Inhibiting Mitochondrial Fission Protects the Heart Against Ischemia/Reperfusion Injury
Background— Whether alterations in mitochondrial morphology affect the susceptibility of the heart to ischemia/reperfusion injury is unknown. We hypothesized that modulating mitochondrial morphology protects the heart against ischemia/reperfusion injury.
Methods and Results— In response to ischemia, mitochondria in HL-1 cells (a cardiac-derived cell line) undergo fragmentation, a process that is dependent on the mitochondrial fission protein dynamin-related protein 1 (Drp1). Transfection of HL-1 cells with the mitochondrial fusion proteins mitofusin 1 or 2 or with Drp1K38A, a dominant-negative mutant form of Drp1, increased the percentage of cells containing elongated mitochondria (65±4%, 69±5%, and 63±6%, respectively, versus 46±6% in control: n=80 cells per group; P<0.05), decreased mitochondrial permeability transition pore sensitivity (by 2.4±0.5-, 2.3±0.7-, and 2.4±0.3-fold, respectively; n=80 cells per group; P<0.05), and reduced cell death after simulated ischemia/reperfusion injury (11.6±3.9%, 16.2±3.9%, and 12.1±2.9%, respectively, versus 41.8±4.1% in control: n=320 cells per group; P<0.05). Treatment of HL-1 cells with mitochondrial division inhibitor-1, a pharmacological inhibitor of Drp1, replicated these beneficial effects. Interestingly, elongated interfibrillar mitochondria were identified in the adult rodent heart with confocal and electron microscopy, and in vivo treatment with mitochondrial division inhibitor-1 increased the percentage of elongated mitochondria from 3.6±0.5% to 14.5±2.8% (P=0.023). Finally, treatment of adult murine cardiomyocytes with mitochondrial division inhibitor-1 reduced cell death and inhibited mitochondrial permeability transition pore opening after simulated ischemia/reperfusion injury, and in vivo treatment with mitochondrial division inhibitor-1 reduced myocardial infarct size in mice subject to coronary artery occlusion and reperfusion (21.0±2.2% with mitochondrial division inhibitor-1 versus 48.0±4.5% in control; n=6 animals per group; P<0.05).
Conclusion— Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury, suggesting a novel pharmacological strategy for cardioprotection.
Received August 1, 2009; accepted March 15, 2010.
Innovative treatment strategies for protecting the heart from ischemia/reperfusion injury (IRI) are needed to improve clinical outcomes in patients with coronary heart disease. Previous studies suggest that mitochondria are highly dynamic and that changes in mitochondrial shape can affect a variety of biological processes such as apoptosis, respiration, mitosis, and development.1,2 Mitochondria change their morphology by undergoing either fusion or fission, resulting in either elongated, tubular, interconnected mitochondrial networks or fragmented, discontinuous mitochondria, respectively.1,2 These 2 opposing processes are regulated by the mitochondrial fusion proteins mitofusin (Mfn) 1, Mfn2, and optic atrophy protein 1 and the mitochondrial fission proteins dynamin-related protein 1 (Drp1) and human mitochondrial fission protein 1 (hFis1).1,2 The fine balance between mitochondrial fusion and fission within a cell may be upset by a variety of factors, including oxidative stress3 and simulated ischemia,4 which can predispose the cell to apoptosis,5 and the opening of the mitochondrial permeability transition pore (mPTP),6 critical mediators of IRI.7 Therefore, on the basis that changes in mitochondrial morphology appear to affect biological processes that may be fundamental to cardioprotection, we hypothesized that modulating mitochondrial morphology may protect the heart against IRI.
Clinical Perspective on p 2022
HL-1 Cell Plasmid Transfection
The HL-1 cardiac cell line (derived from murine atrial cardiomyocytes) was cultured according to previously published methods.8 Using Fugene 6 (Roche Molecular Biochemicals, Basel, Switzerland), we transfected cells with plasmids expressing Mfn1, Mfn2,9 Drp1K38A, the dominant-negative mutant form of the mitochondrial fission protein Drp1,5 hFis1, or empty vector.
