Wild type or Hq mice [2–3 months of age (22–28 g), male] were anesthetized with pentobarbital sodium (100 mg/g i.p.) and anti-coagulated with heparin (1 IU/g i.p.) (Chen et al., 2011). In this study, only male mice were used because Hq mice had a gender-dependent response in an experimental stroke model (Yuan et al., 2009). Compared to male Hq mice, female Hq mice exhibited markedly decreased brain injury after experimental stroke (Yuan et al., 2009). Since the AIF gene is located on the X chromosome, using male mice will avoid gender-dependent gene dosage effects on cardioprotection. Hearts were excised and retrograde perfused via the aorta in the Langendorff mode (constant pressure, 75 mmHg) with modified Krebs-Henseleit buffer (composition, in mM: 115 NaCl, 4.0 KCl, 2.0 CaCl₂, 25 NaHCO₃, 1.1 MgSO₄· H₂O, 0.9 KH₂PO₄, and 5.5 glucose) oxygenated with 95% O₂+5% CO₂. Cardiac function was monitored with a balloon inserted into the left ventricle using Powerlab (AD Instruments, Colorado Springs, CO). Heart rate was maintained at 420 bpm with pacing during the equilibration period. Pacing was stopped during global ischemia and restored at 15 min reperfusion. In the IR group, hearts were buffer-perfused for 15 min, followed by 30 min global ischemia at 37°C within 30 min reperfusion (mitochondrial isolation) or 1 h reperfusion (infarction measurement). In the time control group, hearts were buffer-perfused without IR. Myocardial infarct size was determined using TTC staining (Chen et al., 2006). Coronary effluent was collected to determine LDH activity in each group (Chen et al., 2006).

Trypsin was used in the mitochondrial isolation protocol in order to remove potential contamination from AIF located in the cytosol (Chen et al., 2011). The mouse heart was harvested and immediately placed in cold buffer A [100 mM KCl, 50 mM 3-(N-morpholino) propanesulfonic acid (MOPS), 1 mM EGTA, 5 mM MgSO₄, 1 mM ATP]. Cardiac tissue was homogenized with a polytron at 10,000 rpm and incubated with trypsin (5 mg/g) for 15 min. Cold buffer B (0.2% BSA + buffer A) was then added into the homogenate. The homogenate was centrifuged at 500 × g for 10 min. The supernatant was centrifuged at 3000 × g for 10 min. to pellet mitochondria. The mitochondrial pellet was washed and suspended in 100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA.

The polytron pellet was used to isolate nuclear components using a commercial kit from Thermo Scientific (Pittsburgh, PA, catalog # 78835) using supplied solutions and differential centrifugation according to instructions provided.

Oxygen consumption by mitochondria was measured using a Clark-type oxygen electrode at 30°C using glutamate + malate (complex I substrate) or succinate + rotenone (complex II substrate) as donors (Lesnefsky et al., 1997). Respiratory enzyme activities [NADH-decylubiquinol oxidoreductase, rotenone sensitive (complex I)]; NADH ferricyanide oxioreductase (NFR, flavoprotein portion of complex I); Succinate-decylubiquinone oxidoreductase (complex II); and citrate synthase were measured in detergent solubilized mitochondria according to the method of Dr. Hoppel as previously described (Krahenbuhl et al., 1991; Lesnefsky et al., 1997; Chen et al., 2008).

H₂O₂ production by isolated mitochondria was measured using the oxidation of the fluorogenic indicator Amplex red in the presence of horseradish peroxidase without exogenous SOD (Chen et al., 2003). Glutamate + malate and succinate + rotenone were used as complex I and complex II substrates, respectively. Rotenone and antimycin A were used to detect the maximal H₂O₂ generation from complex I and complex III, respectively.

Mitochondrial calcium retention capacity (CRC) was used to reflect opening of the mitochondrial permeability transition pore (MPTP) in isolated mitochondria (Chen et al., 2012a). CRC was studied in the single cell fluorimeter (PerkinElmer, Waltham, Massachusetts) using repetitive calcium pulses (Chen et al., 2012a). Freshly isolated mitochondria (0.25 mg) were incubated in buffer (150 mM sucrose, 50 mM KCl, 2 mM KPi, and 20 mM Tris/HCl, pH 7.4) for 90 s with stirring at 30°C with 0.5 μM calcium green. Succinate (5 mM) was used as substrate. Pulses of calcium (5 nmoles) were added at 60 s intervals. The number of pulses that resulted in calcium release indicated the onset of MPTP.

