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.

Background: Mitochondrial protein O-GlcNAcylation is not well understood.Results: Eighty eight mitochondrial proteins, involved in diverse pathways, are O-GlcNAcylated, and an overall increased O-GlcNAcylation leads to altered mitochondrial function. Conclusion: O-GlcNAcylation is on many mitochondrial proteins within the oxidative phosphorylation system, modulating cardiac mitochondrial function. Significance: O-GlcNAc cycles on many proteins within mitochondria, leading to altered function.

Summary Mitochondrial volume regulation depends on K+ movement across the inner membrane and a mitochondrial Ca2+-dependent K+ channel (mitoKCa) reportedly contributes to mitochondrial K+ uniporter activity. Here we utilize a novel KCa channel activator, NS11021, to examine the role of mitoKCa in regulating mitochondrial function by measuring K+ flux, membrane potential (ΔΨm), light scattering, and respiration in guinea-pig heart mitochondria. K+ uptake and the influence of anions were assessed in mitochondria loaded with the K+ sensor PBFI by adding either the chloride (KCl), acetate (KAc) or phosphate (KH2PO4) salts of K+ to energized mitochondria in a sucrose-based medium. K+ fluxes saturated at ~10mM for each salt, attaining maximal rates of 172±17, 54±2.4 and 33±3.8 nmol K+/min/mg in KCl, KAc or KH2PO4, respectively. NS11021 (50nM) increased the maximal K+ uptake rate by 2.5- fold in the presence of KH2PO4 or KAc, and increased mitochondrial volume, with little effect on ΔΨm. In KCl, NS11021 increased K+ uptake by only 30% and did not increase volume. The effects of NS11021 on K+ uptake were inhibited by the KCa toxins charybdotoxin (200nM) or paxilline (1μM). 50nM NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH2PO4, but not in KCl; however, above 1μM, NS11021 decreased RCR and depolarized ΔΨm. A control compound lacking KCa activator properties did not increase K+ uptake or volume, but had similar nonspecific (toxin insensitive) effects at high concentrations. The results indicate that activating K+ flux through mitoKCa mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained ΔΨm.