Determining Mitochondrial Morphology in HL-1 Cells
Mitochondrial morphology was determined in HL-1 cells transfected with red fluorescent protein targeted to the mitochondrial matrix with a Zeiss 510 CLSM confocal microscope equipped with 63× oil immersion objective (Plan-Apochromat, NA 1.3). Eighty randomly chosen transfected cells per treatment group were designated as containing either predominantly (>50%) elongated or predominantly (>50%) fragmented mitochondria by 3 investigators blinded to the treatment (Figure 1).9
Real-Time Changes in Mitochondrial Morphology During Ischemia and Reperfusion
HL-1 cells containing predominantly (>50%) elongated mitochondria under basal conditions were identified and subjected to 120 minutes of simulated ischemia and 30 minutes of simulated reperfusion (SIRI) with a Warner PM-2 heated perfusion chamber (Harvard Apparatus, Holliston, Mass) mounted on the confocal microscope. The real-time effects of SIRI on changes in mitochondrial morphology were determined in HL-1 cells overexpressing red fluorescent protein targeted to the mitochondrial matrix and either empty vector or Drp1K38A. Over 3 independent experiments, 10 cells for each treatment group were analyzed.
HL-1 Cell Death After SIRI
To determine the effect of inducing mitochondrial elongation on the susceptibility to SIRI, HL-1 cells were subjected to 12 hours of simulated ischemia in an air-tight hypoxic chamber and 1 hour of simulated reperfusion, at the end of which cell death was assessed by propidium iodide staining. For each treatment group, 80 cells taken from 4 randomly selected fields of view were counted. This experiment was repeated on at least 4 separate occasions.
Induction and Detection of mPTP Opening
To determine the effect of inducing mitochondrial elongation on the susceptibility to mPTP opening in HL-1 cells, we used a well-characterized and validated model of oxidative stress to induce and detect mPTP opening in which confocal laser-induced activation of tetramethylrhodamine methyl ester (TMRM) generates oxidative stress within mitochondria and induces mPTP opening, which is indicated by mitochondrial membrane depolarization.10,11 mPTP opening was confirmed in this model through the use of the known mPTP inhibitors ciclosporin A (CsA) and sanglifehrin-A and investigation of the movement of mitochondrial-loaded calcein (see the online-only Data Supplement).10,12 Twenty transfected cells were randomly selected for each treatment group, and this was repeated in at least 4 independent experiments. For the adult cardiomyocytes, we used a different model to assess mPTP opening in which its opening is measured after 45 minutes of simulated ischemia and 30 minutes of simulated reperfusion by measuring the resultant TMRM fluorescence. Twenty cells were randomly selected for each treatment group, and this was repeated in at least 4 independent experiments.
Pharmacological Inhibition of Drp1 to Induce Mitochondrial Fusion
HL-1 cells were incubated with a small-molecule Drp1, inhibitor mitochondrial division inhibitor-1 (mdivi-1)13 (Key Organics Ltd, Camelford, Cornwall, UK), for 40 minutes (at 10 or 50 μmol/L) to investigate the effects of Drp1 inhibition on mitochondrial morphology, susceptibility to mPTP opening, and cell death in HL-1 cells after SIRI as described above.
Confocal Microscopy of Adult Cardiomyocytes
All animal experiments were carried out in accordance with the UK Home Office Guide on the Operation of Animal (Scientific Procedures) Act of 1986. Adenoviral vectors carrying the mitochondrial matrix targeted photoactivatable green fluorescent protein (mtPA-GFP) expression cassette were generated with the AdEasy XL Adenoviral Vector System (Stratagene, La Jolla, Calif). Ventricular cardiomyocytes were isolated from adult Sprague-Dawley rats by perfusion and digestion of ventricles with collagenase according to a previously described method.10 The cells were incubated with plating medium containing an appropriate titer of virus for 4 hours and were imaged 72 hours later. mtPA-GFP within a prespecified region of interest (ROI) was photoactivated by scanning adult rat cardiomyocytes with the 405-nm wavelength ultraviolet laser. The cell was immediately reimaged at 488 nm, and the difference in the intensity of green fluorescence between the 2 images before and after photoactivation was determined with Image J software (National Institutes of Health, Bethesda, Md). The spread of GFP beyond the ROI was expressed as a fold increase relative to the intensity within the ROI. Results were obtained from 30 randomly chosen cells isolated from 3 rats.
