- About this Journal
- Abstracting and Indexing
- Aims and Scope
- Annual Issues
- Article Processing Charges
- Articles in Press
- Author Guidelines
- Bibliographic Information
- Citations to this Journal
- Contact Information
- Editorial Board
- Editorial Workflow
- Free eTOC Alerts
- Publication Ethics
- Reviewers Acknowledgment
- Submit a Manuscript
- Subscription Information
- Table of Contents
Journal of Aging Research
Volume 2012 (2012), Article ID 753949, 9 pages
Blockade of Electron Transport at the Onset of Reperfusion Decreases Cardiac Injury in Aged Hearts by Protecting the Inner Mitochondrial Membrane
1Pauley Heart Center, Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA
2Department of Biochemistry, Virginia Commonwealth University, Richmond, VA 23298, USA
3Cardiology Section, Medical Service 111 (J), McGuire VA Medical Center, Richmond, VA 23249, USA
Received 12 September 2011; Revised 23 November 2011; Accepted 27 December 2011
Academic Editor: Paula Bickford
Copyright © 2012 Qun Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Myocardial injury is increased in the aged heart following ischemia-reperfusion (ISC-REP) compared to adult hearts. Intervention at REP with ischemic postconditioning decreases injury in the adult heart by attenuating mitochondrial driven cell injury. Unfortunately, postconditioning is ineffective in aged hearts. Blockade of electron transport at the onset of REP with the reversible inhibitor amobarbital (AMO) decreases injury in adult hearts. We tested if AMO treatment at REP protects the aged heart via preservation of mitochondrial integrity. Buffer-perfused elderly Fischer 344 24 mo. rat hearts underwent 25 min global ISC and 30 min REP. AMO (2.5 mM) or vehicle was given for 3 min at the onset of REP. Subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated after REP. Oxidative phosphorylation (OXPHOS) and mitochondrial inner membrane potential were measured. AMO treatment at REP decreased cardiac injury. Compared to untreated ISC-REP, AMO improved inner membrane potential in SSM and IFM during REP, indicating preserved inner membrane integrity. Thus, direct pharmacologic modulation of electron transport at REP protects mitochondria and decreases cardiac injury in the aged heart, even when signaling-induced pathways of postconditioning that are upstream of mitochondria are ineffective.
The aged heart sustains increased injury during ischemia-reperfusion in both experimental models [1–4] and in elderly patients . Aging hearts are also resistant to the powerful endogenous protections provided by ischemic preconditioning and postconditioning [6–12]. Pharmacological conditioning such as anesthetic preconditioning also does not protect the aging heart during ischemia-reperfusion . Aging causes dysfunction in interfibrillar mitochondria . The aging defect in complex III and cytochrome oxidase decreases oxidative phosphorylation and increases the generation of reactive oxygen species [14–16]. Improvement of age-induced mitochondrial dysfunction with supplementation of acetylcarnitine decreases myocardial injury during ischemia-reperfusion , supporting that the electron transport chain defects present in the aged heart contribute to the increased myocardial injury.
Cardiac ischemia damages the electron transport chain and leads to the increased generation of reactive oxygen species (ROS) and mitochondrial calcium over load [18–22]. Opening of the mitochondrial permeability transition pore (MPTP) is a critical step in the onset of cardiomyocyte death [23, 24]. The increased generation of ROS and calcium overload are key factors that induce MPTP opening during cellular stress [25–28]. Although the increased production of ROS and calcium overload occur during ischemia , MPTP opening occurs mostly during reperfusion. Ischemia induces intracellular acidification that blunts MPTP opening [25–28]. Ischemic postconditioning decreases cardiac injury and prevents MPTP opening in adult hearts , supporting that MPTP mainly opens during reperfusion. Postconditioning leads to activation of cytoprotective signaling cascades, including the reperfusion injury salvage kinase pathway cascade (RISK)  and the tumor necrosis factor-STAT3 cascade (SAFE) . Intervention during early reperfusion is still able to reduce cardiac injury although ischemic damage to the electron transport chain has already occurred [21, 31]. In fact, in the adult heart, postconditioning appears to work via protection against MPTP in mitochondria that have sustained damage from ischemia . Unfortunately, ischemic postconditioning does not protect aged myocardium [6, 33, 34]. Alternative approaches must be found to decrease cardiac injury in the aged heart.
Blockade of electron transport with amobarbital during ischemia protects cardiac mitochondria and decreases myocardial injury in adult hearts [18, 19, 35]. Amobarbital given before ischemia also decreases cardiac injury in aged rat hearts measured after reperfusion . These findings suggest that direct manipulation of mitochondrial function is an alternative approach to protect aged hearts that lack effective endogenous cytoprotective mechanisms.
