Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca²⁺ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca²⁺ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition, and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes, and that Bnip3 triggers induction of autophagy independent of Ca²⁺, ROS generation, and mPTP opening.     

Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca²⁺ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca²⁺ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes and that Bnip3 triggers induction of autophagy independent of Ca²⁺, ROS generation and mPTP opening.Key words: Bnip3, autophagy, cardiac myocytes, mitochondria, permeability transition pore, cyclophilin D     

Physiological roles of the mitochondrial permeability transition pore

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca²⁺ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F₁F_O ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.     

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca⁺⁺ and inhibited by Mg⁺⁺, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol. 258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.Abbreviations used: PT: permeability transition; PTP: permeability transition pore; MMC: mitochondrial megachannel; IMAC: inner membrane anion channel. P_A: permeability of ion A. CSP: Cyclosporin A.     

Electrophysiology clarifies the megariddles of the mitochondrial permeability transition pore

After a brief review of the early history of mitochondrial electrophysiology, the contribution of this approach to the study of the mitochondrial permeability transition (MPT) is recapitulated. It has for example provided evidence for a dimeric nature of the MPT pore, allowed the distinction between two levels of control of its activity, and underscored the relevance of redox events for the phenomenon. Single-channel recording provides a means to finally solve the riddle of the biochemical entity underlying it by comparing the characteristics of the pore with those of channels formed by candidate molecules or complexes. The possibility that this entity may be the protein import machinery of the inner mitochondrial membrane is emphasized.     

Influence of Tl+ on mitochondrial permeability transition pore in Ca2+-loaded rat liver mitochondria

The Tl⁺-induced opening of the MPTP in Ca²⁺-loaded rat liver mitochondria energized by respiration on the substrates succinate or glutamate plus malate was recorded as increased swelling and dissipation of mitochondrial membrane potential as well as decreased state 4, or state 3, or 2,4-dinitrophenol-stimulated respiration. These effects of Tl⁺ increased in nitrate media containing monovalent cations in the order of Li⁺ < NH₄⁺ ≤ Na⁺ < K⁺. They were potentiated by inorganic phosphate and diminished by the MPTP inhibitors (ADP, CsA, Mg²⁺, Li⁺, rotenone, EGTA, and ruthenium red) both individually and more potently in their combinations. Maximal swelling of both non-energized and energized Ca²⁺-loaded mitochondria in rotenone-free media is an indication of Ca²⁺ uptake driven by respiration on mitochondrial endogenous substrates. It is suggested that Tl⁺ (distinct from Cd²⁺, Hg²⁺, and other heavy metals and regardless of the used respiratory substrates) can stimulate opening of the MPTP only in the presence of Ca²⁺. We discuss the possible participation of Ca²⁺-binding sites, located near the respiratory complex I and the adenine nucleotide translocase, in inducing opening of the MPTP.     

Interaction of mitochondrial potassium channels with the permeability transition pore

Three types of potassium channels cooperate with the permeability transition pore (PTP) in the inner mitochondrial membranes of various tissues, mtK_(ATP), mtBK, and mtKv1.3. While the latter two share similarities with their plasma membrane counterparts, mtK_(ATP) exhibits considerable differences with the plasma membrane K_(ATP)-channel. One important function seems to be suppression of release of proapototic substances from mitochondria through the PTP. Open potassium channels tend to keep the PTP closed thus acting as antiapoptotic. Nevertheless, in their mode of action there are considerable differences among them. This review introduces three K⁺-channels and the PTP, and discusses known facts about their interaction.     

Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore

Mitochondria isolated from engineered mice lacking Cyclophilin D (CypD), a component of the Permeability Transition Pore (PTP) complex, can still undergo a Ca2+ -dependent but Cyclosporin A-insensitive permeabilization of the inner membrane. Higher Ca2+ concentrations are required than for wild-type controls. The characteristics of the pore formed in this system were not known, and it has been proposed that they might differ substantially from those of the normal PTP. To test this hypothesis, we have characterized the PTP of isogenic wild-type and CypD- mouse liver mitochondria in patch clamp experiments, which allow biophysical characterization. The pores observed in the two cases, very similar to those of rat liver mitochondria, are indistinguishable according to a number of criteria. The only clear difference is in their sensitivity to Cyclosporin A. CypD is thus shown to be an auxiliary, modulatory component of the "standard" PTP, which forms and has essentially the same properties even in its absence. The observations suggest that Ca2+, CypD, and presumably other inducers and inhibitors act at the level of an activation or assembly process. Activation is separate and upstream of the gating observable on a short or medium-term time scale. Once the pore is activated, its molecular dynamics and biophysical properties may thus be predicted not to depend on the details of the induction process.     

Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore

Mitochondria isolated from engineered mice lacking Cyclophilin D (CypD), a component of the Permeability Transition Pore (PTP) complex, can still undergo a Ca^2?+?-dependent but Cyclosporin A-insensitive permeabilization of the inner membrane. Higher Ca^2?+? concentrations are required than for wild-type controls. The characteristics of the pore formed in this system were not known, and it has been proposed that they might differ substantially from those of the normal PTP. To test this hypothesis, we have characterized the PTP of isogenic wild-type and CypD^? mouse liver mitochondria in patch clamp experiments, which allow biophysical characterization. The pores observed in the two cases, very similar to those of rat liver mitochondria, are indistinguishable according to a number of criteria. The only clear difference is in their sensitivity to Cyclosporin A. CypD is thus shown to be an auxiliary, modulatory component of the “standard” PTP, which forms and has essentially the same properties even in its absence. The observations suggest that Ca^2?+?, CypD, and presumably other inducers and inhibitors act at the level of an activation or assembly process. Activation is separate and upstream of the gating observable on a short or medium-term time scale. Once the pore is activated, its molecular dynamics and biophysical properties may thus be predicted not to depend on the details of the induction process.     

Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane

The mitochondrial permeability transition pore allows solutes with a m.w. 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential () and pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to . Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.     

Acute stress delays brain mitochondrial permeability transition pore opening

Since emotional stress elicits brain activation, mitochondria should be a key component of stressed brain response. However, few studies have focused on mitochondria functioning in these conditions. In this work, we aimed to determine the effects of an acute restraint stress on rat brain mitochondrial functions, with a focus on permeability transition pore (PTP) functioning. Rats were divided into two groups, submitted or not to an acute 30‐min restraint stress (Stress, S‐group, vs. Control, C‐group). Brain was removed immediately after stress. Mitochondrial respiration and enzymatic activities (complex I, complex II, hexokinase) were measured. Changes in PTP opening were assessed by the Ca²⁺ retention capacity. Cell signaling pathways relevant to the coupling between mitochondria and cell function (adenosine monophosphate‐activated protein kinase, phosphatidylinositol 3‐kinase, glycogen synthase kinase 3 beta, MAPK, and cGMP/NO) were measured. The effect of glucocorticoids was also assessed in vitro. Stress delayed (43%) the opening of PTP and resulted in a mild inhibition of complex I respiratory chain. This inhibition was associated with significant stress‐induced changes in adenosine monophosphate‐activated protein kinase signaling pathway without changes in brain cGMP level. In contrast, glucocorticoids did not modify PTP opening. These data suggest a rapid adaptive mechanism of brain mitochondria in stressed conditions, with a special focus on PTP regulation.

     

Sphingosine impairs mitochondrial function by opening permeability transition pore

Growing evidence suggest that, in the heart, sphingosine participates to contractile dysfunction by altering calcium transients and mitochondria function. However, mechanisms underlying sphingosine-induced cardiac mitochondria dysfunction are poorly understood. Here, we studied the effects of sphingosine on isolated cardiac mitochondria of either wild-type or Bcl-2 overexpressing transgenic mice. Sphingosine induced reductions in ADP-coupled respiration, membrane potential, mitochondrial cytochrome c content and ATP production, which were partially prevented by cyclosporine A and mitochondrial Bcl-2 overexpression. These data suggest that sphingosine promotes mitochondrial permeability transition pore opening, which may result in uncoupled respiration and participate in cardiac contractile dysfunction.     

Effects of curcumin and curcumin derivatives on mitochondrial permeability transition pore

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a natural compound with antiproliferative properties. Recent studies suggest that these properties might be due to the ability of curcumin to induce apoptosis in tumor cells by increasing the permeability of the mitochondrial membrane. In the present study, we confirm these observations and provide a molecular mechanism for the action of curcumin in rat liver mitochondria. Curcumin induced mitochondrial swelling, the collapse of Deltapsi, and the release of cytochrome C, events associated with the opening of the permeability transition pore (PTP). Experiments were performed with chemically substituted curcumin derivatives. Some derivatives were obtained by modification of groups on the terminal aromatic rings, and others were obtained by substitution of the diketone function with the cyclohexanone function. They demonstrated that phenol and methoxy groups were essential to promote PTP opening. Curcumin and curcumin derivatives that open the PTP were able to oxidize thiol groups. In addition, PTP opening was abolished in medium devoid of O2 and decreased in the presence of catalase, ferrozine, o-phenanthroline, mannitol, or N-ethylmaleimide. These data suggest that the mechanism by which curcumin promotes PTP opening involves the reduction of Fe3+ to Fe2+, inducing hydroxyl radical (HO*) production and oxidation of thiol groups in the membrane, leading to pore opening.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2 transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2 transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mClCR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mClCR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mClCR and PTP opening. mClCR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2 transport and PTP opening are largely dependent on electron transport and energy coupling.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2+ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2+ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2+ transport and PTP opening are largely dependent on electron transport and energy coupling.     

