Bnip3 mediates permeabilization of mitochondria and release of cytochrome c via a novel mechanism

Abstract

Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with loss of cardiac myocytes after a myocardial infarction. Previous studies have demonstrated that Bnip3 induces mitochondrial dysfunction, but the mechanisms involved in this process remain unknown. In this study, we demonstrate that Bnip3 induces permeabilization of the mitochondria via a novel mechanism that is different from other BH3-only proteins. We found that Bnip3 induced mitochondrial swelling and cytochrome c release in isolated heart mitochondria in vitro. Another BH3-only protein, tBid, also caused release of cytochrome c but failed to induce swelling of mitochondria. Swelling of mitochondria is a characteristic of mitochondrial permeability transition pore (mPTP) opening, but Bnip3-mediated mitochondrial swelling was insensitive to cyclosporine A, an inhibitor of the mPTP and independent of cyclophilin D (cypD), an essential component of the mPTP. Bnip3 also induced permeabilization of the mitochondrial membranes as evident by calcein release from the matrix in both wild type (WT) and cypD deficient mouse embryonic fibroblasts (MEFs). Moreover, Bnip3 induced mitochondrial matrix remodeling and large amplitude swelling of the inner membrane, which led to disassembly of OPA1 complexes and release from the mitochondria. Thus, these studies suggest that Bnip3 mediates mitochondrial permeabilization by a novel mechanism that is different from other BH3-only proteins.

Figure 1

Bnip3 induces swelling of isolated heart mitochondria via a different mechanism than Ca²⁺. A. Mitochondria isolated from rat hearts were incubated with recombinant Bnip3 (1 μg) or Ca²⁺ (250 μM) and reduction in absorbance at 520 nm was measured. Swelling assay is representative of three independent experiments. B. The degree of swelling (amplitude) and rate (V_max) of swelling was significantly different between Ca²⁺ and Bnip3. Results are means±S.E.M. (n=3,*p<0.05).

Figure 2

Bnip3-mediated swelling and cytochrome c release are not inhibited by the mPTP inhibitor cyclosporine A. A. Incubation of mitochondria with 1 μM CsA inhibits swelling mediated by 250 μM Ca²⁺. B. CsA has no effect on swelling mediated by Bnip3 (1 μg). C. Bnip3 (1 μg) and Ca²⁺ (250 μM) induce release of cytochrome c from isolated mitochondria. CsA (1 μM) reduces Ca²⁺-mediated cytochrome c release but has no effect on Bnip3-mediated release. Western blot is representative of three independent experiments. D. Isolated mitochondria were treated with 0, 0.1, 0.5, or 1 μg Bnip3 in the presence or absence of 1 μM CsA.

Figure 3

Mitochondrial morphological changes and matrix remodeling in Ca²⁺ and Bnip3-treated mitochondria. Electron microscopy analysis of isolated mitochondria after incubation with (A) buffer, (B) 250 μM Ca²⁺ or (C) 1 μg Bnip3 for 45 min.

Figure 4

Bnip3-mediated swelling induces disruption of OPA1 complex. A. After treatment with buffer, Ca²⁺ (250 μM) or Bnip3 (1 μg) for 30 min, mitochondria were incubated with DMSO or 10 mM BMH for 30 min at 37°C to cross link proteins. Proteins were separated by SDS-PAGE and analyzed by Western blotting for OPA1. B. The presence of PEG prevents Bnip3-mediated disruption of OPA1 complex. C. PEG prevents Bnip3-mediated release of OPA1 from mitochondria. D. PEG partially prevents Bnip3-mediated cytochrome c release. Data shown are representative of three independent experiments.

Figure 5

The BH3-only protein tBid induces cytochrome c release in the absence of mitochondrial swelling. A. Bnip3 (1 μg) and tBid (100 nM), but not Bnip3ΔTM (1 μg), induce release of cytochrome c release. B. Incubation of heart mitochondria with recombinant tBid (100 ng) or Bnip3ΔTM (1 μg) do not induce swelling of mitochondria. C. Incubation of heart mitochondria with recombinant Bcl-2 (1 μg) does not prevent Bnip3-mediated swelling. D. Bcl-2 does not inhibit cytochrome c release by Bnip3. Data shown are representative of three independent experiments.

Figure 6

Heart mitochondria isolated from cypD deficient mice are resistant to Ca²⁺-mediated swelling but not to Bnip3. A. Mitochondrial swelling of cypD deficient mitochondria treated with 0, 0.1, 0.5, or 1 μg Bnip3. B. Cytochrome c release in cypD deficient mitochondria by Bnip3._C. Mitochondria lacking cypD were incubated with 250 μM Ca²⁺ or 1 μg Bnip3 and swelling was assessed by measuring a decrease in absorbance. D. Western blot analysis of cypD deficient mitochondria for release of cytochrome c. E. Swelling of cypD deficient mitochondria was determined by measuring a decrease in absorbance. F. CypD deficient mitochondria were incubated with Bnip3 (1 μg), Bnip3ΔTM (1 μg), or tBid (100 ng) and analyzed for release of cytochrome c by Western blotting. Data shown are representative of three independent experiments.

Figure 7

Bnip3 induces cell death in both wild type (WT) and cypD^-/- MEFs. A. Cells were infected with an adenovirus encoding β-gal or Bnip3 for 24 h. Cell death was determined by (A) assessing release of LDH into the culture media or (B) TUNEL assay. Results are means±S.E.M. (n=3, p<0.001). C. Expression of Bnip3 in WT and cypD-/- MEFs infected with an adenovirus encoding β-gal or Bnip3 for 24 h was determined by Western blot analysis.

Figure 8

Bnip3, but not Ca²⁺, induces release of calcein from mitochondrial matrix in CypD^-/- MEFs. A. Cells infected with β-gal or Bnip3, or treated with 250 μM H₂O₂ were loaded with 1 μM calcein-AM and 5 mM CoCl₂. B. Quantitation of cells with cytosolic calcein. Results are means±S.E.M. (n=3, p<0.05).

Figure 9

H₂O₂, but not Bnip3, promotes release of calcein from mitochondria in Bax/Bak DKO MEFs. A. Cells infected with β-gal or Bnip3, or treated with 250 μM H₂O₂ were loaded with 1 μM calcein-AM and 5 mM CoCl₂. B. Quantitation of cells with cytosolic calcein. Results are means±S.E.M. (n=3, p<0.05).