Mitochondrial Matrix Swelling Research Articles

Association Between L-OPA1 Cleavage and Cardiac Dysfunction During Ischemia-Reperfusion Injury in Rats.

Background/Aims:Structural and functional alterations in mitochondria, particularly, the inner mitochondrial membrane (IMM) plays a critical role in mitochondria-mediated cell death in response to cardiac ischemia-reperfusion (IR) injury. The integrity of IMM can be affected by two potential intra-mitochondrial factors: i) mitochondrial matrix swelling, and ii) proteolytic cleavage of the long optic atrophy type 1 (L-OPA1), an IMM-localized dynamin-like GTPase engaged in the regulation of structural organization and integrity of the mitochondrial cristae. However, the relationship between these two factors in response to oxidative stress remains unclear. Here, we elucidated the effects of cardiac IR injury on L-OPA1 cleavage and OMA1 activity.Methods:Langendorff-mode perfused isolated rat hearts were subjected to 25-min of global ischemia followed by 90-min reperfusion in the presence or absence of XJB-5-131 (XJB, a mitochondria-targeting ROS scavenger) and sanglifehrin A (SfA, a permeability transition pore inhibitor).Results:XJB in combination with SfA increased post-ischemic recovery of cardiac function and reduced mitochondrial ROS production at 30- and 60-min reperfusion and affected mitochondrial swelling. L-OPA1 levels were reduced in IR hearts; however, neither XJB, SfA, and their combination prevented IR-induced reduction of L-OPA1 cleavage. Likewise, IR increased the OMA1 enzymatic activity, which remained unchanged in the presence of XJB and/or SfA.Conclusion:IR-induced cardiac and mitochondrial dysfunctions are associated with OMA1 activation and L-OPA1 cleavage. However, XJB, SfA, and their combination do not prevent these changes despite improved heart and mitochondria function, thus, suggesting that different mechanisms can be implicated in L-OPA1 processing in response to cardiac IR injury.

C1orf194 deficiency leads to incomplete early embryonic lethality and dominant intermediate Charcot-Marie-Tooth disease in a knockout mouse model.

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy and shows clinical and genetic heterogeneity. Mutations in C1orf194 encoding a Ca2+ regulator in neurons and Schwann cells have been reported previously by us to cause CMT disease. In here, we further investigated the function and pathogenic mechanism of C1or194 by generating C1orf194 knockout (KO) mice. Homozygous mutants of C1orf194 mice exhibited incomplete embryonic lethality, characterized by differentiation abnormalities and stillbirth on embryonic days 7.5-15.5. Heterozygous and surviving homozygous C1orf194 KO mice developed motor and sensory defects at the age of 4months. Electrophysiologic recordings showed decreased compound muscle action potential and motor nerve conduction velocity in the sciatic nerve of C1orf194-deficient mice as a pathologic feature of dominant intermediate-type CMT. Transmission electron microscopy analysis revealed demyelination and axonal atrophy in the sciatic nerve as well as swelling and loss of mitochondrial matrix and other abnormalities in axons and Schwann cells. A histopathologic examination showed a loss of motor neurons in the anterior horn of the spinal cord and muscle atrophy. Shorter internodal length between nodes of Ranvier and Schmidt-Lanterman incisures was detected in the sciatic nerve of affected animals. These results indicate that C1orf194 KO mice can serve as an animal model of CMT with a severe dominant intermediate CMT phenotype that can be used to investigate the molecular mechanisms of the disease and evaluate the efficacy of therapeutic strategies.

Abstract 854: The Role of Mitochondrial Permeability Transition in the Regulation of ETC Supercomplexes Assembly by OPA1