Although prevention of mitochondrial damage during ischemia by amobarbital treatment [18, 19, 35] or ischemic preconditioning [6–8, 34] is an optimal strategy for cardioprotection, the clinical relevance of these treatments is limited due to the unpredictable occurrence of ischemic events. Interventions applied at the onset of reperfusion are practical for therapeutic use in the treatment of acute myocardial infarction [21, 37]. Direct blockade of electron transport by amobarbital at the onset of reperfusion decreases myocardial injury in the adult heart [19, 31]. In the present study, we tested if blockade of electron transport at the onset of reperfusion was able to decrease cardiac injury in the aged heart. We also evaluated if protection by direct modulation of mitochondrial function during early reperfusion is mediated by the protection of mitochondrial inner membrane integrity.
2.1. Preparation of Rat Hearts for Perfusion
The Animal Care and Use Committees of the McGuire VA Medical Center and Virginia Commonwealth University approved the protocol. Male Fischer rats (24 mo.) were anesthetized with pentobarbital sodium (100 mg/kg i.p.) and anticoagulated with heparin (1000 IU/kg i.p.). Hearts were excised and perfused retrograde via the aorta in the Langendorff mode with modified Krebs-Henseleit (K-H) buffer oxygenated with 95% O2/5% CO2 as previously described . Cardiac function was monitored with a balloon inserted into the left ventricle.
2.2. Protocol for Heart Ischemia and Reperfusion and Amobarbital Perfusion
In untreated hearts, the heart was perfused for 15 min with K-H buffer, followed by 25 min global ischemia at 37°C and 30 min reperfusion. In the amobarbital treated group, hearts followed the same perfusion protocol except that amobarbital (2.5 mM) was perfused in identical K-H buffer for three min at onset of reperfusion (Figure 1, upper panel) . Hearts were paced at 300 beats per min during the 15 min equilibration period and again after 10 min reperfusion. Coronary effluent was collected during the 30 min reperfusion period, and LDH activity in coronary effluent was determined to reflect myocardial injury .
2.3. Determination of Myocardial Infarct Size
In order to further evaluate the amobarbital-mediated cardiac protection, myocardial infarct size was measured in buffer-perfused mouse hearts in the absence and presence of amobarbital treatment at the onset of reperfusion. In untreated hearts, the heart was perfused for 15 min with K-H buffer, followed by 30 min global ischemia at 37°C and 60 min reperfusion . In the amobarbital-treated group, hearts followed the same perfusion protocol except that amobarbital was perfused in identical K-H buffer for three min at onset of reperfusion (Figure 1, lower panel) . Hearts were paced at 420 beats per min during the 15 min equilibration period and again after 10 min reperfusion. Coronary effluent was collected during the 30 min reperfusion period, and LDH activity in coronary effluent was determined to reflect myocardial injury . Myocardial infarct size was determined using TTC staining .
2.4. Mitochondrial Isolation and Functional Assessment
Subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated from hearts at the end of the experiment . Oxidative phosphorylation was determined using a Clark-type oxygen electrode at 30°C . H2O2 production from intact mitochondria was measured using the oxidation of the fluorogenic indicator Amplex Red in the presence of horseradish peroxidase (HRP) .
2.5. Measurement of Mitochondrial Inner Membrane Potential
Mitochondrial inner membrane potential was assessed using the fluorescence intensity of the indicator TMRM (tetramethylrhodamine, methyl ester) . TMRM is a lipophilic cation accumulated by mitochondria in proportion to Δψ (emission at 590 nM, excitation at two wavelengths: 573 nm and 546 nm). Accumulated TMRM in mitochondria causes a fluorescence “red shift,” and fluorescence intensity was quantified by calculating the ratio of intensity from 573 to intensity from 546 (573/546). TMRM was selected to reflect membrane potential since TMRM did not inhibit oxidative phosphorylation at the concentration (0.3 μM) used . The Δψ was reflected by Δ change of fluorescence intensity of TMRM in the presence of ADP (2 mM), oligomycin (1 μM), and DNP (0.2 mM/each) using glutamate (10 mM) as a complex I substrate (Figure 4).
2.6. Determination of Calcium Tolerance Capacity (CRC)
Mitochondrial tolerance to calcium loading was studied in the single-cell fluorometer using repetitive calcium pulses [21, 41]. Freshly isolated SSM and IFM (0.2 mg/mL) were incubated in buffer (150 mM sucrose, 50 mM KCl, 2 mM KPi, and 20 mM Tris/HCl, pH 7.4) for 90 sec. with stirring at 30°C with 0.5 M calcium green. Succinate (5 mM) was used as substrate. Pulses of calcium (20 nmoles) were added at 1 min intervals. The number of pulses that resulted in calcium release (MPTP opening) was used to calculate CRC (Figure 6(a)).