Adult Cardiomyocyte Death After SIRI
Ventricular cardiomyocytes were isolated from adult C57BL/6 male mice by perfusion and digestion of ventricles with collagenase according to a previously described method.14 The cells were then randomized to receive pretreatment with either vehicle control or mdivi-1 treatment at either 10 or 50 μmol/L (n>250 cells per experiment for 4 experiments) before being subjected to 45 minutes of simulated ischemia followed by 30 minutes of simulated reperfusion, at the end of which cell death was measured by propidium iodide staining.14
In Vivo Model of IRI
C57BL/6 male mice were anesthetized by intraperitoneal injection (0.01 mL/g) of a solution containing ketamine 10 mg/mL, xylazine 2 mg/mL, and atropine 0.06 mg/mL and were subjected to in vivo 30 minutes of regional myocardial ischemia followed by 120 minutes of myocardial reperfusion, at the end of which myocardial infarct size was determined by triphenyltetrazolium staining.14 Mice were randomly assigned to receive by intravenous injection either vehicle control (0.1 mL of 0.1% dimethyl sulfoxide) or mdivi-1 (at either 0.24 or 1.2 mg/kg, doses that were equivalent to the ex vivo concentrations of 10 and 50 μmol/L, respectively) 15 minutes before myocardial ischemia (n=6 mice per treatment group). The in vivo doses were calculated to reflect as closely as possible the in vitro concentrations used; however, the pharmacokinetic properties of the drug in vivo are unknown.
Hearts were excised from C57BL/6 male mice after an intravenous injection of either vehicle control (0.1 mL of 0.1% dimethyl sulfoxide) or mdivi-1 (0.24 mg/kg) after 15 minutes of stabilization (n=4 mice) or after 15 minutes of stabilization followed by 20 minutes of regional ischemia (n=4 mice). The excised hearts were perfused with a fixative overnight, following which a 2-mm transverse slice, 3 mm from the apex, was obtained from each heart. Ultrathin sections were viewed with a Joel 1010 transition electron microscope (Joel Ltd, Warwickshire, UK). In 6 randomly selected electron micrographs of longitudinally arranged cardiomyocytes, the proportions of interfibrillar mitochondria with lengths that were <2, 2, or >2 μm (the length of a single sarcomere) were determined. For each heart, the lengths of >500 to 600 interfibrillar mitochondria were assessed.
All values are expressed as mean±SEM. Data were analyzed by 1-way ANOVA followed by a Tukey multiple-comparison posthoc test. Differences were considered significant at values of P<0.05.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Inducing Mitochondrial Fusion in the HL-1 Cells
Transfecting HL-1 cells with Mfn1, Mfn2, or Drp1K38A increased the percentage of cells containing predominantly elongated mitochondria: 46±6% in the vector control versus 65±4% with Mfn1 (P<0.05), 69±5% with Mfn2 (P<0.01), and 63±6% with Drp1K38A (P<0.05; Figure 1F). Conversely, overexpressing hFis1 reduced the percentage of cells containing predominantly elongated mitochondria (46±6% in the vector control to 11±4% with hFis1; P<0.001; Figure 1F).
Mitochondrial Fission Is Induced by Ischemia and Is Dependent on Drp1
Mitochondria in the majority of HL-1 cells underwent fission in response to simulated ischemia (Figure 2A). However, this effect was largely prevented in cells overexpressing Drp1K38A (Figure 2A), suggesting that this fission process was dependent on Drp1. By the end of the simulated ischemia period, the percentage of cells displaying elongated mitochondria had fallen from 100% to 11.0±11.0% in control cells and to 91.0±5.6% in the Drp1K38A-transfected cells (P<0.05; Figure 2B), a difference that persisted into simulated reperfusion (Figure 2B). Of note, it would have been preferable to have performed this experiment with an unselected population of HL-1 cells rather than choosing cells displaying only elongated mitochondria.