Optic atrophy type 1 (OPA1), a dynamin-related GTPase is the inner mitochondrial membrane (IMM) protein, which, in addition to mitochondrial fusion, participates in maintaining the structural integrity of the IMM. The function and topography of the IMM is also regulated by changes in the matrix volume. Opening of the mitochondrial permeability transition pores (PTP) is the leading cause of mitochondrial matrix swelling. However, the cause-and-effect relationship between OPA1 proteolytic cleavage and PTP induction remains unknown. Here, we elucidated the role of PTP in OPA1-mediated regulation of electron transport chain (ETC) supercomplexes (SCs) assembly. First, intact mitochondria isolated from the rat heart were used to analyze the effect of PTP induction on the cleavage of long (L)-OPA1. Calcium-induced mitochondrial swelling increased L-OPA1 cleavage (66%, P<0.001) associated with accumulation of short (S)-OPA1. Inhibition of swelling by sanglifehrin A (a PTP inhibitor) prevented L-OPA cleavage whereas tert-butyl hydroperoxide (TBH) had no effect on L-OPA levels. Second, OPA1 knockdown (KD) by siRNA in HEK cells resulted in a 74% decrease (P<0.001) in OPA1 protein expression but did not change the proportion of L-OPA1 to total OPA. Mitochondria isolated from OPA1 KD cells showed reduced calcium-stimulated swelling with no changes in protein levels of cyclophilin D, a major PTP regulator. OPA1 KD cells demonstrated a 20% increase (P<0.05) in calcium-induced ROS generation. Analysis of individual ETC complexes revealed significant reduction of protein levels of the complexes I, III, and IV in OPA1 KD cells. Likewise, activities of complexes I and IV were reduced (48% and 37%, respectively, P<0.01 for both). OPA1 KD cells contained 38% (P<0.05) less ATP whereas the citrate synthase activity reflecting mitochondria mass was not affected. Blue native PAGE analysis demonstrated a 35% decrease (P<0.01) of high molecular weight proteins (>100 kDa) and a 24% decrease (P<0.05) of the respirasome, the main SC, levels in OPA1 KD mitochondria. We conclude that mitochondrial swelling by PTP opening stimulates OPA1 cleavage, and reduced OPA1 levels result in cristae malformation including reduced respirasome levels and the ETC complexes activity.

Metformin Has Direct Protective Effect on Human Cardiac Mitochondria

ObjectiveMetformin (Metf), an oral antihyperglycemic agent, is widely used to treat type 2 diabetes mellitus. The primary action of the drug is to lower hepatic glucose synthesis and enhance peripheral glucose uptake via activation of the insulin receptor. It also exerts a cardioprotective effect independent of its glucose lowering action; however, mechanisms underlying cardioprotection in the human heart remain poorly defined. We hypothesize that the cardioprotective effect of Metf is associated with mild inhibition of mitochondrial oxidative phosphorylation (OXPHOS) and reduction of oxidative stress that protect mitochondria against permeability transition pore (PTP) opening.MethodsLeft atrial appendage tissue was collected from middle‐aged and elderly patients (n=21) undergoing open‐heart surgery. Mitochondria were isolated from fresh tissue using differential centrifugation and exposed to different concentrations of Metf (0–10 mM). The functional activity of mitochondrial OXPHOS complexes (CI‐V) was measured spectrophotometrically using specific OXPHOS inhibitors, electron donors, and acceptors. Superoxide production was measured by monitoring changes in fluorescence intensity of dihydroethidium (DHE) in the absence and presence of antimycin A (AA, 5 μM). The sensitivity of mitochondria toward PTP opening was assessed by exposing isolated mitochondria to 10 μM of Ca2+ pulses and monitoring abrupt mitochondrial Ca2+ release (Fluo‐5N fluorescence) and swelling of the matrix (decrease in absorbance). The study was approved by the Aurora Institutional Review Board and adhered to the Health Insurance Portability and Accountability Act and Aurora Health Care patient privacy and security guidelines. The study followed the principles of the Declaration of Helsinki.ResultsMetf had a dose‐dependent mild inhibitory effect on the activity of CI, CIV, and CV without affecting CII and CIII (Figure A). This was associated with reduction of superoxide production before and after exposure of the mitochondria to AA (Figure B). Metf in a dose‐dependent manner also attenuated Ca2+‐induced mPTP opening (Figure C) with delayed swelling of mitochondrial matrix (Figure D).ConclusionMetf improves cardioprotection by increasing tolerance to stress‐induced PTP opening due to its mild inhibitory effect on the OXPHOS and reduction of superoxide production. This can be a beneficial alternative to patients with impaired endogenous cardioprotective responses.Support or Funding InformationN 570‐3657, Cardiac Research Award, Aurora Health CareThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of hypoxia/reoxygenation on the biological effect of IGF system and the inflammatory mediators in cultured synoviocytes

Hypoxia/reoxygenation (H/R) plays an important role in the pathogenesis of osteoarthritis. Fibroblast-like synoviocytes (FLS), which are highly sensitive to H/R, are thought to be associated with cartilage degradation during osteoarthritis development. In this study, we investigated the biological effects of insulin-like growth factor (IGF) system and the expression of inflammatory mediators in FLS. We also pretreated FLS with tumor necrosis factor-α (TNF-α) before H/R in order to observe the response of FLS with the background of inflammatory cytokines. H/R increased the levels of TNF-α-induced C-C chemokine ligand 5 (CCL5), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in cell-free culture supernatants; H/R also increased the expression of TNF-α-induced insulin-like growth factor binding protein 3 (IGFBP-3), downregulated the expression of insulin-like growth factor 1 (IGF-1), promoted the loss of mitochondrial membrane potential (MMP), the openness of mitochondrial permeability transition pore (MPTP), the release of intracellular reactive oxygen species (ROS), and mitochondrial matrix swelling, outer membrane rupture and decrease in cristae. Furthermore, H/R induced the expression of catabolic factors and activated the NF-κB signaling pathway in FLS. We therefore concluded that H/R may play a role in inducing inflammation and increase the TNF-α-induced inflammatory effect in FLS, contributing to osteoarthritis pathogenesis.

