is an effective and promising strategy for the protection of the heart against IR injury.6,7,9

We have previously reported that inhibition of NHE-1 with EMD-87580 attenuates mitochondrial PTP opening and mitochondrial dysfunction associated with its anti-remodelling effect in rat hearts 12 and 18 weeks after coronary artery ligation. Furthermore, mitochondria isolated from the EMD-87580-treated hearts demonstrated reduced sensitivity to Ca2+ addition.11 One possible mechanism for this may represent a pH-dependent process since intracellular protons can inhibit the PTP opening.6,7 Accordingly, we examined the effect of the new NHE-1 specific inhibitor AVE-4890 (AVE) on cardiac performance and mitochondrial function by measuring ex vivo and in vitro PTP opening, respiration rates, and the release of mitochondrial proteins AIF and EndoG following IR in isolated rat hearts. Our study demonstrates that AVE improves postischaemic recovery of heart function and lactate dehydrogenase (LDH) release. Furthermore, these findings were associated with inhibition of mitochondrial PTP opening, improvement of mitochondrial respiration and inhibition of mitochondrial release of AIF and EndoG following myocardial IR.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
Male Sprague-Dawley rats weighing 250–280 g were purchased from Charles River (St Constant, Quebec, Canada). All experiments were performed according to protocols approved by the University Animal Care and Use Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1. Langendorff heart perfusion and animal groups
Isolated rat hearts were perfused by the Langendorff mode as described in detail previously.11 Briefly, isolated hearts were placed in Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.8 KCl, 1.2 KH2PO4, 1.25 CaCl2, 1.2 MgSO4, 25 NaHCO3 and 11 glucose equilibrated at pH 7.4 with 95%O2–5%CO2. Hearts were perfused with 37°C Krebs-Henseleit buffer at a constant flow of 10–12 mL/min and paced at 300 beats/min with an electrical stimulator (SD-9, Grass Instruments, Quincy, MA, USA). A water-filled latex balloon was inserted into the left ventricle for continuous monitoring of developed pressure (LVDP). Initial left ventricular end-diastolic pressure (LVEDP) was set to ~5 mmHg before the start of the experiment by adjusting the volume of the balloon. All determinations of ventricular performance and coronary pressure were obtained online using an IOX data analysis system (EMKA Technologies, Paris, France).

Animals were randomly assigned to the following treatment groups: (i) hearts perfused with Krebs-Henseleit solution (control group); (ii) hearts perfused with Krebs-Henseleit solution containing 5 µM AVE (control+AVE group); (iii) hearts subjected to global ischaemia followed by reperfusion (IR group); or (iv) hearts subjected to global ischaemia followed by reperfusion in the presence of 5 µM AVE (IR+AVE group). Protocols of experiments are illustrated schematically in Figure 1. Global normothermic ischaemia was induced by switching off the pump and electrical stimulation simultaneously and immersing the heart in buffer maintained at 37°C after a total pre-ischaemic period of 60 min. In all experiments, the ischaemic period was 40 min after which flow and pacing were restored to pre-ischaemic levels and reperfusion was followed for 60 min. When the NHE-1 inhibitor was present, the hearts were perfused for 10 min before ischaemia and throughout the reperfusion period with AVE dissolved in Krebs-Henseleit solution. AVE was a gift from Sanofi-Aventis (Frankfurt, Germany). To determine the role of the NCE, in select experiments, 1 µM KB-R7943 (KBR, Tocris Bioscience, Ellisville, MO, USA), a selective inhibitor of the reverse mode of the NCE was administered exactly as described for AVE. Samples of perfusate were collected prior to ischaemia and during reperfusion at indicated time points to measure LDH activity. The hearts were used to isolate mitochondria after the corresponding protocols.


Figure 1
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 Perfusion protocols. Experimental groups – Control: hearts perfused with normal Krebs-Henseleit solution; Control + AVE (AVE-4890): hearts perfused with Krebs-Henseleit solution containing 5 µM AVE; IR (ischaemia/reperfusion): hearts subjected to global ischaemia followed by reperfusion; IR + AVE: hearts subjected to global ischaemia followed by reperfusion in the presence of 5 µM AVE.

 
LDH activity in perfusate was assessed by an enzymatic method as previously described.12

2.2. Measurement of the mitochondrial permeability transition pore opening in isolated heart
The method for measurement of the mitochondrial PTP opening using 2-deoxy-[3H]-glucose ([3H]-DOG) entrapment technique has previously been described.11,13 Briefly, after a 20 min equilibration period the hearts were perfused in a recirculating manner with 50 mL of Krebs-Henseleit buffer containing 0.5 mM [3H]-DOG (0.1 µCi/mL) for 30 min. Perfusion then returned to non-recirculating mode with normal buffer alone or containing 5 µM AVE for 10 min followed by the 40-min global ischaemia and 60-min reperfusion with or without AVE (Figure 1). At the end of the corresponding perfusion protocol myocardial mitochondria were isolated.