Increased Mitochondrial Fusion Protects HL-1 Cells Against SIRI
The overexpression of the mitochondrial fusion proteins (Mfn1 or Mfn2) or Drp1K38A significantly reduced the percentage cell death after SIRI (assessed after 1 hour of reperfusion): 41.8±4.1% in the vector control to 11.6±3.0% with Mfn1 (P<0.0001), 16.2±3.9% with Mfn2 (P<0.0001), and 12.1±2.9% with Drp1K38A (P<0.001; Figure 2C). In contrast, overexpression of hFis1 increased the percentage cell death (assessed after 1 hour of reperfusion): 41.8±4.1% in the vector control to 65.5±2.1% with hFis1 (P<0.05; Figure 2C). The percentage of dead cells under normoxic conditions was <5.0%, and this was not significantly altered by transgene expression.
Inducing Mitochondrial Fusion in HL-1 Cells Decreases the Susceptibility to mPTP Opening
Overexpression of Mfn1, Mfn2, or Drp1K38A delayed the time taken to induce mPTP opening compared with the vector control group by 2.4±0.5-fold with Mfn1 (P<0.01), 2.3±0.7-fold with Mfn2 (P<0.05), and 2.4±0.3-fold with Drp1K38A (P<0.05; Figure 3B). This was similar to the effect of the known mPTP inhibitor, CsA, which delayed the time taken to induce mPTP opening by 2.2±0.4-fold (P<0.05). The overexpression of hFis1 did not significantly influence the time taken to induce mPTP opening compared with the vector control group (0.89±0.70-fold with hFis1; P>0.05). To further verify that the observed mitochondrial membrane depolarization was due to mPTP opening, we demonstrated that its onset coincided with the redistribution into the cytosol of mitochondrion-loaded calcein (see the online-only Data Supplement).12,15
The Beneficial Effects of the Drp1 Inhibitor mdivi-1 in HL-1 Cells
The optimal treatment regimen for mdivi-1 was first determined in HL-1 cells identified as containing predominantly fragmented mitochondria only. At 50 μmol/L (the concentration previously demonstrated as having a maximal effect on mitochondrial elongation)13 but not at 10 μmol/L, mdivi-1 significantly increased the percentage of HL-1 cells displaying elongated mitochondria after 40 minutes of drug incubation. After 20 minutes, the percentage of untreated cells containing elongated mitochondria rose from 0 to a new steady state of 19±11%, which was not significantly altered after a further 20-minute incubation (13±11%). After 20 minutes in 50 μmol/L mdivi-1, the percentage of elongated mitochondria was similar (19±3%), but by 40 minutes, this had increased significantly to 60±8% (Figure 4A). The presence of 10 μmol/L mdivi-1 had no significant effect (8±8% at 20 minutes and 19±3% at 40 minutes).
In a separate set of experiments, the incubation of HL-1 cells containing either elongated or fragmented mitochondria under basal conditions with 50 μmol/L mdivi-1 for 40 minutes resulted in an increase in the percentage of cells displaying elongated mitochondria from 26±7% at baseline to 67±4% after 40 minutes (P<0.05). There was no significant change in the percentage of cells displaying elongated mitochondria in cells treated with vehicle control (34±9% at baseline versus 38±8% after 40 minutes; P>0.05) or in cells treated with mdivi-1 at 10 μmol/L (33±1% at baseline versus 33±11% after 40 minutes; P>0.05; Figure 4B).
Pretreatment of HL-1 cells with mdivi-1 for 40 minutes decreased the percentage cell death after subsequent exposure to SIRI at 50 μmol/L (42.1±4.8% in the vehicle control versus 20.0±2.6% with mdivi-1; P<0.05) but not at 10 μmol/L (42.1±4.8% in the vehicle control versus 45.5±3.5% with mdivi-1; P>0.05; Figure 4C). Similarly, pretreatment of HL-1 cells with mdivi-1 for 40 minutes delayed the time taken to induce mPTP opening at 50 μmol/L (2.1±0.5-fold delay; P<0.05) but not at 10 μmol/L (0.6±0.1-fold delay; P>0.05; Figure 4D). Again, we confirmed that the model was sensitive to CsA (2.3±0.5-fold delay; P<0.05; Figure 4D).