Effects of baicalin on mitochondria apoptotic pathway of trophoblast cells in a preeclampsia rat model

Objective To explore the effects of baicalin in the treatment of a preeclampsia (PE) rat model by detecting the expression of X-linked inhibitor of apoptosis protein (XIAP) and cysteine containing aspartate-9 (Caspase-9) and observing the ultrastructure of mitochondria in trophoblast cells. Methods Forty-eight pregnant Wistar rats were randomly divided into two groups: 12 in the control group and 36 in the PE model group. The PE model was established with subcutaneous injection of l-nitro arginine methyl ester with 100 mg/kg per day from the 13th day of pregnancy. Beginning from the 16th day of pregnancy, the PE rats were injected with different doses of baicalin till cesarean section, and divided into three groups: non-intervention PE model group treated with saline (NIP group), low-dose baicalin intervention group (IDB group) at 50 mg/kg per day, and high-dose baicalin intervention group (HDB group) at 100 mg/kg per day. The rat tail artery blood pressure and 24-h urine protein level were measured at pregnant day 10, 16 and 20. The levels of XIAP and Caspase-9 in placenta were measured by immunohistochemistry. The ultrastructure of mitochondria of trophoblast cells of the rat placenta was observed under electron microscope. T test, F test and LSD-t test were applied for statistical analysis. Results (1) On pregnant day 10, no significant differences were observed in rat tail artery systolic blood pressure, diastolic blood pressure and 24-h urine protein level between the control group and PE model group (all P>0.05). On pregnant day 16 and 20, the systolic blood pressure, diastolic blood pressure and 24-h urine protein level of NIP, IDB and HDB groups were significantly higher than those of control group [pregnant day 16: systolic blood pressure: (137.74±5.21),(136.15±4.86),(138.28±4.79) and (110.57±3.79) mmHg (1 mmHg=0.133 kPa), diastolic blood pressure:(89.58±5.50), (88.45±8.59), (89.42±6.29) and (80.28±7.36) mmHg, 24-h urine protein: (7.78±0.45), (7.53±0.54), (7.86±0.57) and (6.45±0.56) mg; pregnant day 20: systolic blood pressure: (145.26±4.67), (131.28±4.34), (130.93±5.33) and (110.40±6.92) mmHg, diastolic blood pressure: (89.87±6.55), (85.34±7.33), (84.64±7.36) and (80.19±7.34) mmHg, 24-h urine protein: (11.18±0.42), (9.65±0.54), (9.06±0.56) and (6.31±0.45) mg] (all P 0.05). (2)Compared with NIP group, the expression of XIAP in control group, IDB and HDB groups were significantly increased(210.39±0.78,180.56±0.82,195.36±0.96 and 192.84±1.06, all P<0.01). There was no significant difference in the expression of XIAP between IDB and HDB groups (P=0.66). The expression of Caspase-9 in control group, IDB and HDB groups were significantly decreased compared with NIP group (210.36±0.55, 195.53±0.96, 198.42±1.01 and 185.25±0.64, all P<0.01). There was no significant difference in the expression of Caspase-9 between IDB and HDB groups (P=0.65). Ultrastructure of mitochondria in NIP group showed different degrees of damage, matrix swelling, and mitochondrial cristae bresk or disappearance. In IDB group, mitochondrial matrix swelling was not obvious, and mitochondrial cristae were visible. In HDB group, mitochondrial cristae were neat and clear. Conclusions Baicalin may play an important role in the treatment of preeclampsia by reversing the trophoblast apoptosis and improving the ultrastructure of mitochondria through its regulation of XIAP expression and downregulation of Caspase-9 expression. Key words: Pre-eclampsia; Baicalin; Trophoblasts; X-Linked inhibitor of apoptosis protein; Caspase-9; Apoptosis; Rats

Upconversion nano-photosensitizer targeting into mitochondria for cancer apoptosis induction and cyt c fluorescence monitoring

