Bax regulates primary necrosis through mitochondrial dynamics

Abstract

The defining event in apoptosis is mitochondrial outer membrane permeabilization (MOMP), allowing apoptogen release. In contrast, the triggering event in primary necrosis is early opening of the inner membrane mitochondrial permeability transition pore (mPTP), precipitating mitochondrial dysfunction and cessation of ATP synthesis. Bcl-2 proteins Bax and Bak are the principal activators of MOMP and apoptosis. Unexpectedly, we find that deletion of Bax and Bak dramatically reduces necrotic injury during myocardial infarction in vivo. Triple knockout mice lacking Bax/Bak and cyclophilin D, a key regulator of necrosis, fail to show further reduction in infarct size over those deficient in Bax/Bak. Absence of Bax/Bak renders cells resistant to mPTP opening and necrosis, effects confirmed in isolated mitochondria. Reconstitution of these cells or mitochondria with wild-type Bax, or an oligomerization-deficient mutant that cannot support MOMP and apoptosis, restores mPTP opening and necrosis, implicating distinct mechanisms for Bax-regulated necrosis and apoptosis. Both forms of Bax restore mitochondrial fusion in Bax/Bak-null cells, which otherwise exhibit fragmented mitochondria. Cells lacking mitofusin 2 (Mfn2), which exhibit similar fusion defects, are protected to the same extent as Bax/Bak-null cells. Conversely, restoration of fused mitochondria through inhibition of fission potentiates mPTP opening in the absence of Bax/Bak or Mfn2, indicating that the fused state itself is critical. These data demonstrate that Bax-driven fusion lowers the threshold for mPTP opening and necrosis. Thus, Bax and Bak play wider roles in cell death than previously appreciated and may be optimal therapeutic targets for diseases that involve both forms of cell death.

Fig. 1.

Deletion of Bax and Bak markedly reduces necrotic injury during myocardial infarction in vivo. (A) Infarct size following 45 min of left coronary artery occlusion followed by 24 h of reperfusion (I/R). AAR/LV, area at risk/left ventricle; INF/AAR, infarct size normalized to AAR; (Left graph) WT, wild-type mice; DKO, double knockout mice lacking Bax and Bak; TKO, triple knockout mice lacking Bax, Bak, and cyclophilin D. (Right graph) WT, wild-type mice; Ppif KO, mice lacking cyclophilin D. Numbers of animals indicated in circles. Confirmation of knockouts in Fig. S1. (B) Apoptosis assessed within the AAR of heart sections from mice subjected to sham operation or 45 min ischemia/10 h reperfusion using TUNEL and costaining with troponin I to identify cardiac myocytes and DAPI. (C) Transmission electron microscopy of infarct zone and remote myocardium following 45 min ischemia/24 h reperfusion. Key features of myocardial necrosis including amorphous mitochondrial densities (red arrow), poorly defined cristae (black arrow), mitochondria swelling, and rupture, sarcomeric disorganization (on lower power images). Representative of at least 10 randomly selected fields for each genotype. Data mean ± SEM. ***P < 0.001, *P < 0.05, compared with WT. ^†No significant difference compared with DKO.

Fig. 2.

Absence of Bax and Bak inhibits mPTP opening and necrosis. (A) mPTP opening in WT and Bax/Bak DKO MEFs treated with ionomycin (Iono) (10 μM) or staurosporine (STS) (2 μM) as assessed by loss of Δψ_m using flow cytometry of live cells stained with tetramethyl rhodamine ethyl ester (TMRE). ***P < 0.001 compared with zero time point. (B) Cytochrome c (cyt c) assessed by immunoblot of the cytosolic fraction of WT and Bax/Bak DKO MEFs following stimulation with STS (2 μM) or Iono (10 μM). GAPDH (cytosolic) and complex Vα (CVα) (inner mitochondrial membrane) markers. (C) LDH release by enzymatic assay of the media of WT and Bax/Bak DKO MEFs following stimulation with STS (2 μM) or Iono (10 μM). **P < 0.01 compared with zero time point. (D) Isolated cardiac mitochondria loaded by repetitive additions of CaCl₂ (35 μM first addition, 25 μM subsequent additions) to the cuvettete as shown by green spikes. Dotted lines, WT; solid lines, Bax/Bak DKO; green, Ca Green; red, Δψ_m; gray, mitochondrial swelling. Δψ_m lost in WT during Ca²⁺ load 5 and in DKO after load 9. n ≥ 3 independent experiments for each panel.

Fig. 3.

Reconstitution of Bax/Bak-null MEFs or mitochondria with wild type or oligomerization-deficient Bax restores mPTP opening. (A) Size fractionation of cellular lysates from wild-type MEFs not treated or treated for 8 h with staurosporine (1 μM) or ionomycin (10 μM) [all conditions in the presence of z-VADfmk (50 μM)] followed by immunoblotting of fractions for endogenous Bax. Representative of two independent experiments. (B, Left) Reconstitution of Bax/Bak-null MEFs by transfection with GFP, GFP-Bax (WT), or GFP-Bax(63-65)A in the presence of z-VADfmk (40 μM). Flow cytometric analysis of Δψ_m loss in transfected cells gated for GFP. (Right) Flow cytometric analysis of GFP intensities indicate similar levels of expression of GFP-Bax (WT) and GFP-Bax(63-65)A. ***P < 0.001, compared with no ionomycin. (C) Measurement of Ca²⁺ load required for Δψ_m loss and swelling following reconstitution of Bax/Bak DKO isolated cardiac mitochondria with 100 nM recombinant WT Bax or Bax(63-65)A. *P < 0.05, compared with no treatment. n ≥ 3 independent experiments.

Fig. 4.

Cells with fragmented mitochondria are resistant to mPTP opening, which can be reversed by restoration of fused morphology. (A) Analysis of mitochondrial morphology by confocal microscopy of WT, Bax/Bak DKO, and Mfn2 KO MEFs not treated or treated for 6 h with Mdivi-1 (50 μM). Cells stained with MitoTracker Red and DAPI. Quantification of fused mitochondrial morphology by flow cytometry of mitochondria. FSC-A, forward scatter area. (B) Ionomycin-induced mPTP opening in the same groups except ionomycin (10 μM) added 2 h before analysis. n = 3 independent experiments. ***P < 0.001, compared with wild-type cells that were treated with ionomycin but not Mdivi-1. ^§§§P < 0.001 compared with cells of the same genotype that were treated with ionomycin but not Mdivi-1.