Elongated Mitochondria Are Present in Adult Cardiomyocytes
Using confocal microscopy, we were able to visualize mitochondrial morphology in adult rat cardiomyocytes loaded with TMRM and the adenoviral construct expressing mtPA-GFP, which is nonfluorescent but can be activated in a highly localized manner by scanning with the ultraviolet confocal laser line (Figure 5A). The photoactivated GFP diffuses rapidly within the mitochondrial matrix to the full extent of the inner mitochondrial membrane and can therefore be used to “tag” individual mitochondria.16 Using this experimental approach, we were able identify elongated, interfibrillar mitochondria in primary adult cardiomyocytes. The length of these elongated mitochondria ranged from 2 to 6 μm (corresponding to 1 to 3 sarcomeres). In addition, we observed the spread of photoactivated mitochondrial GFP outside the initial area of photoactivation (2.17±0.06-fold increase in the area of GFP; Figure 5A). This finding was confirmed by observing the changes in mitochondrial membrane potential taking place in individual mitochondria in response to low levels of oxidative stress generated by laser scanning of TMRM. This results in “flickering” of individual mitochondria as they depolarize and repolarize, with coincident loss and reaccumulation of TMRM dye.17 Both mitochondrial membrane depolarization and repolarization were found to occur in a synchronous fashion along the entire length of the elongated mitochondria (Movie I of the online-only Data Supplement), implying a single, elongated mitochondrion. After 72 hours of culture to allow expression of the adenoviral DNA constructs, there was some cell death and some rounding of cell ends, although the changes appear to be relatively minor and the healthy cells remain typically rod shaped (Figure 5A).
The Beneficial Effects of Pharmacologically Inhibiting Drp1 in the Adult Heart
Forty minutes of pretreatment of adult murine cardiomyocytes with 50 μmol/L mdivi-1 decreased the percentage cell death after SIRI (46.0±1.1% in the vehicle control compared with 34.0±1.9% with mdivi-1; P<0.05), whereas pretreatment with 10 μmol/L mdivi-1 had no significant effect (46.0±1.1% in the vehicle control to 48.0±3.2% with mdivi-1; P>0.05; Figure 6A). There was no significant effect of mdivi-1 on percentage cell death during normoxia (data not shown). Furthermore, 40 minutes of pretreatment with 50 μmol/L mdivi-1 decreased mPTP sensitivity after SIRI as evidenced by preservation of mitochondrial membrane potential detected with TMRM fluorescence (0.78±0.06 SIRI control versus 1.04±0.04 mdivi-1; P<0.05; Figure 6B).
We examined longitudinal sections of adult murine hearts by electron microscopy to determine the arrangement and morphology of the interfibrillar subpopulation of mitochondria (Figure 5B). Interestingly, we observed a significant number of elongated interfibrillar mitochondria with lengths that extended beyond 2 μm and in some cases were up to 6 μm, allowing them to align next to up to 3 adjacent sarcomeres (each ≈2 μm in length; Figure 5C and 5D). We found that the in vivo treatment of adult murine hearts with mdivi-1 (50 μmol/L) for 15 minutes had no effect on mitochondrial length before ischemia (Figure 6C), although it significantly increased the proportion of elongated mitochondria (>2 μm or 1 sarcomere in length) after 15 minutes of stabilization plus 20 minutes of myocardial ischemia from 3.6±0.5% in control to 14.5±2.8% with mdivi-1 (P=0.023) as visualized and assessed by electron microscopy (Figure 6D and 6E).