2.3. Isolation of mitochondria
To isolate mitochondria, the ventricles were cut, weighed, and homogenized with a Polytron homogenizer in 5 mL of ice-cold sucrose buffer containing (in mM): sucrose 300, Tris–HCl 10, EGTA 2; pH 7.4. Mitochondria were isolated from the homogenate by centrifugation for 2 min at 2000 x g to remove cell debris, followed by centrifugation of the supernatant at 10 000 x g for 5 min. The pellet was then washed two times at 10 000 x g for 5 min. The final pellet was resuspended in 500 µL of sucrose buffer and a 50-µL sample was taken to assay citrate synthase (CS) activity. A 200-µL sample was used to measure mitochondrial respiration rates and protein analysis. To quantify the entrapped 3H, an equal volume of 5% perchloric acid was added to 250 µL of mitochondria and centrifuged at 10 000 x g for 2 min. The supernatant was added to 10 mL of scintillant (Ecolite+, ICN) to measure the radioactivity. Mitochondrial entrapment of [3H]-DOG as an indicator of PTP opening is expressed in DOG units as a ratio of 100 000 times mitochondrial [3H] dpm per unit CS activity to the total heart [3H] dpm per gram wet weight.12

2.4. Measurement of respiration, citrate synthase activity and permeability transition pore opening in isolated mitochondria
Measurement of mitochondrial respiration was performed using a Gilson Oxygraph equipped with a Clark-type oxygen electrode at 30°C as described previously.11 Mitochondria were suspended in a buffer containing (in mM): 125 KCl, 20 MOPS, 10 Tris, 0.5 EGTA, 2 KH2PO4, pH 7.2, supplemented with either of the following two substrate combinations to measure complex I- and complex II-mediated respiration rates, respectively: (i) 2.5 mM 2-oxoglutarate + 1 mM L-malate or (ii) 2.5 mM succinate + 1 µM rotenone. Respiration rates were measured in the absence (state 2) or presence (state 3) of 1 mM ADP. At the end of each run, 0.5 µM antimycin A and 10 mM ascorbate + 0.3 mM N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) were added and the new rate of respiration measured. Additionally, 20 µM cytochrome c was added at state 3 to measure the cytochrome c-induced stimulation of mitochondrial respiration. The respiration rates were normalized to mg of mitochondrial protein.

CS activity was assayed spectrophotometrically by measuring coenzyme A formation at 412 nm as described previously.14

For measurement of in vitro PTP opening in isolated mitochondria, swelling of de-energized mitochondria in the presence or absence of calcium was determined by monitoring the decrease in light scattering at 520 nm as described previously.15 Mitochondria were incubated at 25°C in 3 mL buffer containing (mM) KSCN 150, MOPS 20, Tris 10 and nitrilotriacetic acid 2, supplemented with 0.5 µM rotenone, 0.5 µM antimycin and 2 µM A23187. [GenBank] Nitrilotriacetic acid was present to allow buffered calcium concentrations of 50–500 µM to be employed. A23187 [GenBank] was added to ensure complete equilibration of Ca2+ across the mitochondrial inner membrane under de-energized conditions.

2.5. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blotting
Protein concentration in isolated mitochondria was determined by the Bradford protein assay (Bio-Rad, Hercules, CA). Twenty or 30 µg of mitochondrial (for AIF, adenine nucleotide translocase, ANT, EndoG and cytochrome c) and cytosolic [for cleaved poly-(ADP-ribose) polymerase, PARP and actin] protein was resolved on 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membrane (Amersham Biosciences Inc., Piscataway, NJ, USA). The PARP antibody detects full length PARP as well as 89 and 24 kDa fragments produced primarily from caspase cleavage. The membranes were incubated with actin, EndoG (Calbiochem, CA, USA), AIF, PARP, cytochrome c (Cell Signalling Technology Inc., Beverly, MA, USA) or ANT (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies for 1 h followed by secondary antibody for 1 h, and the signal was visualized using a chemiluminescent detection system, ECLTM (Amersham BioSciences, Cleveland, OH, USA).

2.6. Immunostaining for apoptosis-inducing factor, endonuclease G and cytochrome c oxidase subunit IV
Tissue samples were fixed in 10% neutral formalin, dehydrated and embedded in paraffin. The 4-µm sections were pre-treated with proteinase K (20 µg/mL) and blocked with staining buffer (1% BSA, 0.05% Tween-20, PBS). The sections were incubated with EndoG, AIF or cytochrome c oxidase subunit IV (COX IV) antibodies for 2 h and probed with secondary antibody conjugates: anti-rabbit IgG-AlexaFluor 647, anti-goat IgG-AlexaFluor 488 or anti-mouse-AlexaFluor 594 (Invitrogen) for 1 h, respectively. Slides were analysed under 600x magnification using an Olympus IX-81 microscope (Olympus America Inc., Center Valley, PA, USA). Images and acquisitions were made with a digital camera HAMAMATSU ORCA-ER (Hamamatsu Photonics K.K., Hamamatsu City, Japan) and software Slidebook 4.1 (SciTech Pty Ltd., Australia). The distribution of proteins in cellular compartments was determined as described previously.16 Optical density of COX IV signal was used as the marker of mitochondrion localization and subtracted from the EndoG of AIF optical signals. To minimize the effect of readings obtained at different wavelengths, the brightness of the fluorofores bound to the antibodies was determined prior to the image capturing, and the sensitivity of the camera was adjusted to equilibrate the sintensities of the signals. The result of the subtraction was considered ‘non-mitochondrial EndoG’ or ‘non-mitochondrial AIF’, while the rest of the signals were considered ‘mitochondrial’ EndoG or AIF.

TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s manual.

2.7. Caspase-3 activity
The activity of caspase-3 was measured in 20 µg cytosol using a colorimetric caspase-3 assay kit (Assay Designs, Ann Arbor, MI, USA) according to the manufacturer’s protocol.

2.8. Statistical analysis
Data were analysed using a two-way ANOVA and group differences were detected using a Tukey post-hoc test. In experiments on LDH release and mitochondrial respiration in the presence of exogenous cytochrome c where only two groups were studied, group differences were detected using a two-tailed Student’s t-test. Differences were considered to be statistically significant at a level of P < 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1. Heart function
To study the effect of AVE on cardiac function, hearts (n = 8/group) were perfused with Krebs-Henseleit solution in the presence or absence of AVE prior to being subjected to 40 min global ischaemia and 60 min reperfusion. AVE did not affect LVDP or LVEDP in control hearts. Pre-ischaemic values for LVDP were 57.9 ± 5.6 mmHg and 55.9 ± 3.4 mmHg in IR and IR + AVE groups, respectively. As shown in Figure 2A, AVE significantly improved the functional recovery of hearts on reperfusion: LVDP was increased from 37.9 ± 1.5 to 53.3 ± 3.4 mmHg (IR + AVE: 94% of the pre-ischemic LVDP vs. IR: 66%, P < 0.05, 60 min after the beginning of reperfusion). Beneficial effects of AVE were also noted in the significant reduction of LVEDP recovered from 46.6 ± 6.4 to 30.5 ± 7.0 mmHg (P < 0.05) after 60 min reperfusion in hearts treated with AVE (Figure 2B). Coronary pressure was not significantly affected by AVE treatment (data not shown). The improvement in functional recovery in the presence of AVE was associated with a significant decrease in LDH release, a marker of necrotic cell death, throughout reperfusion (Figure 2C).


Figure 2
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 The effect of AVE (AVE-4890) on functional recovery and LDH (lactate dehydrogenase) release in hearts. (A) LVDP (left ventricular developed pressure) in perfused hearts in the absence (IR – ischaemia/reperfusion, circles) or presence (IR + AVE, squares) of AVE. Data are expressed as percent of pre-ischaemic values. (B) LVEDP (left ventricular end-diastolic pressure) in perfused hearts in absence (IR, circles) or presence (IR + AVE, squares) of AVE. Data are expressed in mm Hg. (C) LDH activity in perfusate. Data are shown as means ± SEM of eight hearts in each group. *P < 0.05, **P < 0.01 IR + AVE vs. IR.

 
3.2. Mitochondrial permeability transition pore opening
The effect of AVE on IR-induced PTP opening was examined in isolated hearts by the [3H]-DOG technique and the data are summarized in Figure 3. PTP opening was increased from 20.1 ± 2.5 in control group to 87.1 ± 9.9 (P < 0.01) DOG units in IR group that was reduced by 28% (to 62.9 ± 7.0) with AVE (P < 0.05 vs. IR group). AVE had no effect on mitochondrial PTP opening in control hearts that were not subjected to IR (Figure 3).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 The effect of AVE (AVE-4890) on mitochondrial PTP (permeability transition pore) opening. PTP opening in hearts from control (C) or IR (ischaemia/reperfusion) group in the presence or absence of AVE was measured by the [3H]-DOG (2-deoxy-[3H]-glucose) entrapment method. Results are expressed in DOG units, which indicate a ratio of 100 000 times mitochondrial [3H] dpm per unit CS (citrate synthase) activity to the total heart [3H] dpm per gram wet weight. Data are shown as means ± SEM of seven (C + AVE group) and eight (C, IR, IR + AVE groups) hearts in each group. *P < 0.01 IR vs. C; +P < 0.05 IR + AVE vs. IR.

 
Since the AVE-induced inhibition of mitochondrial PTP opening could be attributable to a direct effect of AVE on pore formation, we next studied the direct effect of AVE on de-energized mitochondrial swelling following the addition of 200 µM Ca2+ (Figure 4A). In parallel, 0.6 µM cyclosporin A was used as a positive control for pore opening inhibition. It is important to note that we have measured PTP in isolated mitochondria with complete equilibration of Ca2+ across the mitochondrial inner membrane under de-energized conditions and thus eliminated the possible effects of calcium loading of the mitochondria and/or differences in membrane potential, which are potent PTP inducers.6,7 When added directly to mitochondria, AVE was not able to inhibit Ca2+-induced mitochondrial matrix swelling and thus did not change in vitro PTP opening. However, mitochondria isolated from AVE-treated hearts were less sensitive to [Ca2+] than control mitochondria (Figure 4B and C). Taken together, these data suggest that reduced pore opening in AVE-treated hearts in vivo was not due to a direct effect of the NHE-1 inhibitor on the mitochondrial PTP.


