A deficiency of apoptosis inducing factor (AIF) in Harlequin mouse heart mitochondria paradoxically reduces ROS generation during ischemia-reperfusion

Abstract

Background and aims: AIF (apoptosis inducing factor) is a flavin and NADH containing protein located within mitochondria required for optimal function of the respiratory chain. AIF may function as an antioxidant within mitochondria, yet when released from mitochondria it activates caspase-independent cell death. The Harlequin (Hq) mouse has a markedly reduced content of AIF, providing an experimental model to query if the main role of AIF in the exacerbation of cell death is enhanced mitochondrial generation of reactive oxygen species (ROS) or the activation of cell death programs. We asked if the ROS generation is altered in Hq heart mitochondria at baseline or following ischemia-reperfusion (IR).

Methods: Buffer perfused mouse hearts underwent 30 min ischemia and 30 min reperfusion. Mitochondrial function including oxidative phosphorylation and H2O2 generation was measured. Immunoblotting was used to determine the contents of AIF and PAR [poly(ADP-ribose)] in cell fractions.

Results: There were no differences in the release of H2O2 between wild type (WT) and Hq heart mitochondria at baseline. IR increased H2O2 generation from WT but not from Hq mitochondria compared to corresponding time controls. The complex I activity was decreased in WT but not in Hq mice following IR. The relocation of AIF from mitochondria to nucleus was increased in WT but not in Hq mice. IR activated PARP-1 only in WT mice. Cell injury was decreased in the Hq mouse heart following in vitro IR.

Conclusion: A deficiency of AIF within mitochondria does not increase ROS production during IR, indicating that AIF functions less as an antioxidant within mitochondria. The decreased cardiac injury in Hq mouse heart accompanied by less AIF translocation to the nucleus suggests that AIF relocation, rather than the AIF content within mitochondria, contributes to cardiac injury during IR.

Keywords: apoptosis; electron transport chain; poly(ADP-ribose); reactive oxygen species.

Figure 1

Ischemia-reperfusion (IR) increases the net H₂O₂ production in wild type but not in Hq mouse cardiac mitochondria. There were no differences in the net release of H₂O₂ between wild type and Harlequin mouse heart mitochondria from non-ischemic hearts. IR increased the generation of H₂O₂ in wild type but not in Harlequin mice compared to time control (A). The maximal H₂O₂ generation from complex I was measured using rotenone to inhibit complex I. An AIF deficiency in Harlequin mice did not alter the maximal H₂O₂ generation from complex I compared to wild type in control heart (B). The maximal H₂O₂ generation from complex I was also decreased in Harlequin mice following IR compared to wild type (B). Knock down of AIF in Harlequin mice did not alter the H₂O₂ generation using succinate + rotenone as complex II substrates compared to wild type control. The H₂O₂ generation was decreased in Harlequin mice following IR compared to wild type (C). The maximal H₂O₂ generation from complex III in the presence of antimycin A was not decreased in Harlequin mice following IR compared to wild type (D). Data are expressed as mean ± s.e.m.; ^*p < 0.05 vs. time control; ^†p < 0.05 vs. wild type IR.

Figure 2

IR led to decreased cardiac injury in Harlequin mice compared to wild type. Heart rate was maintained at 420 bpm by pacing during 15 min equilibration perfusion. The pacing was stopped during ischemia and resumed at 15 min reperfusion. The recovery of myocardial contractile function during reperfusion, shown by the improvement in left ventricular developed pressure (LVDP mmHg, A), was improved in Harlequin mice compared to wild type. The diastolic function was also improved in Harlequin mice following IR, reflected by a decrease in left ventricular end-diastolic pressure (LVEDP) at the end of reperfusion (B). The cardiac injury during IR was decreased in Harlequin mice as shown by a smaller infarct size (C) and less LDH release into coronary effluent. Data are expressed as mean ± s.e.m.; ^*p < 0.05 vs. time control (TC); ^†p < 0.05 vs. wild type untreated IR.

Figure 3

IR decreased the CRC in wild type and Harlequin cardiac mitochondria. Original tracings of CRC measurement in wild type heart mitochondria were shown in panel (A). The CRC was decreased in control Harlequin mouse heart mitochondria compared to wild type (B), suggesting that the sensitivity to MPTP opening was increased in Harlequin mice in the basal condition. IR led to decreased CRC in both wild type and Harlequin mice compared to time control (B). Compared to wild type, IR led to a slight further decrease in CRC in Harlequin mice (B). Data are expressed as mean ± s.e.m.; ^*p < 0.05 vs. time control; ^†p < 0.05 vs. wild type IR.

Figure 4

IR increases AIF translocation from mitochondria to nucleus in wild type. The content of poly (ADP-ribose) (PAR) was markedly increased in wild type mice following IR compared to time control, indicating that IR activated PARP-1 (A–C). There were no differences in the PAR content in Hq mice between time control and mice following IR (A–C). GAPDH was used as a cytosol marker for loading control. The precursor form of AIF (67 kd) and the mature form of AIF (62 kd) were detected in wild type mouse heart mitochondria (D). Compared to control, IR markedly decreased the content of the mature form of AIF (62 kd band) in wild type mice (D). The mature AIF content in Harlequin mice was of course decreased compared to wild type (D). In wild type, IR increased AIF content (62 kd) in nucleus compared to time control, indicating a translocation of AIF from mitochondria to nucleus (E). Subunit 4 of cytochrome oxidase and lamin were used as protein loading control for mitochondria and nucleus, respectively.

Figure 5

Depiction of the PARP-1 activation and AIF translocation during IR. IR damages the electron transport chain and increases the ROS generation from complex I and complex III and activates mitochondrial μ-calpain (M-μ-Cal) to induce release of AIF from the inner mitochondrial membrane. Translocation of AIF from mitochondria to nucleus enhances DNA damage and PARP-1 activation, in turn further reducing NAD⁺ content. Oxidative stress during IR leads to DNA damage DNA that activates the PARP-1 that leads to decreased NAD⁺ content in cytosol and increased generation of PAR within nucleus. The PAR is released into cytosol and relocates to mitochondria and facilitates AIF release from the outer mitochondrial membrane. The activated PARP-1 may contribute to complex I defect through an indirect mechanism. IR may activate mitochondrial localized PARP-1 to induce direct complex I damage in cardiac mitochondria. CI, complex I; CIII, complex III; M-μ-Cal, mitochondrial μ-calpain.