2.7. Statistical Analysis
Data are expressed as the mean ± standard error of the mean. Differences among groups were compared by two-tailed -test (SigmaStat 3.5, ProgramPaketet, Gothenburg, Sweden). Two-way ANOVA was used to analyze the hemodynamic data and inner mitochondrial membrane potential, and post hoc test was used to show statistical difference between groups. A difference of was considered significant.
3.1. Reversible Blockade of Electron Transport at Onset of Reperfusion Decreased Myocardial Injury
There were no differences in hemodynamic data (left ventricular developed pressure-LVDP and left ventricular end diastolic pressure-LVEDP) between the two groups at the end of the equilibration period before ischemia (Figure 2). Ischemia markedly increased diastolic pressure (LVEDP) in both groups (Figure 2). There was no difference in LVEDP between the two groups at the end of ischemia, indicating that hearts in both groups suffered the same degree of ischemic contracture before treatment. The diastolic pressure remained elevated during reperfusion in untreated hearts. Amobarbital given during early reperfusion had minimal effect on diastolic pressure (Figure 2). In untreated hearts, LVDP is markedly decreased during reperfusion compared to the preischemic value. Amobarbital given during reperfusion tended to improve the recovery of LVDP, but it did not reach statistical significance (Figure 2). In contrast to cardiac function, amobarbital given at the onset of reperfusion markedly decreased LDH release into coronary effluent compared to untreated hearts, indicating that modulation of electron transport during early reperfusion provided solid cardioprotection in aged hearts following reperfusion (Figure 3(a)). Amobarbital given during early reperfusion also protected buffer-perfused mouse hearts as shown by decreased LDH release (Figure 3(b)) and myocardial infarct size (Figure 3(c)). These data further support that reversible blockade of electron transport during early reperfusion decreased myocardial injury.
3.2. Reversible Blockade of Electron Transport at the Onset of Reperfusion Did Not Improve Mitochondrial Oxidative Phosphorylation
Amobarbital given during reperfusion did not improve oxidative phosphorylation in SSM or IFM (glutamate, succinate, and TMPD-ascorbate as complex I, II, and IV substrates, resp.) compared to untreated hearts (Table 1). Amobarbital treatment also did not alter uncoupled respiration induced by dinitrophenol (DNP) compared to untreated hearts (Table 1). Thus, manipulation of respiration during early reperfusion did not protect the electron transport chain. This result was consistent with our previous findings that the damage to the electron transport chain occurred during ischemia, rather than during reperfusion [21, 35, 42].
3.3. Reversible Blockade of Electron Transport at the Onset of Reperfusion Improved Mitochondrial Inner Membrane Potential
Mitochondrial inner membrane potential was used to assess inner membrane permeability during reperfusion. An original tracing of inner membrane potential is shown in Figure 4. Mitochondrial inner membrane potential stimulated by ADP was significantly improved in both SSM and IFM by amobarbital treatment compared to untreated hearts (Figure 5). Amobarbital treatment improved inner membrane potential in the presence of oligomycin (complex V inhibitor) in both SSM and IFM, suggesting that complex V was not the site of the defect (Figure 5). Titration of DNP caused complete depolarization of mitochondria (Figure 4). Amobarbital treatment improved inner membrane potential with DNP titration in both SSM and IFM (Figure 5).
3.4. Reversible Blockade of Electron Transport at Onset of Reperfusion Did Not Improve Mitochondrial Calcium Retention Capacity (CRC)
CRC was used to assess MPTP opening in isolated mitochondria . An original tracing of CRC measurement is shown in Figure 6(a). Amobarbital treatment during reperfusion did not significantly improve CRC in SSM and IFM compared to untreated hearts (Figure 6(b)).
3.5. Blockade of Electron Transport at the Onset of Reperfusion Decreases H2O2 Generation
Mitochondrial electron transport is a major source of ROS generation [22, 43]. Using glutamate as a complex I substrate, amobarbital treatment decreased the generation of H2O2 in SSM compared to untreated hearts (Figure 7). The amount of H2O2 generated from IFM from amobarbital treated hearts also was lower than that in the untreated group (). There was no difference in H2O2 production between the two groups in both SSM and IFM using succinate + rotenone as a complex II substrate (H2O2 pmol/mg/min Mean ± SEM: SSM,untreated versusamobarbital; IFM,untreated versusamobarbital, P = NS, in each group).