Finally, the effect of pretreatment with mdivi-1 was investigated in vivo in the whole murine heart subjected to myocardial infarction using 2 doses, 0.24 mg/kg (dose 1) or 1.2 mg/kg (dose 2), which were equivalent to the in vitro concentrations of 10 and 50 μm, respectively. The hemodynamic profiles during coronary artery occlusion and reperfusion (in terms of heart rate and blood pressure) were comparable between the treatment groups (Figure 7A and 7B). However, the observed basal heart rates of 400 bpm in the control and treated groups were depressed to a similar extent by the anesthetic protocol used in this study. The area at risk, expressed as a percentage of the left ventricular volume, was also comparable between treatment groups: 53.6±5.6% in vehicle control versus 54.5±6.5% with mdivi-1 (dose 1) and 55.8±3.9% with mdivi-1 (dose 2; Figure 7C). A single intravenous bolus of mdivi-1 (dose 1) administered 10 minutes before acute coronary occlusion of the left main coronary artery failed to significantly reduce myocardial infarct size, expressed as a percentage of the area at risk (42.0±4.9% with mdivi-1 versus 48.0±4.5% in vehicle control; P>0.05), whereas a single intravenous bolus of mdivi-1 (dose 2) significantly reduced myocardial infarct size in the in vivo murine heart (21.0±2.2% with mdivi-1 versus 48.0±4.5% in vehicle control; P<0.05; Figure 7D and 7E).
The present study has shown that modulating mitochondrial morphology protects the heart against IRI. We found that inducing mitochondrial elongation with either genetic or pharmacological manipulation decreased mPTP sensitivity and reduced cell death after SIRI in HL-1 cells. Crucially, in the adult rodent heart in which the spatial organization of interfibrillar mitochondria differs from HL-1 cells, elongated mitochondria were identified with the use of confocal and electron microscopy. Furthermore, in vivo treatment with the Drp1 inhibitor mdivi-1 significantly increased the proportion of elongated interfibrillar mitochondria in the ischemic adult murine heart. Finally, pharmacological Drp1 inhibition with mdivi-1 protected adult cardiomyocytes against SIRI, inhibited mPTP opening in cardiomyocytes, and reduced myocardial infarct size in an in vivo murine model.
Changes in mitochondrial morphology may affect the susceptibility of the cell to apoptotic cell death, a contributory cause for the cell death incurred during IRI.18 In the kidney, renal injury has been reported to occur via the induction of Drp1-dependent mitochondrial fragmentation and apoptosis, and prevention of this process was found to be beneficial.19 The limited number of studies that have examined mitochondrial morphology in cardiovascular cells have, on the whole, been confined to either cell lines or neonatal cardiomyocytes and have tended to focus on the effect on apoptosis (reviewed elsewhere20). Mfn2 has been reported to exert a diverse range of effects that appear to be unrelated to its ability to elongate mitochondria (reviewed elsewhere21). Therefore, we used the dominant-negative mutant form of the mitochondrial fission protein Drp1 (Drp1K38A) to induce mitochondrial elongation; this had the same effects as Mfn2, suggesting that the observed cardioprotection and mPTP inhibition were due to mitochondrial elongation.
The cardioprotective effect elicited by mitochondrial elongation was linked to the inhibition of mPTP opening in the present study. The mPTP is a critical mediator of cell death induced by IRI.7 Overexpressing Drp1 or hFis1 has been reported to increase the susceptibility to calcium-induced mPTP opening in COS epithelial cells,6 whereas in our study, the overexpression of hFis1 in HL-1 cells did not influence mPTP opening sensitivity. This may be due to a difference in models used, ie, a limitation in the resolution of our model to detect increased mPTP opening sensitivity. An alternative explanation may be that hFis1 increased death without a detectable effect on mPTP opening, suggesting that it may have damaging effects that are independent of the mPTP. Inhibiting Drp1 has been reported to increase mPTP resistance in response to hyperglycemia.22 Neuspiel and coworkers11 also found that the overexpression of Mfn2 prevented mPTP opening induced by free radicals in COS-7 cells. The mechanism through which increased mitochondrial elongation prevents mPTP opening is not clear, although one may speculate that elongated mitochondria may accommodate a greater burden of mitochondrial calcium load and oxidative stress before undergoing mPTP opening compared with fragmented mitochondria and that mitochondrial elongation may generate mitochondria with greater respiratory capacity that are better equipped to withstand the metabolic and biochemical stresses associated with IRI. In this regard, it has previously been demonstrated that altering mitochondrial fusion protein expression similarly alters oxygen consumption, mitochondrial membrane potential, and mitochondrial respiration in myotubes and other cells.23,24 It must be noted that the model of mPTP opening used in the present study may not accurately reflect the conditions of ischemia and reperfusion. Another limitation of the present study is that we used a number of different experimental models to investigate the effect of modulating mitochondrial morphology on the susceptibility to IRI when a single model may have been preferable.