Figure 4
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 Ca2+ sensitivity of the PTP (permeability transition pore) opening in mitochondria treated with AVE (AVE-4890) in vitro or in mitochondria isolated from AVE-treated hearts. Measurement of the PTP under de-energized conditions was performed by monitoring the calcium-induced decrease in light scattering (A520). (A) Original traces are shown for one mitochondrial preparation derived from hearts of control, IR (ischaemia/reperfusion) or IR + AVE groups. Additionally, mitochondria isolated from hearts of IR group were treated in vitro with 5 µM AVE (IR + AVE in vitro) or 0.6 µM cyclosporine A (IR + CsA in vitro). To measure the effect of AVE or CsA on PTP opening in vitro, the inhibitors were added directly to the cuvette, and swelling of mitochondria was monitored. Matrix swelling was induced by 200 µM or 1 mM (maximal swelling) Ca2+. (B) Maximum mitochondrial swelling induced by 1 mM CaCl2. Data are shown as a change of optical density ({Delta}OD/s). (C) Rate of swelling of mitochondria. Data are expressed relative to the maximum rate determined at 1 mM Ca2+ and were determined by differentiation of the traces shown to obtain the maximum rate of change of A520. Data are shown as means ± SEM of 5–6 mitochondrial preparations in each group. *P < 0.05 IR + AVE or IR + CsA in vitro vs. IR.

 
3.3. Respiratory function of mitochondria
To further examine the potential role of mitochondria in mediating the protective effects of AVE, we next determined mitochondrial respiration rates following AVE treatments. As shown in Table 1, state 3 respiration was reduced by 48% at complex I (L-malate in the presence of 2-oxoglutarate) and by 36% at complex II (succinate) by IR (P < 0.01). Of interest, AVE treatment significantly improved state 3 respiration in hearts subjected to IR (Table 1). IR decreased respiratory control index (RCI) for oxidation at both complex I and complex II. AVE increased RCI, although it was not statistically significant for complex I. The rate of ascorbate oxidation was also significantly increased by AVE in hearts from the IR group. Since our working hypothesis is that the beneficial effect of AVE on hearts subjected to IR is mediated through attenuation of Ca2+ overload in cytoplasm and mitochondria we also examined the effect of KBR on mitochondrial respiration. Our results show that KBR significantly improves respiration rates for oxidation of substrates at both complex I and II, although the increase of ascorbate oxidation was not statistically significant (Table 1). The improved mitochondrial respiration was associated with significantly less LDH release in KBR-treated hearts during reperfusion (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 1 The effect of AVE or KBR treatment on mitochondrial respirationa

 
A decrease in respiration rates in mitochondria isolated from IR hearts may be associated with disruption of the inner mitochondrial membrane. As shown in Figure 5A and B, activity of the matrix localized enzyme CS in both the raw homogenate and the mitochondrial fraction of IR hearts was 9.8 and 18.2% (P < 0.05 for both) less, respectively, than in control hearts. Treatment with AVE significantly increased CS in both fractions. A large difference in the change of CS activity in mitochondria compared to homogenate might be explained by a loss of damaged and fragile mitochondria during isolation. Significant difference between groups disappears if we re-express the respiration rate (state 3) per unit of CS activity as opposed to per mg of mitochondrial protein (Table 1). Furthermore, it was likely that a subpopulation of mitochondria isolated from hearts subjected to IR still have an intact matrix but ruptured outer membrane. To confirm this, we measured respiration rates (state 3) in the presence of 20 µM cytochrome c. The results demonstrate that cytochrome c induced a 49% increase (P < 0.01) in state 3 respiration in mitochondria isolated from hearts subjected to IR (Figure 6). Cytochrome c added to mitochondrial suspension from AVE-treated hearts induced only a 16% increase (P < 0.06) in state 3 respiration, which indicates a preservation of outer membrane in the presence of the inhibitor (Figure 6). Taken together, these data demonstrate that AVE attenuates mitochondrial dysfunction by improvement in respiration rates and CS activity.


Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5 Citrate synthase activity in homogenate (A) or mitochondria (B). Enzyme activity is shown as units per gram wet weight (units/gww) of myocardium for homogenate or units per mg of mitochondrial protein for mitochondria. Data are shown as means ± SEM of seven to eight hearts in each group. *P < 0.05 IR (ischaemia/reperfusion) vs. control (C).

 


Figure 6
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6 The effect of exogenous cytochrome c on mitochondrial respiration rates (state 3). Cytochrome c (cyt c, 20 µM) was added directly to the cuvette to measure cytochrome c-induced stimulation of mitochondrial respiration in the presence of 1 mM ADP (state 3). 2.5 mM 2-oxoglutarate and 1 mM L-malate were used as substrates for complex I. The respiration rates were normalized to mg of mitochondrial protein. Data are shown as means ± SEM of eight to nine hearts in each group. *P < 0.01 IR + cyt c vs. IR – cyt c.

 
3.4. Indices of apoptosis
Under normal physiological conditions AIF and EndoG are confined to mitochondria but are released into the cytoplasm as a consequence of cellular injury with further translocation into the nucleus to induce caspase-independent apoptosis.8,10 As shown in Figure 7A and B, IR decreased both AIF and EndoG in mitochondria by 47% (P < 0.01) and 29% (P < 0.05), respectively. However, these changes were prevented by AVE treatment. As a consequence, AVE decreased AIF and EndoG levels in the cytosolic fraction, although the change in cytosolic EndoG did not reach statistical significance (data not shown).


Figure 7
View larger version (42K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7 The effect of AVE (AVE-4890) on apoptosis-inducing factor (AIF, A) and endonuclease G (EndoG, B) in mitochondria, and caspase-3 activity (C) and cleaved poly-(ADP-ribose) polymerase (PARP) levels (D). Data are expressed as percent increase relative to control groups except for caspase-3 activity, which is expressed as units per mg of cytosol. Representative western blots are shown as appropriate. Data are presented as means ± SEM from seven to eight hearts in each group. *P < 0.05, **P < 0.01 IR vs. C; +P < 0.05 IR + AVE vs. IR.