The novel findings of the present study are (1) reversible blockade of electron transport at the onset of reperfusion decreases myocardial injury in aged hearts; (2) the protection by amobarbital treatment during reperfusion was mediated by protection of the inner mitochondrial membrane. The present study indicates that modulation of mitochondrial respiration at the onset of reperfusion is able to decrease cardiac injury in the aged heart even when oxidative phosphorylation was already compromised from ischemia . Thus, the aged heart can be protected in a manner similar to the adult heart by using this translationally relevant pharmacologic strategy to attenuate cardiac injury during reperfusion.
Aging leads to impaired mitochondrial function that augments myocardial injury during ischemia-reperfusion [15, 17, 42]. The loss of endogenous protective mechanisms in the aged heart provides a greater challenge to protect the aged heart during acute myocardial infarction and its treatment. The endogenous protective mechanisms can be restored in aging hearts by treatments such as caloric restriction , exercise , and a pharmacologic strategy to inhibit protein phosphatase 2A activity . Supplementation of acetylcarnitine before ischemia decreases myocardial injury in aged hearts via restoration of mitochondrial respiration to an adult-phenotype . However, these approaches must all be instituted before ischemia and require variable pretreatment periods in order to be effective. Due to the unpredictable occurrence of acute ischemic events, these strategies are unfortunately less clinically relevant to protect the ischemic-reperfused aged heart. In the present study, amobarbital given at the onset of reperfusion decreased myocardial injury. Compared to the pre-ischemia strategies, amobarbital treatment at reperfusion has therapeutic potential since amobarbital could be regionally infused during primary stent-mediated reperfusion treatment of ST elevation acute infarcts in the high risk elderly patient population .
Amobarbital given before ischemia decreases cell injury in isolated rabbit , rat [35, 47], and guinea pig hearts . Amobarbital given before ischemia prevents electron transport chain damage measured following reperfusion in isolated adult  and aged rat hearts . In the present study, amobarbital given at the onset of reperfusion does not protect the electron transport chain in aged hearts. Taken together, these results further support the notion that the damage of mitochondrial electron transport chain mainly occurs during ischemia, rather than during reperfusion , including in the aged heart . In the adult heart, amobarbital treatment during reperfusion decreases cardiac injury without improving oxidative phosphorylation  indicating that the mechanism of myocyte death during reperfusion is not solely dependent on the function of mitochondrial respiration. Ischemic postconditioning, another well known method applied during early reperfusion, also fails to improve oxidative phosphorylation in the adult heart [21, 50]. Thus, manipulation of ischemia-damaged mitochondria either via activation of cytoprotective signaling systems in the adult heart [29, 50–52] or this novel direct manipulation of mitochondrial function in the aged heart is able to decrease myocardial injury during reperfusion.
MPTP opening contributes to myocyte death during ischemia-reperfusion [23, 24, 53]. Although the exact structure of the MPTP remains elusive, the effect of MPTP opening is clear: its opening leads to increased permeability of both mitochondrial inner and outer membranes [23, 26]. Mitochondrial inner membrane potential (Ψ) is commonly used to reflect the permeability of the inner membrane [25, 53], including from MPTP. In the present study, inner membrane potential was used to reflect inner membrane integrity in isolated mitochondria. Amobarbital treatment preserves inner membrane potential in both SSM and IFM compared to untreated hearts, suggesting that amobarbital decreases the permeability of inner membrane during reperfusion. In the presence of oligomycin , inner membrane potential remains lower in mitochondria from untreated hearts compared to amobarbital treatment, indicating that there is a consistent proton leakage site other than complex V. Ischemia-reperfusion may activate uncoupling proteins that allow proton backflow into the matrix . If this was the case, we anticipate that the difference of Ψ between untreated and amobarbital-treated mitochondria would be eliminated in the presence of uncoupler, DNP. Amobarbital improves inner membrane potential in the presence of DNP, suggesting that uncoupling proteins are a less likely mechanism for the proton leak. Another potential proton leak site is the adenine nucleotide translocase (ANT) . ANT was once considered a key component of the MPTP, but the role of ANT in the pore is now uncertain . We found that inhibition of ANT using bongkrekic acid did not restore inner membrane potential in mitochondria following ischemia-reperfusion (data not shown). These results indicate that the impairment of inner membrane potential in mitochondria isolated following ischemia-reperfusion in the aged heart is not through the specific sites discussed above.