Silencing of cardiac mitochondrial NHE1 prevents mitochondrial permeability transition pore opening

Inhibition of Na(+)/H(+) exchanger 1 (NHE1) reduces cardiac ischemia-reperfusion (I/R) injury and also cardiac hypertrophy and failure. Although the mechanisms underlying these NHE1-mediated effects suggest delay of mitochondrial permeability transition pore (MPTP) opening, and reduction of mitochondrial-derived superoxide production, the possibility of NHE1 blockade targeting mitochondria has been incompletely explored. A short-hairpin RNA sequence mediating specific knock down of NHE1 expression was incorporated into a lentiviral vector (shRNA-NHE1) and transduced in the rat myocardium. NHE1 expression of mitochondrial lysates revealed that shRNA-NHE1 transductions reduced mitochondrial NHE1 (mNHE1) by ～60%, supporting the expression of NHE1 in mitochondria membranes. Electron microscopy studies corroborate the presence of NHE1 in heart mitochondria. Immunostaining of rat cardiomyocytes also suggests colocalization of NHE1 with the mitochondrial marker cytochrome c oxidase. To examine the functional role of mNHE1, mitochondrial suspensions were exposed to increasing concentrations of CaCl(2) to induce MPTP opening and consequently mitochondrial swelling. shRNA-NHE1 transduction reduced CaCl(2)-induced mitochondrial swelling by 64 ± 4%. Whereas the NHE1 inhibitor HOE-642 (10 μM) decreased mitochondrial Ca(2+)-induced swelling in rats transduced with nonsilencing RNAi (37 ± 6%), no additional HOE-642 effects were detected in mitochondria from rats transduced with shRNA-NHE1. We have characterized the expression and function of NHE1 in rat heart mitochondria. Because mitochondria from rats injected with shRNA-NHE1 present a high threshold for MPTP formation, the beneficial effects of NHE1 inhibition in I/R resulting from mitochondrial targeting should be considered.     

The role of the mitochondrial permeability transition pore in heart disease

Like Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty.     

谷氨酰胺对心肌细胞线粒体膜PTP开放干预作用的实验研究

目的:探讨谷氨酰胺(Gln)对过度训练状态下心肌线粒体膜通透性转换孔(PTP)开放的干预作用及其可能机制。方法:30只SD大鼠随机分为3组(n=10):对照组(CG组)、过度训练组(OG组)和补充Gln+过度训练组(GOG组)。采用分光光度法检测大鼠心肌线粒体PTP开放程度,电化学法检测心肌丙二醛(MDA)、还原型谷胱苷肽(GSH)含量和磷脂酶A2(PLA2)活性。结果:OG组与GOG组比较,吸光度(A0)显著下降(P<0.05),吸光度变化(△A)值显著降低(P<0.05);荧光剂罗丹明123(Rh123)的荧光强度(F0)显著增强(P<0.05),Rh123荧光强度变化(△F)值明显降低(P<0.05)。与GOG组比较,线粒体GSH含量显著降低(P<0.05),PLA2活性显著增加(P<0.05);MDA含量显著升高(P<0.05)。结论:过度训练可导致心肌细胞线粒体PTP开放增加,过度训练状态下线粒体活性氧生成增多,PLA2活性增加及GSH的含量下降,补充外源性的Gln对这些变化有显著的干预作用。     

The role of mitochondrial permeability transition pore in transmembrane Ca2+-exchange in mitochondria

Ca2+-uptake accompanied with mitochondrial permeability transition pore (MPTP) opening is studied in rat liver mitochondria. In conditions of MPTP opening, as well as in conditions of MPTP blockage by cyclosporine A (CsA), Ca2+-uptake in mitochondria is counterbalanced by proton efflux into incubation medium. Independent of MPTP opening, observed stoichiometry of this exchange is 1Ca2+ : 1H+. MPTP opening dramatically decreases Ca2+-uptake in mitochondria: from approximately 400 nmol/mg protein in the presence of CsA to approximately 80-100 nmol/mg protein due to the increased mitochondrial membrane permeability. In the absence of CsA Ca2+-uptake is accompanied by the insensitive to Ca2+-uniporter blocker, ruthenium red (RR), release of Ca2+ from mitochondria which corresponds to as well RR-insensitive, but sensitive to CsA uptake of H+ into mitochondrial matrix. This calcium-proton exchange resulting from MPTP opening is observed only when Ca2+ uptake into matrix exceeds some basal level. The data are consistent with an assumption that, contrary to Ca2+-uniporter, MPTP has its own proton conductance. MPTP opening provides exchange of Ca2+ between mitochondria and medium which is coupled to the counterflow of protons into matrix space. Obtained data elucidate the physiological role of MPTP as regulatory mechanism for control of Ca2+-uptake level and intramitochondrial pH.     

BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.