 
To confirm these results, intracellular translocation of AIF and EndoG was examined by immunostaining of heart sections. In control hearts AIF and EndoG mostly localized in mitochondria and only traces are seen in cytoplasm. In hearts subjected to IR both apoptotic proteins are released into cytoplasm and as a consequence, their level decreases in mitochondria. However, AVE-treated hearts exhibited a lower leakage of both AIF and EndoG, most likely due to protection of the integrity of the outer mitochondrial membrane (data not shown). In agreement with the western blotting results, these findings demonstrate that AIF and EndoG release from the mitochondria following IR can be attenuated by AVE treatment.

Additionally, to investigate the contribution of the caspase-dependent pathway to apoptosis, cytochrome c levels in mitochondria and caspase-3 activity in the cytosol were examined. Western blot analysis revealed an 18% decrease (P < 0.05) of cytochrome c in mitochondria isolated from hearts in the IR group that was not observed in hearts treated with AVE (data not shown). Caspase-3 assay in the cytosol did not reveal any significant difference between groups (Figure 7C). Although nuclei of cells from hearts subjected to IR were TUNEL negative (data not shown), western blot analysis demonstrated a 46% (P < 0.05) increase in levels of cleaved PARP in hearts subjected to IR, which was abrogated by AVE (Figure 7D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
This study was designed to address the potential involvement of mitochondrial function in the cardioprotective effects of NHE-1 inhibition in isolated hearts subjected to IR. The main findings of this study are as follows: (i) the NHE-1 inhibitor AVE improved postischaemic recovery of cardiac function and decreased LDH release in coronary effluent; (ii) AVE preserved mitochondrial respiratory function following IR via augmented substrate oxidation at complexes I, II, and IV; (iii) mitochondrial PTP opening induced by IR was significantly attenuated by treatment with AVE and mitochondria isolated from AVE-treated hearts after IR demonstrated a reduced sensitivity to Ca2+-induced matrix swelling in vitro; and (iv) AVE attenuated IR-induced release of the apoptotic proteins AIF, EndoG, and cytochrome c from mitochondria as well as the increased levels of cleaved PARP.

Mitochondrial PTP opening acts as the end-effector of mitochondrial dysfunction to trigger cell death through both apoptosis and necrosis. We have observed for the first time that AVE inhibits mitochondrial PTP opening measured by [3H]-DOG uptake in the isolated heart. Most likely, the effect of AVE on mitochondrial PTP is indirect since the drug did not affect Ca2+-induced PTP opening in vitro when it was added directly to isolated mitochondria (Figure 4). This is in agreement with our recent finding that the NHE-1 specific inhibitor EMD-87580 fails to alter anoxia/reoxygenation-induced depression in respiratory function of isolated mitochondria.17 The inhibition of PTP opening during IR by AVE may involve several mechanisms. The most likely mechanism involves the attenuation of the IR-induced Ca2+overload. Activation of the NHE-1 due to intracellular acidosis during ischaemia leads to [Na+] elevation in the cytoplasm that, in turn, causes the NCE to uptake less Na+ or to actively increase Ca2+ entry via reverse mode NCE activity.1,3 Numerous studies using various models of IR and oxidative stress have shown that inhibition of NHE-1 attenuates Na+- and Ca2+-overload.1,3 Tissue, cytoplasmic and/or mitochondrial Ca2+-overload induced by IR in isolated hearts has been attenuated or prevented in the presence of a number of NHE-1 inhibitors including SM-20550,18 eniporide19 and KR-33028.20 The NHE-1 specific inhibitor cariporide prevented cytoplasmic and mitochondrial Ca2+ overload in neonatal cardiomyocytes in response to H2O2-induced oxidative stress21 and hypoxia/reoxygenation.22 The results of these studies are consistent with our findings, which show decreased Ca2+-induced matrix swelling in mitochondria isolated from AVE-treated hearts. Furthermore, our data demonstrate that KBR, a selective inhibitor of the reverse mode of NCE, significantly decreases LDH release in coronary effluent and improves respiratory function of mitochondria in hearts subjected to IR. The cardioprotective effect of KBR has been shown previously using similar models of IR in isolated and perfused rat heart.23