The improved inner membrane potential following amobarbital treatment suggests that treatment decreases MPTP opening during reperfusion. However, amobarbital treatment does not improve mitochondrial calcium tolerance (calcium retention capacity—CRC), another common index used to reflect MPTP [21, 56]. These results indicate that the mechanism by which amobarbital treatment decreases inner mitochondrial membrane permeability is not solely through prevention of MPTP opening. Cardiolipin is a unique phospholipid located in the inner membrane [48, 57, 58], and insufficient cardiolipin content or the presence of oxidized cardiolipin increases inner membrane permeability [59, 60]. Aging does not alter cardiolipin content  but results in the enhanced formation of oxidized cardiolipin species in both SSM and IFM following ischemia compared to adult hearts . The increases in oxidized cardiolipin content persist during reperfusion in the aged heart , suggesting ongoing production during the oxidative stress of reperfusion. Amobarbital treatment during reperfusion may decrease the permeability of the inner membrane by attenuating the ongoing production of oxidized cardiolipin during reperfusion. This concept is an area of ongoing study in our laboratory.
The burst of ROS formation at the onset of reperfusion contributes to myocardial injury . The ischemia-damaged electron transport chain increases the generation of ROS during reoxygenation in isolated mitochondria . Protection of mitochondrial electron transport during ischemia decreases ROS generation during reperfusion [35, 36]. In the present study, amobarbital given during early reperfusion decreases ROS generation from isolated mitochondria obtained from the aged heart, suggesting that amobarbital decreases myocardial injury by decreasing ROS formation during reperfusion, perhaps leading to decreased production of oxidized cardiolipin and thereby protecting the inner mitochondrial membrane. Ischemia increases ROS generation from complex I and complex III . In the present study, amobarbital treatment at the onset of reperfusion was likely protective by two complimentary mechanisms. First, amobarbital decreased electron flow into complex III, which produces increased oxidative injury from both age-related [14–16] and ischemia-induced  defects, the latter observed online in the intact heart . Second, amobarbital treatment decreases complex-I-mediated complex I damage [19, 35] with decreased production of ROS from complex I as observed during reperfusion in the current study.
Reperfusion in clinical settings usually occurs following at least moderate periods of ischemia that result in mitochondrial damage. Transient interruption of mitochondrial oxidative metabolism only during early reperfusion decreases myocardial injury in aged hearts providing a relevant approach to limit myocardial cell death during reperfusion in the high-risk elderly population.
This work was supported by the program Project 2PO1AG15885 from the National Institutes of Health, the Office of Research and Development, Medical Research Service, the Department of Veterans Affairs, and The Pauley Heart Center of Virginia Commonwealth University.
- K. Ataka, D. Chen, S. Levitsky, E. Jimenez, and H. Feinberg, “Effect of aging on intracellular Ca2+, pHi, and contractility during ischemia and reperfusion,” Circulation, vol. 86, no. 5, pp. 371–376, 1992.
- V. V. Frolkis, R. A. Frolkis, L. S. Mkhitarian, and V. E. Fraifeld, “Age-dependent effects of ischemia and reperfusion on cardiac function and Ca2+ transport in myocardium,” Gerontology, vol. 37, no. 5, pp. 233–239, 1991.
- E. J. Lesnefsky, D. S. Gallo, J. Ye, T. S. Whittingham, and W. D. Lust, “Aging increases ischemia-reperfusion injury in the isolated, buffer-perfused heart,” The Journal of Laboratory and Clinical Medicine, vol. 124, no. 6, pp. 843–851, 1994.
- D. T. Lucas and L. I. Szweda, “Cardiac reperfusion injury: aging, lipid peroxidation, and mitochondrial dysfunction,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 2, pp. 510–514, 1998.
- E. J. Lesnefsky, C. F. Lundergan, J. M. Hodgson et al., “Increased left ventricular dysfunction in elderly patients despite successful thrombolysis: the GUSTO-I angiographic experience,” Journal of the American College of Cardiology, vol. 28, no. 2, pp. 331–337, 1996.
- D. A. Vessey, M. Kelley, L. Li, and Y. Huang, “Sphingosine protects aging hearts from ischemia/reperfusion injury: superiority to sphingosine 1-phosphate and ischemic pre- and post-conditioning,” Oxidative Medicine and Cellular Longevity, vol. 2, no. 3, pp. 146–151, 2009.
- K. Boengler, I. Konietzka, A. Buechert et al., “Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43,” American Journal of Physiology. Heart and Circulatory Physiology, vol. 292, no. 4, pp. H1764–H1769, 2007.
- D. Schulman, D. S. Latchman, and D. M. Yellon, “Effect of aging on the ability of preconditioning to protect rat hearts from ischemia-reperfusion injury,” American Journal of Physiology. Heart and Circulatory Physiology, vol. 281, no. 4, pp. H1630–H1636, 2001.