Whether the spatial organization of interfibrillar mitochondria in adult cardiomyocytes restricts their movement and prevents them from undergoing fusion or fission has been the subject of recent debate. The machinery required for regulating mitochondrial morphology in terms of the mitochondrial fusion proteins and fission proteins is present in the adult heart.20 It has recently been demonstrated that interfibrillar mitochondria in adult rat cardiomyocytes are dynamic structures that are capable of undergoing rapid low-amplitude fluctuations, although they appear isolated with respect to electric activity,25 and it is generally assumed that mitochondria are restricted to 2-μm lengths alongside each sarcomere in adult cardiomyocytes. However, using both confocal and electron microscopy, we were readily able to identify elongated mitochondria extending up to 2 to 3 sarcomeres (4 to 6 μm) in length. The synchronous depolarization and repolarization of mitochondrial membrane potential in response to low levels of oxidative stress occurred throughout the whole length of the elongated mitochondrion, providing strong confirmation that we were visualizing a single functional unit. Interestingly, previous electron microscopy studies have observed individual mitochondria >2 to 3 sarcomeres in length,26,27 and as far back as 1969, Sun and coworkers28 demonstrated elongated mitochondria ranging from 3 to 7 sarcomeres in length in isolated perfused adult rat hearts in response to short periods of hypoxia (3 to 7 minutes). This raises the interesting, although at this point speculative, possibility that short nonlethal periods of hypoxia may have induced mitochondrial elongation as an endogenous protective mechanism against further hypoxic or ischemic insult. In this regard, a recently published study suggests that mitochondrial elongation may be an initial response to a low level of stress, which provides a protective response against a future insult.29
We report here that inducing mitochondrial elongation protects the HL-1 cardiac cell line from IRI by inhibiting mPTP opening. We have been able to detect changes in mitochondrial morphology in the adult heart despite their distinctive arrangement. Crucially, we have demonstrated that pharmacological inhibition of the mitochondrial fission protein Drp1 protected adult murine cardiomyocytes against SIRI, inhibited mPTP opening, and more important, reduced myocardial infarct size in the in vivo murine heart. These findings suggest that manipulating mitochondrial morphology may provide a novel therapeutic strategy for cardioprotection.
We would like to thank Mark Turmaine for the preparation of the electron micrographs. Dr Davidson acknowledges the support of the Medical Research Council.
Sources of Funding
S.-B. Ong is funded by a Dorothy Hodgkin Postgraduate Award (Biotechnology and Biological Sciences Research Council). We thank the British Heart Foundation for their continued support. This work was undertaken at University College London Hospital/University College London, which received a portion of funding from the Department of Health’s National Institute of Health Research Biomedical Research Centres funding scheme.
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Despite optimal therapy, clinical outcomes in patients with coronary heart disease can be further improved by the discovery of new therapeutic strategies for protecting the heart from ischemia/reperfusion injury. In this respect, in the present study, we have demonstrated that the modulation of mitochondrial morphology in the heart may provide a novel therapeutic strategy for cardioprotection. Specifically, we have demonstrated that inhibiting mitochondrial fission protected the heart by inhibiting the opening of the mitochondrial permeability transition pore, a critical mediator of ischemia/reperfusion injury. As a cardioprotective strategy, inhibition of mitochondrial fission may be induced through the pharmacological manipulation of mitochondrial morphology. In the present study, the in vivo pharmacological inhibition of the mitochondrial fission protein dynamin-related protein 1 with the small molecule inhibitor mdivi-1 reduced myocardial infarct size in the adult murine heart. The discovery of other novel compounds capable of inhibiting mitochondrial fission proteins such as dynamin-related protein 1 or activating mitochondrial fusion proteins such as mitofusin 1, mitofusin 2 or optic atrophy protein 1 may in the future be used to block mitochondrial fission in the clinical settings of acute ischemia/reperfusion injury to reduce myocardial injury and infarct size in patients undergoing cardiac bypass surgery or patients presenting with a myocardial infarction, respectively, and to improve clinical outcomes in patients with coronary heart disease.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.906610/DC1.