Besides Ca2+, PTP formation is also regulated by H+ and a low pH is known to inhibit pore opening.6,7 This may explain why maintenance of the acidic intracellular pH through inhibition of NHE in neonatal cardiomyocytes and myocardium offers protection against pH-dependent reperfusion injury and facilitates full recovery of cell and heart function.24,25 It has been previously shown that the PTPs remain closed during ischaemia but open during the first 5 min of reperfusion, a time period coincident with pHi normalization.26 It is interesting that inhibition of NHE-1 inhibition does not affect pHi during ischaemia, but significantly retards pHi recovery during the first 2–5 min of reperfusion.24,25 Thus, attenuation of PTP opening by NHE-1 inhibition very likely occurs during the first few minutes after the onset of reperfusion when attenuation of pHi recovery is clearly evident. The cardioprotective effect of NHE-1 inhibition appears to be complex involving a delay of pHi recovery and attenuation of Ca2+-overload and oxidative stress during reperfusion. In support of this, we have found that a low (1 µM) AVE failed to inhibit PTP opening and was protective only during early reperfusion (data not shown). Cardiac injury progresses with prolongation of the reperfusion time as shown for LDH release (Figure 2C) due to an increase in ROS generation and Ca2+ overload, both of which are potent inducers of PTP.6,7,9 Oxidative stress also modifies thiol groups on the ANT and thus increases Ca2+-sensitivity of the PTP.15 In our study, measurement of PTP opening in isolated mitochondria demonstrated less swelling of mitochondria in AVE-treated hearts. Since we measured PTP opening in buffer containing A23187 [GenBank] and buffered calcium to ensure that hearts from both IR and IR + AVE groups were exposed to the same [Ca2+] and pH, the protection induced by AVE might be a result of reduced oxidative stress in IR. In this regard, we27 and others28 have previously reported that inhibition of NHE-1 decreases superoxide production in mitochondria and total ROS levels in cardiomyocytes.

Finally, attenuation of PTP opening in AVE-treated hearts may result from improved respiratory function and oxidative phosphorylation in mitochondria. The protective effects of the NHE-1 inhibitors may act by reducing the mitochondrial dysfunctions observed in the intact heart during IR20,29 and post-infarction remodelling11 as well as in cultured cardiomyocytes subjected to oxidative stress21 and hypoxia.20 Specifically, these mitochondrial dysfunctions include: a decrease of respiration rates and ATP level, depolarization of mitochondrial membrane ({Delta}{psi}m loss) and cytochrome c release. We observed an improved respiratory function in mitochondria isolated from AVE-treated hearts after IR, which might inhibit PTP opening due to partial recovery of oxidative phosphorylation and ATP synthesis. Attenuation of PTP opening in the presence of AVE may also reflect enhanced activation of mitochondrial KATP channel. NHE-1 inhibition has been shown to open mitochondrial KATP channels in rabbit hearts subjected to in vivo or ex vivo myocardial infarction.30 On the other hand, a previous study from our laboratory demonstrated that mitochondrial KATP channel activation suppresses NHE-1 expression in neonatal cardiomyocytes and attenuates phenylephrine-induced hypertrophy.31 Thus, mitochondrial KATP channels may play a critical role in mediating the effects of NHE-1 inhibition, although a cause-effect relationship between these two processes needs to be elucidated.

We further studied the protective effect of NHE-1 inhibition on outer mitochondrial membrane integrity by determining the effect of AVE on AIF and EndoG release from mitochondria and sought to evaluate whether release of AIF and EndoG was associated with DNA fragmentation-mediated apoptosis in our model of IR. Our results demonstrate that IR was a potent stimulus for the release of both AIF and EndoG from mitochondria. A reduced release of EndoG from mitochondria during IR in comparison with AIF might be due to its localization in the matrix or the fact that it was tightly bound to the inner mitochondrial membrane.32 Finally, AIF and EndoG might be released from the cytoplasm to the effluent during reperfusion and not translocated to the nucleus. Indeed, using immunostaining we observed only traces of both proteins in nuclei (data not shown). AIF and EndoG have been shown to translocate into nuclei following an apoptotic signal and induce a caspase-independent DNA fragmentation and apoptosis.8,10 On the other hand, studies performed in EndoG knockout mice demonstrated that inactivation of this gene does not attenuate apoptosis during early development,33 although in these studies EndoG levels in wild-type cells were not measured. In our experiments, although we have seen some nuclei positively stained for both AIF and EndoG after IR, these nuclei were TUNEL-negative. To investigate the contribution of the caspase-dependent pathway to apoptosis we measured cytochrome c levels in mitochondria as well as caspase-3 activity and PARP cleavage in the cytosol. We observed a decrease in the cytochrome c protein level in the mitochondrial fraction isolated from IR hearts. Caspase-3 activity was unaffected by IR although we observed an increased level of cleaved PARP thus suggesting the presence of apoptosis. Increased levels of cleaved PARP were seen in the absence of positive TUNEL staining or increased caspase-3 activity. This may reflect the mild severity of IR used in the present study and/or the higher sensitivity of PARP cleavage to assay apoptotic cell death. Furthermore, increased levels of cleaved PARP in the absence of increased caspase-3 activity suggest that PARP cleavage and apoptosis occurred through a caspase-3-independent mechanism as has been reported by other investigators.34–36

Taken together, our study demonstrates for the first time that inhibition of NHE-1 by AVE attenuates mitochondrial PTP opening that is associated with improved heart contractility as well as respiratory function and integrity of mitochondria in the reperfused postischaemic heart. Although the molecular mechanisms underlying the mitochondria-mediated cardioprotective effects of NHE-1 inhibition against ischaemia remain to be elucidated, our results strongly demonstrate that inhibition of NHE-1 is associated with attenuation of the PTP opening and improvement of mitochondrial function as well as an attenuation of pro-apoptotic factors.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
Canadian Institutes of Health Research (to M. K.); in part, National Institutes of Health (PO1 DK58324-01A1 to A. B.).


   Acknowledgements
 
M. K. holds a Canada Research Chair in Experimental Cardiology. The authors thank E. Apostolov for immunohistology and L. A. Kirshenbaum for helpful comments.