- M. Tani, Y. Honma, H. Hasegawa, and K. Tamaki, “Direct activation of mitochondrial K(ATP) channels mimics preconditioning but protein kinase C activation is less effective in middle-aged rat hearts,” Cardiovascular Research, vol. 49, no. 1, pp. 56–68, 2001.
- D. J. Hausenloy, G. Baxter, R. Bell et al., “Translating novel strategies for cardioprotection: the Hatter Workshop Recommendations,” Basic Research in Cardiology, vol. 105, no. 6, pp. 677–686, 2010.
- L. Schwartz Longacre, R. A. Kloner, A. E. Arai et al., “New horizons in cardioprotection: recommendations from the 2010 national heart, lung, and blood institute workshop,” Circulation, vol. 124, no. 10, pp. 1172–1179, 2011.
- K. Boengler, R. Schulz, and G. Heusch, “Loss of cardioprotection with ageing,” Cardiovascular Research, vol. 83, no. 2, pp. 247–261, 2009.
- R. Sniecinski and H. Liu, “Reduced, efficacy of volatile anesthetic preconditioning with advanced age in isolated rat myocardium,” Anesthesiology, vol. 100, no. 3, pp. 589–597, 2004.
- E. J. Lesnefsky, T. I. Gudz, S. Moghaddas et al., “Aging decreases electron transport complex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site,” Journal of Molecular and Cellular Cardiology, vol. 33, no. 1, pp. 37–47, 2001.
- S. W. Fannin, E. J. Lesnefsky, T. J. Slabe, M. O. Hassan, and C. L. Hoppel, “Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria,” Archives of Biochemistry and Biophysics, vol. 372, no. 2, pp. 399–407, 1999.
- S. Moghaddas, C. L. Hoppel, and E. J. Lesnefsky, “Aging defect at the QO site of complex III augments oxyradical production in rat heart interfibrillar mitochondria,” Archives of Biochemistry and Biophysics, vol. 414, no. 1, pp. 59–66, 2003.
- E. J. Lesnefsky, D. He, S. Moghaddas, and C. L. Hoppel, “Reversal of mitochondrial defects before ischemia protects the aged heart,” The FASEB Journal, vol. 20, no. 9, pp. 1543–1545, 2006.
- M. Aldakkak, D. F. Stowe, Q. Chen, E. J. Lesnefsky, and A. K. S. Camara, “Inhibited mitochondrial respiration by amobarbital during cardiac ischaemia improves redox state and reduces matrix Ca2+ overload and ROS release,” Cardiovascular Research, vol. 77, no. 2, pp. 406–415, 2008.
- G. Ambrosio, J. L. Zweier, C. Duilio et al., “Evidence that mitochondrial respiration is a source of potentially toxic oxygen free radicals in intact rabbit hearts subjected to ischemia and reflow,” Journal of Biological Chemistry, vol. 268, no. 25, pp. 18532–18541, 1993.
- Q. Chen, A. K. S. Camara, D. F. Stowe, C. L. Hoppel, and E. J. Lesnefsky, “Modulation of electron transport protects cardiac mitochondria and decreases myocardial injury during ischemia and reperfusion,” American Journal of Physiology. Cell Physiology, vol. 292, no. 1, pp. C137–C147, 2007.
- M. Paillard, L. Gomez, L. Augeul, J. Loufouat, E. J. Lesnefsky, and M. Ovize, “Postconditioning inhibits mPTP opening independent of oxidative phosphorylation and membrane potential,” Journal of Molecular and Cellular Cardiology, vol. 46, no. 6, pp. 902–909, 2009.
- J. F. Turrens, “Mitochondrial formation of reactive oxygen species,” Journal of Physiology, vol. 552, no. 2, pp. 335–344, 2003.
- D. J. Hausenloy, S. B. Ong, and D. M. Yellon, “The mitochondrial permeability transition pore as a target for preconditioning and postconditioning,” Basic Research in Cardiology, vol. 104, no. 2, pp. 189–202, 2009.
- G. Heusch, K. Boengler, and R. Schulz, “Inhibition of mitochondrial permeability transition pore opening: the holy grail of cardioprotection,” Basic Research in Cardiology, vol. 105, no. 2, pp. 151–154, 2010.
- A. B. Gustafsson and R. A. Gottlieb, “Heart mitochondria: gates of life and death,” Cardiovascular Research, vol. 77, no. 2, pp. 334–343, 2008.
- A. P. Halestrap, S. J. Clarke, and S. A. Javadov, “Mitochondrial permeability transition pore opening during myocardial reperfusion—a target for cardioprotection,” Cardiovascular Research, vol. 61, no. 3, pp. 372–385, 2004.