Particle free cytosol, purified mitochondria, and nucleus were boiled for 5 min. in buffer including 4% (w/v) SDS, 1 mM 2-mercaptoethanol, 10 mM Tris/HCl (pH 6.8) and 10% (w/v) glycerol. Equal amounts of protein were loaded onto 4–15% or 4–20% SDS-PAGE (dependent on molecular weight of proteins), electrophoresed and transferred to a PVDF membrane. The membranes were first blocked by 5% non-fat milk for 1 h followed by exposure to primary antibodies overnight (Chen et al., 2011). Antibodies to AIF, PARP-1, GAPDH, lamin, and subunit IV of cytochrome oxidase were purchased from Cell Signaling Technology (Danvers, MA). Monoclonal PAR antibody was purchased from Millipore (Billerica, MA). The blots were incubated with peroxidase conjugated anti-rabbit or anti-mouse secondary antibody for 1 h prior to ECL detection (GE Healthcare Life Science, Pittsburgh, PA). The intensity of blotting was quantified by Fuji Film Image station (Edison, NJ).

Data were expressed as the mean ± standard error of the mean. Differences among four groups were compared by one-way analysis of variance with post-hoc comparisons performed using the Student-Newman-Keuls test of multiple comparisons (Sigmastat 3.5, ProgramPaketet, Gothenburg, Sweden). Differences in CRC and H₂O₂ between wild type and Hq mice were compared by unpaired student t-test. A difference of p < 0.05 was considered significant.

There were no differences in the rate of oxidative phosphorylation between time control wild type and Hq mitochondria when glutamate + malate (complex I) or succinate + rotenone (complex II) were used as substrates. IR decreased the ADP-stimulated respiration in mitochondria from both wild type and Hq using either substrate (Table 1). The rate of dinitrophenol (DNP) uncoupled respiration was also decreased in both wild type and Hq mice following IR (Table 1), supporting that IR damages the electron transport chain. Interestingly, the rate of oxidative phosphorylation with glutamate + malate in Hq hearts was decreased following IR compared to corresponding wild type (Table 1), whereas the rate of succinate oxidation was similar in Hq and wild type hearts following IR (Table 1). These results indicated that IR led to additional decreases in respiration in Hq mouse heart mitochondria when NADH-dependent substrates were used.

In order to test if IR led to further damage to complex I; NADH:decylubiquinol oxidoredutase, NFR, and complex II activities were measured. Complex I activity [shown as the ratio of complex I/CS (citrate synthase), Table 2] was decreased in wild type following IR compared to time control. However, complex I activity was not decreased in Hq mitochondria following IR compared to its corresponding time control. The NADH dehydrogenase activity (NFR) was not decreased in either wild type or Hq mice following IR (Table 2), consistent with previous study (Chen et al., 2007; Szczepanek et al., 2011). NADH dehydrogenase activity was slightly higher in Hq mouse heart mitochondria following IR compared to corresponding wild type (Table 2). The physiological significance of this subtle difference is unclear. IR did not alter complex II activity in wild type or Hq mice (Table 2). These results support that IR leads to a complex I defect in wild type mouse heart mitochondria.

There were no differences in H₂O₂ generation between time control wild type and Hq heart mitochondria using complex I (Figure 1A) or complex II substrates (Figure 1C). IR markedly increased the production of H₂O₂ in wild type but not in Hq mouse heart mitochondria with either a complex I or complex II substrate (Figures 1A,C). Compared to time control, inhibition of complex I using rotenone dramatically increased H₂O₂ generation in wild type mouse heart following IR (Figure 1B). In contrast, rotenone inhibition did not increase the H₂O₂ generation in Hq mouse heart following IR (Figure 1B). The maximal ROS generation from mitochondria was induced with antimycin A inhibition. Inhibition of complex III using antimycin A increased the H₂O₂ generation in both wild type and Hq mouse heart following IR vs. time control (Figure 1D). However, there were no differences in ROS generation between wild type and Hq mice with or without IR.

In the buffer perfused hearts, myocardial injury was decreased in the Hq mouse heart following IR compared to wild type. Knock down of AIF content in Hq mouse heart did not affect the cardiac function before ischemia (Figure 2A). IR decreased left ventricular developed pressure (LVDP) in both wild type and Hq hearts vs. time control. Systolic function was improved in Hq hearts vs. wild type during reperfusion (Figure 3A). Strikingly, the infarct size was also much smaller in Hq mice than in wild type (Figure 2B). The release of LDH into coronary effluent was much lower in Hq mice than in wild type (Figure 2C), also indicating less necrosis.

The CRC (Figure 3A) was decreased in mitochondria from non-ischemic Hq mice compared to wild type (Figure 3B), suggesting that a decrease in AIF content within mitochondria sensitizes to calcium-stimulated MPTP opening. Although there was a slight difference in the CRC between Hq and wild type following IR (Figures 3A,B), the small magnitude of this difference may not exert a significant impact on cardiac injury during IR.