Conflict of interest: None declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

  1. Karmazyn M, Sostaric JV, Gan XT. The myocardial Na+/H+ exchanger: a potential therapeutic target for the prevention of myocardial ischaemic and reperfusion injury and attenuation of postinfarction heart failure. Drugs (2001) 61:375–389.[CrossRef][Web of Science][Medline]
  2. Karmazyn M. Amiloride enhances postischaemic ventricular recovery: possible role of Na+-H+ exchange. Am J Physiol (1988) 255:H608–H615.[Web of Science][Medline]
  3. Avkiran M, Marber MS. Na(+)/H(+) exchange inhibitors for cardioprotective therapy: progress, problems and prospects. J Am Coll Cardiol (2002) 39:747–753.[Abstract/Free Full Text]
  4. Gumina RJ, Gross GJ. If ischaemic preconditioning is the gold standard, has a platinum standard of cardioprotection arrived? Comparison with NHE inhibition. J Thromb Thrombolysis (1999) 8:39–44.[CrossRef][Web of Science][Medline]
  5. Haist JV, Hirst CN, Karmazyn M. Effective protection by NHE-1 inhibition in ischaemic and reperfused heart under preconditioning blockade. Am J Physiol Heart Circ Physiol (2003) 284:H798–H803.[Abstract/Free Full Text]
  6. Javadov S, Karmazyn M. Mitochondrial permeability transition pore opening as an endpoint to initiate cell death and as a putative target for cardioprotection. Cell Physiol Biochem (2007) 20:1–22.[Web of Science][Medline]
  7. Di Lisa F, Bernardi P. Mitochondria and ischaemia-reperfusion injury of the heart: fixing a hole. Cardiovasc Res (2006) 70:191–199.[Abstract/Free Full Text]
  8. Regula KM, Kirshenbaum LA. Apoptosis of ventricular myocytes: a means to an end. J Mol Cell Cardiol (2005) 38:3–13.[CrossRef][Web of Science][Medline]
  9. Honda HM, Korge P, Weiss JN. Mitochondria and ischaemia/reperfusion injury. Ann NY Acad Sci (2005) 1047:248–258.[CrossRef][Web of Science][Medline]
  10. Zamzami N, Susin SA, Marchetti P, Hirsch T, Gomez-Monterrey I, Castedo M, et al. Mitochondrial control of nuclear apoptosis. J Exp Med (1996) 183:1533–1544.[Abstract/Free Full Text]
  11. Javadov S, Huang C, Kirshenbaum L, Karmazyn M. NHE-1 inhibition improves impaired mitochondrial permeability transition and respiratory function during postinfarction remodelling in the rat. J Mol Cell Cardiol (2005) 38:135–143.[CrossRef][Web of Science][Medline]
  12. Hohorst HJ. L-(+)-Lactate. In: Methods of Enzymatic Analysis—Bergmeyer HU, ed. (1963) New York: Academic Press. 215–219.
  13. Griffiths EJ, Halestrap AP. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem J (1995) 307:93–98.[Web of Science][Medline]
  14. Srere PA. Citrate synthase. Methods Enzymol (1969) 13:3–11.[CrossRef]
  15. Halestrap AP, Woodfield KY, Connern CP. Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase. J Biol Chem (1997) 272:3346–3354.[Abstract/Free Full Text]
  16. Yin X, Apostolov EO, Shah SV, Wang X, Bogdanov K, Buzder T, et al. Induction of renal endonuclease G by cisplatin is reduced in DNase I-deficient mice. J Am Soc Nephrol (2007) 18:2544–2553.[Free Full Text]
  17. Javadov S, Purdham DM, Zeidan A, Karmazyn M. NHE-1 inhibition improves cardiac mitochondrial function through regulation of mitochondrial biogenesis during postinfarction remodeling. Am J Physiol Heart Circ Physiol (2006) 291:H1722–H1730.[Abstract/Free Full Text]
  18. Yamamoto S, Matsui K, Ohashi N. Protective effect of Na+ /H+ exchange inhibitor, SM-20550, on impaired mitochondrial respiratory function and mitochondrial Ca2+ overload in ischaemic/reperfused rat hearts. J Cardiovasc Pharmacol (2002) 39:569–575.[CrossRef][Web of Science][Medline]
  19. Camara AK, An J, Chen Q, Novalija E, Varadarajan SG, Schelling P, et al. Na+/H+ exchange inhibition with cardioplegia reduces cytosolic [Ca2+] and myocardial damage after cold ischaemia. J Cardiovasc Pharmacol (2003) 41:686–698.[CrossRef][Web of Science][Medline]
  20. Jung YS, Kim MY, Kim MJ, Oh KS, Yi KY, Lee S, et al. Pharmacological profile of KR-33028, a highly selective inhibitor of Na+/H+ exchanger. Eur J Pharmacol (2006) 535:220–227.[CrossRef][Web of Science][Medline]
  21. Teshima Y, Akao M, Jones SP, Marban E. Cariporide (HOE642), a selective Na+-H+ exchange inhibitor, inhibits the mitochondrial death pathway. Circulation (2003) 108:2275–2281.[Abstract/Free Full Text]
  22. Sun HY, Wang NP, Halkos ME, Kerendi F, Kin H, Wang RX, et al. Involvement of Na+/H+ exchanger in hypoxia/re-oxygenation-induced neonatal rat cardiomyocyte apoptosis. Eur J Pharmacol (2004) 486:121–131.[CrossRef][Web of Science][Medline]