- J. N. Weiss, P. Korge, H. M. Honda, and P. Ping, “Role of the mitochondrial permeability transition in myocardial disease,” Circulation Research, vol. 93, no. 4, pp. 292–301, 2003.
- G. Heusch, J. Musiolik, N. Gedik, and A. Skyschally, “Mitochondrial STAT3 activation and cardioprotection by ischemic postconditioning in pigs with regional myocardial ischemia/reperfusion,” Circulation Research, vol. 109, no. 11, pp. 1302–1308, 2011.
- D. J. Hausenloy and D. M. Yellon, “Reperfusion injury salvage kinase signalling: taking a RISK for cardioprotection,” Heart Failure Reviews, vol. 12, no. 3-4, pp. 217–234, 2007.
- S. Lecour, “Activation of the protective Survivor Activating Factor Enhancement (SAFE) pathway against reperfusion injury: does it go beyond the RISK pathway?” Journal of Molecular and Cellular Cardiology, vol. 47, no. 1, pp. 32–40, 2009.
- S. Stewart, E. J. Lesnefsky, and Q. Chen, “Reversible blockade of electron transport with amobarbital at the onset of reperfusion attenuates cardiac injury,” Translational Research, vol. 153, no. 5, pp. 224–231, 2009.
- Q. Chen, M. Paillard, L. Gomez, H. Li, Y. Hu, and E. J. Lesnefsky, “Postconditioning modulates ischemia-damaged mitochondria during reperfusion,” Journal of Cardiovascular Pharmacology, vol. 59, no. 1, pp. 101–108, 2012.
- K. Przyklenk, M. Maynard, C. E. Darling, and P. Whittaker, “Aging mouse hearts are refractory to infarct size reduction with post-conditioning,” Journal of the American College of Cardiology, vol. 51, no. 14, pp. 1393–1398, 2008.
- K. Boengler, A. Buechert, Y. Heinen et al., “Cardioprotection by ischemic postconditioning is lost in aged and STAT3-deficient mice,” Circulation Research, vol. 102, no. 1, pp. 131–135, 2008.
- Q. Chen, S. Moghaddas, C. L. Hoppel, and E. J. Lesnefsky, “Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion,” Journal of Pharmacology and Experimental Therapeutics, vol. 319, no. 3, pp. 1405–1412, 2006.
- C. C. Tanaka-Esposito, Q. Chen, and E. J. Lesnefsky, “Blockade of electron transport before ischemia protects mitochondria and decreases myocardial injury during reperfusion in aged rat hearts,” Translational Research. In press.
- C. Piot, P. Croisille, P. Staat et al., “Effect of cyclosporine on reperfusion injury in acute myocardial infarction,” New England Journal of Medicine, vol. 359, no. 5, pp. 473–481, 2008.
- Q. Chen, M. Paillard, L. Gomez, et al., “Activation of mitochondrial mu-calpain increases AIF cleavage in cardiac mitochondria during ischemia-reperfusion,” Biochemical and Biophysical Research Communications, vol. 415, no. 4, pp. 533–538, 2011.
- Q. Chen, E. J. Vazquez, S. Moghaddas, C. L. Hoppel, and E. J. Lesnefsky, “Production of reactive oxygen species by mitochondria: central role of complex III,” Journal of Biological Chemistry, vol. 278, no. 38, pp. 36027–36031, 2003.
- R. C. Scaduto and L. W. Grotyohann, “Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives,” Biophysical Journal, vol. 76, no. 1 I, pp. 469–477, 1999.
- L. Gomez, H. Thibault, A. Gharib et al., “Inhibition of mitochondrial permeability transition improves functional recovery and reduces mortality following acute myocardial infarction in mice,” American Journal of Physiology. Heart and Circulatory Physiology, vol. 293, no. 3, pp. H1654–H1661, 2007.
- E. J. Lesnefsky, T. I. Gudz, C. T. Migita et al., “Ischemic injury to mitochondrial electron transport in the aging heart: damage to the iron-sulfur protein subunit of electron transport complex III,” Archives of Biochemistry and Biophysics, vol. 385, no. 1, pp. 117–128, 2001.
- L. B. Becker, T. L. V. Hoek, Z. H. Shao, C. Q. Li, and P. T. Schumacker, “Generation of superoxide in cardiomyocytes during ischemia before reperfusion,” American Journal of Physiology. Heart and Circulatory Physiology, vol. 277, no. 6, pp. H2240–H2246, 1999.
- P. Long, Q. Nguyen, C. Thurow, and T. L. Broderick, “Caloric restriction restores the cardioprotective effect of preconditioning in the rat heart,” Mechanisms of Ageing and Development, vol. 123, no. 10, pp. 1411–1413, 2002.