Activation of PARP-1 during IR increases the generation of PAR [Poly (ADP-ribose) (PAR)] that is transferred to cytosol and mitochondria (Sevrioukova, 2011). The content of PAR was markedly increased in wild type following IR compared to time control (Figures 4A–C), indicating that IR leads to PARP-1 activation. In contrast, IR did not alter the PAR content in Hq mice following IR (Figures 4A–C).

The precursor of AIF (67 kd) is nuclear-encoded and subsequently transported into the mitochondrial matrix via its mitochondrial targeting sequence (Sevrioukova, 2011). The mature form of AIF (62 kd) is formed in the matrix through cleavage of precursor protein via a mitochondrial matrix peptidase (Sevrioukova, 2011). The mature AIF (62 kd) is transferred into the mitochondrial intermembrane space through the Tim23 protein (Sevrioukova, 2011). Consistent with these concepts, two AIF bands are detected in non-ischemic wild type mouse heart mitochondria (Figure 4D). In contrast, the mature form of AIF (62 kd) is almost undetectable in Hq mouse heart mitochondria (Figure 4D), confirming the lower content of AIF within mitochondria. The content of AIF within mitochondria was decreased in wild type mice following IR compared to time control (Figure 4D). The AIF content (62 kd) in nucleus was increased in wild type hearts following IR (Figure 4E).

Complex I activity is decreased in heart mitochondria following both in vivo (Rouslin and Millard, 1980; Rouslin, 1983) and in vitro IR (Lesnefsky et al., 2001; Gustafsson and Gottlieb, 2008; Murphy and Steenbergen, 2008). In the present study, IR leads to decreased complex I activity without alteration in NADH dehydrogenase activity (NFR). These results indicate that ischemia likely damages complex I at the iron sulfur centers (Chen et al., 2008) distal to the flavoprotein, in line with previous studies (Ohnishi and Trumpower, 1980; Chen et al., 2006; Zhou et al., 2006; Szczepanek et al., 2011).

Oxidative modification of complex I by nitrosation (Burwell et al., 2006) or glutathionylation (Hurd et al., 2008) or the modification of its inner membrane environment via depletion of cardiolipin (Paradies et al., 2004) all contribute to decreases in activity. Mitochondrial AIF content also affects complex I activity, especially in brain and retina (Klein et al., 2002; van Empel et al., 2005). Depletion of AIF also decreases complex I activity in heart mitochondria (Pospisilik et al., 2007). However, the effect of lower expression of AIF in Hq mice on heart mitochondrial complex I activity is not consistent (Szczepanek et al., 2013). Thus, an AIF deficiency may affect complex I activity in a tissue-dependent manner. Genetic depletion of PARP-1 protects complex I activity in mouse heart following IR (Zhou et al., 2006), indicating that PARP-1 activation contributes to the complex I defect during IR. In the present study, IR decreases complex I activity accompanied by an activated PARP-1 in wild type mice. In contrast, IR does not decrease complex I activity in Hq mice. PARP-1 is also not activated in Hq mice following IR. These results support that activation of PARP-1 contributes to the complex I defect during IR. Since PARP-1 is considered as a nuclear protein, complex I inhibition by PARP-1 activation appears to present a challenge (Figure 5) (Zhou et al., 2006). Recently, a mitochondrial localized PARP-1 has been identified (Rossi et al., 2009). Thus, activation of PARP-1 may directly regulate complex I activity (Figure 5).

The ischemia-damaged respiratory chain including complex I, is a key source of ROS that increases cardiac injury (Turrens, 2003; Chen et al., 2007). Blockade of proximal electron transport using amobarbital before ischemia protects complex I and decreases cardiac injury during reperfusion, supporting that preservation of complex I activity reduces cardiac injury (Chen et al., 2006). Transient partial (Xu et al., 2014) or complete (Stewart et al., 2009; Chen et al., 2012b) blockade of complex I at the onset of reperfusion decreases myocardial injury in buffer perfused hearts, indicating that a temporary complex I inhibition is also beneficial for cardiac recovery. However, persistent, severe complex I inhibition is detrimental to the heart both at baseline and for recovery during reperfusion (Karamanlidis et al., 2013). In contrast to wild type, the complex I activity is not altered in Hq mice following IR. The lack of damage to complex I during IR in Hq mice may lead to decreased ROS generation that contributes to the observed decrease in cardiac injury in Hq mice. Alteration of mitochondrial antioxidants including thioredoxin reductase-2 significantly affects a release of H₂O₂ from mitochondria. The IR-induced complex I damage may also increase H₂O₂ by inhibiting thioredoxin reductase-2 through alteration of its redox state (Rigobello et al., 2006; Horstkotte et al., 2011; Stanley et al., 2011). The increased oxidative stress will favor activated μ-calpain to cleave AIF and facilitate its release from mitochondria (Norberg et al., 2010).