  23. Schafer C, Ladilov Y, Inserte J, Schafer M, Haffner S, Garcia-Dorado D, et al. Role of the reverse mode of the Na+/Ca2+ exchanger in reoxygenation-induced cardiomyocyte injury. Cardiovasc Res (2001) 51:241–250.[CrossRef][Web of Science][Medline]
  24. Stromer H, de Groot MC, Horn M, Faul C, Leupold A, Morgan JP, et al. Na(+)/H(+) exchange inhibition with HOE642 improves postischaemic recovery due to attenuation of Ca(2+) overload and prolonged acidosis on reperfusion. Circulation (2000) 101:2749–2755.[Abstract/Free Full Text]
  25. Ten Hove M, Van Echteld CJ. Limited effects of post-ischaemic NHE blockade on [Na+]i and pHi in rat hearts explain its lack of cardioprotection. Cardiovasc Res (2004) 61:522–529.[Abstract/Free Full Text]
  26. Kerr PM, Suleiman MS, Halestrap AP. Reversal of permeability transition during recovery of hearts from ischaemia and its enhancement by pyruvate. Am J Physiol (1999) 276:H496–H502.[Web of Science][Medline]
  27. Javadov S, Baetz D, Rajapurohitam V, Zeidan A, Kirshenbaum LA, Karmazyn M. Antihypertrophic effect of Na+/H+ exchanger isoform 1 inhibition is mediated by reduced mitogen-activated protein kinase activation secondary to improved mitochondrial integrity and decreased generation of mitochondrial-derived reactive oxygen species. J Pharmacol Exp Ther (2006) 317:1036–1043.[Abstract/Free Full Text]
  28. Hotta Y, Nishimaki H, Takeo T, Itoh G, Yajima M, Otsuka-Murakami H, et al. Differences in the effects of Na+-H+ exchange inhibitors on cardiac function and apoptosis in guinea-pig ischaemia-reperfused hearts. Eur J Pharmacol (2004) 503:109–122.[CrossRef][Web of Science][Medline]
  29. Ruiz-Meana M, Garcia-Dorado D, Pina P, Inserte J, Agullo L, Soler-Soler J. Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischaemic conditions. Am J Physiol Heart Circ Physiol (2003) 285:H999–H1006.[Abstract/Free Full Text]
  30. Miura T, Liu Y, Goto M, Tsuchida A, Miki T, Nakano A, et al. Mitochondrial ATP-sensitive K+ channels play a role in cardioprotection by Na+-H+ exchange inhibition against ischaemia/reperfusion injury. J Am Coll Cardiol (2001) 37:957–963.[Abstract/Free Full Text]
  31. Xia Y, Rajapurohitam V, Cook MA, Karmazyn M. Inhibition of phenylephrine-induced hypertrophy in rat neonatal cardiomyocytes by the mitochondrial KATP channel opener diazoxide. J Mol Cell Cardiol (2004) 37:1063–1067.[CrossRef][Web of Science][Medline]
  32. Arnoult D, Gaume B, Karbowski M, Sharpe JC, Cecconi F, Youle RJ. Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J (2003) 22:4385–4399.[CrossRef][Web of Science][Medline]
  33. David KK, Sasaki M, Yu SW, Dawson TM, Dawson VL. EndoG is dispensable in embryogenesis and apoptosis. Cell Death Differ (2006) 13:1147–1155.[CrossRef][Web of Science][Medline]
  34. Liu J, Shen HM, Ong CN. Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells. Life Sci. (2001) 69:1833–1850.[CrossRef][Web of Science][Medline]
  35. Yang Y, Zhao S, Song J. Caspase-dependent apoptosis and -independent poly(ADP-ribose) polymerase cleavage induced by transforming growth factor beta1. Int J Biochem Cell Biol (2004) 36:223–234.[CrossRef][Web of Science][Medline]
  36. Ohta T, Eguchi R, Suzuki A, Miyakaze S, Ayuzawa R, Kaji K. Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells. J Cell Physiol (2007) 211:673–681.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
S. Javadov, M. Karmazyn, and N. Escobales
Mitochondrial Permeability Transition Pore Opening as a Promising Therapeutic Target in Cardiac Diseases
J. Pharmacol. Exp. Ther., September 1, 2009; 330(3): 670 - 678.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
C. D. Garciarena, C. I. Caldiz, M. V. Correa, G. R. Schinella, S. M. Mosca, G. E. Chiappe de Cingolani, H. E. Cingolani, and I. L. Ennis
Na+/H+ exchanger-1 inhibitors decrease myocardial superoxide production via direct mitochondrial action
J Appl Physiol, December 1, 2008; 105(6): 1706 - 1713.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
D. Garcia-Dorado, H. M. Piper, and D. A. Eisner
Sarcoplasmic reticulum and mitochondria in cardiac pathophysiology
Cardiovasc Res, January 15, 2008; 77(2): 231 - 233.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
77/2/416    most recent
cvm039v3
cvm039v2
cvm039v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Javadov, S.
Right arrow Articles by Karmazyn, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Javadov, S.
Right arrow Articles by Karmazyn, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?