- P. Abete, G. Testa, G. Galizia et al., “Tandem action of exercise training and food restriction completely preserves ischemic preconditioning in the aging heart,” Experimental Gerontology, vol. 40, no. 1-2, pp. 43–50, 2005.
- R. A. Fenton, E. W. Dickson, and J. G. Dobson, “Inhibition of phosphatase activity enhances preconditioning and limits cell death in the ischemic/reperfused aged rat heart,” Life Sciences, vol. 77, no. 26, pp. 3375–3388, 2005.
- J. W. Park, Y. S. Chun, Y. H. Kim, C. H. Kim, and M. S. Kim, “Ischemic preconditioning reduces generation and prevents respiratory impairment in the mitochondria of post-ischemic reperfused heart of rat,” Life Sciences, vol. 60, no. 24, pp. 2207–2219, 1997.
- E. J. Lesnefsky, Q. Chen, T. J. Slabe et al., “Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin,” American Journal of Physiology. Heart and Circulatory Physiology, vol. 287, no. 1, pp. H258–H267, 2004.
- E. J. Lesnefsky, P. Minkler, and C. L. Hoppel, “Enhanced modification of cardiolipin during ischemia in the aged heart,” Journal of Molecular and Cellular Cardiology, vol. 46, no. 6, pp. 1008–1015, 2009.
- G. Keusch, K. Boengler, and R. Schulz, “Cardioprotection: nitric oxide, protein kinases, and mitochondria,” Circulation, vol. 118, no. 19, pp. 1915–1919, 2008.
- D. M. Yellon and D. J. Hausenloy, “Myocardial reperfusion injury,” New England Journal of Medicine, vol. 357, no. 11, pp. 1074–1135, 2007.
- S. Lecour, N. Suleman, G. A. Deuchar et al., “Pharmacological preconditioning with tumor necrosis factor-α activates signal transducer and activator of transcription-3 at reperfusion without involving classic prosurvival kinases (Akt and extracellular signal-regulated kinase),” Circulation, vol. 112, no. 25, pp. 3911–3918, 2005.
- E. Murphy and C. Steenbergen, “Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury,” Physiological Reviews, vol. 88, no. 2, pp. 581–609, 2008.
- S. M. Nadtochiy, A. J. Tompkins, and P. S. Brookes, “Different mechanisms of mitochondrial proton leak in ischaemia/reperfusion injury and preconditioning: implications for pathology and cardioprotection,” Biochemical Journal, vol. 395, no. 3, pp. 611–618, 2006.
- A. W. C. Leung and A. P. Halestrap, “Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore,” Biochimica et Biophysica Acta, Bioenergetics, vol. 1777, no. 7-8, pp. 946–952, 2008.
- L. Gomez, M. Paillard, H. Thibault, G. Derumeaux, and M. Ovize, “Inhibition of GSK3β by postconditioning is required to prevent opening of the mitochondrial permeability transition pore during reperfusion,” Circulation, vol. 117, no. 21, pp. 2761–2768, 2008.
- E. J. Lesnefsky, T. J. Slabe, M. S. K. Stoll, P. E. Minkler, and C. L. Hoppel, “Myocardial ischemia selectively depletes cardiolipin in rabbit heart subsarcolemmal mitochondria,” American Journal of Physiology. Heart and Circulatory Physiology, vol. 280, no. 6, pp. H2770–H2778, 2001.
- E. J. Lesnefsky, M. S. K. Stoll, P. E. Minkler, and C. L. Hoppel, “Separation and quantitation of phospholipids and lysophospholipids by high-performance liquid chromatography,” Analytical Biochemistry, vol. 285, no. 2, pp. 246–254, 2000.
- G. C. Sparagna and E. J. Lesnefsky, “Cardiolipin remodeling in the heart,” Journal of Cardiovascular Pharmacology, vol. 53, no. 4, pp. 290–301, 2009.
- F. L. Hoch, “Mini review: cardiolipins and mitochondrial protonselective leakage,” Journal of Bioenergetics and Biomembranes, vol. 30, no. 6, pp. 511–532, 1998.
- S. Moghaddas, M. S. K. Stoll, P. E. Minkler, R. G. Salomon, C. L. Hoppel, and E. J. Lesnefsky, “Preservation of cardiolipin content during aging in rat heart interfibrillar mitochondria,” Journals of Gerontology. Series A, vol. 57, no. 1, pp. B22–B28, 2002.
- Q. Chen, S. Moghaddas, C. L. Hoppel, and E. J. Lesnefsky, “Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria,” American Journal of Physiology. Cell Physiology, vol. 294, no. 2, pp. C460–C466, 2008.