The mature form of AIF (62 kd) is anchored at the inner mitochondrial membrane within the intermembrane space (Otera et al., 2005). Release of AIF from the mitochondria and translocation to the nucleus to activate caspase-independent cell death is a multistep process. First, the mature AIF bound within mitochondria on the inner membrane requires liberation. Cleavage of the mature 62 kd form of AIF by activated mitochondrial calpains (Ozaki et al., 2007), t-bid (Cabon et al., 2012) or other proteases can liberate AIF from the inner membrane, with release of a truncated, approximately 57 kd AIF peptide. Next, permeation of the outer mitochondrial membrane is required for AIF release (Ozaki et al., 2007). IR increases MPTP opening as a mechanism of increased outer membrane permeability (Weiss et al., 2003), with oxidative stress is a key contributor to the increased susceptibility to MPTP opening during IR (Weiss et al., 2003; Halestrap et al., 2004; Chen et al., 2012a). In contrast, activation of PARP-1 is not involved in the permeation of the outer mitochondrial membrane during IR (Schriewer et al., 2013). The decreased AIF content in the purified mitochondria following IR supports that IR does lead to a loss of AIF from mitochondria. The increased MPTP opening in wild type mice during reperfusion favors a release of AIF from mitochondria into cytosol with subsequent translocation to the nucleus. It currently appears that even following MPTP, the released AIF is the 57 kd cleaved form, at least based upon calcium activation of mitochondrial calpains concomitant with MPTP. This area of calcium mediated injury deserves further consideration. As discussed above, IR activates PARP-1 in wild type but not Hq mice (Pacher and Szabo, 2007). Although activation of PARP-1 provides a beneficial effect to repair DNA damage, over activation of PARP-1 has a detrimental effect via consumption of NAD⁺ (Pacher and Szabo, 2007). PAR, which is generated by activation of PARP-1 within nucleus, is released into cytosol and subsequently relocates to mitochondria to induce AIF release from mitochondria (Pacher and Szabo, 2007). Interestingly, a portion of the mature AIF is also reported to be loosely attached on the mitochondrial outer membrane (Yu et al., 2006, 2009). The PAR can detach the AIF from the outer membrane (Figure 5) (Wang et al., 2009). Outer membrane bound-AIF has been identified in mouse heart mitochondria (Chen and Lesnefsky, unpublished data). Thus, activation of PARP-1 may increase AIF translocation to the nucleus through detachment of mature AIF from the outer membrane, in addition to release of the pool from the inner membrane via cleavage. The accumulation of AIF in the nucleus accompanied by increased cardiac injury in wild type mice following IR supports the proposal that the translocation of AIF from the mitochondria to the nucleus augments cardiac injury.

Although cardiac injury is increased in Hq mouse heart following in vivo IR compared to wild type controls (van Empel et al., 2005), cardiac injury is actually decreased in Hq mouse heart following in vitro IR. Several key differences likely contribute to these divergent results. In the present study, only male Hq mice were used based upon the rationale discussed in Methods, whereas both female and male Hq mice were included in the previous in vivo study (van Empel et al., 2005). This is important, since gender-related cardiac protection as well as gene dosage issues related to the X chromosome location of the aif gene can introduce variability. In the present study, only 2–3-month-old mice were used whereas middle-aged and elderly mice were used in the in vivo study (van Empel et al., 2005). In vivo, there is the additional impact of exogenous inflammatory cells (with or without AIF deficiency). Furthermore, in vivo, substrate utilization is uncontrolled. The metabolism of fatty acids in vivo, in contrast to glucose utilization in the current study, may have exacerbated the phenotype of mitochondrial defects present. Taken together, gender, age, exogenous cells and the different IR models likely resulted in the differences observed in our current study compared to the previous in vivo study. An isolated heart was used in order to focus on myocyte specific responses in the present study.

In summary, the key contribution of AIF to cardiac injury during IR is related to release from mitochondria and activation of programmed cell death via cytosolic transport, nuclear import and DNA cleavage. The findings in cardiac mitochondria from Hq mice compared to littermate controls support that a decreased content of AIF does not enhance ROS production from mitochondria nor augment cardiac injury at baseline nor during IR. Thus, AIF does not exert significant mitochondrial antioxidant protection during IR. The prevention of AIF translocation to nucleus is a potentially powerful approach to reduce cardiac injury.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.