Disruption of mitochondrial reactive oxygen species (mitoROS) plays a major role in cancer cell apoptosis. Here, we designed a core/shell-structured mitochondria-targeting upconversion-based nano-photosensitizer (TPP-UC(PS)) with a lanthanide-doped upconversion nanoparticle (UCNP) core coated by a photosensitizer (PS)-incorporated dense silica shell. Following irradiation with external near-infrared laser (NIR), TPP-UC(PS) in mitochondria caused serious mitochondrial matrix swelling for the activated upconversion-based photodynamic therapy (UC-PDT), and the mobilization of cytochrome c (cyt c) was amplified in response to the increased mitoROS. Specifically, this heme-containing cyt c could be monitored by varying TPP-UC(PS)’s upconversion luminescence signal (UCL), which may facilitate the in situ detection of cyt c for apoptosis research. As a proof of concept, our designed TPP-UC(PS) may provide significant opportunities for controlling cancer cell apoptosis under NIR stimulation and for studying apoptosis using the dynamic UCL, which is influenced by local cyt c.

Improving mitochondrial bioenergetics under ischemic conditions increases warm ischemia tolerance in the kidney.

Ischemia time during partial nephrectomy is strongly associated with acute and chronic renal injury. ATP depletion during warm ischemia inhibits ATP-dependent processes, resulting in cell swelling, cytoskeletal breakdown, and cell death. The duration of ischemia tolerated by the kidney depends on the amount of ATP that can be produced with residual substrates and oxygen in the tissue to sustain cell function. We previously reported that the rat can tolerate 30-min ischemia quite well but 45-min ischemia results in acute kidney injury and progressive interstitial fibrosis. Here, we report that pretreatment with SS-20 30 min before warm ischemia in the rat increased ischemia tolerance from 30 to 45 min. Histological examination of kidney tissues revealed that SS-20 reduced cytoskeletal breakdown and cell swelling after 45-min ischemia. Electron microscopy showed that SS-20 reduced mitochondrial matrix swelling and preserved cristae membranes, suggesting that SS-20 enhanced mitochondrial ATP synthesis under ischemic conditions. Studies with isolated kidney mitochondria showed dramatic reduction in state 3 respiration and respiratory control ratio after 45-min ischemia, and this was significantly improved by SS-20 treatment. These results suggest that SS-20 increases efficiency of the electron transport chain and improves coupling of oxidative phosphorylation. SS-20 treatment after ischemia also significantly reduced interstitial fibrosis. These new findings reveal that enhancing mitochondrial bioenergetics may be an important target for improving ischemia tolerance, and SS-20 may serve well for minimizing acute kidney injury and chronic kidney disease following surgical procedures such as partial nephrectomy and transplantation.

Arecoline is cytotoxic for human endothelial cells

Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4h with arecoline at 333μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis.

Isoangustone A induces apoptosis in SW480 human colorectal adenocarcinoma cells by disrupting mitochondrial functions

Licorice and its components have been reported to posses various anti-tumor activities, but its active ingredients and underlying mechanisms are not well understood yet. In the present study, a group of representative licorice-derived compounds that could be detected in rat plasma or urine were screened for anti-tumor activity. Among these compounds, isoangustone A (IAA) was found to promptly inhibit the viability of SW480 human colorectal adenocarcinoma cells in a time- and concentration-dependent manner. Further analyses indicate that IAA activated caspase-dependent pro-apoptotic signaling and induced significant apoptosis, while had little effect on cell cycle. IAA strongly inhibited Akt phosphorylation within 5min; however, overexpression of constitutively activated Akt could not rescue IAA-mediated inhibition, indicating that inhibition of Akt was not involved in IAA-induced apoptosis. Further examinations show that IAA induced dissipation of mitochondria membrane potential and release of cytochrome C within 1h, accompanied by swelling of mitochondrial matrix and disrupting of mitochondrial outer membrane, and followed by decreasing of cellular ATP. The above results suggest that IAA induced apoptosis in colorectal cancer cells principally by inducing mitochondrial outer membrane permeabilization, and deserves further investigations as a novel anti-colorectal cancer agent.

Efficacy of PLGA-loaded apigenin nanoparticles in Benzo[a]pyrene and ultraviolet-B induced skin cancer of mice: Mitochondria mediated apoptotic signalling cascades

Skin cancer is increasing at an alarming rate and becoming resistant to conventional chemotherapy necessitating improved drug delivery system. We loaded apigenin (Ap), a dietary flavonoid having anti-cancer property, with poly (lactic-co-glycolide) (PLGA) nanoparticles (NAp) to explore if nano-encapsulation could enhance anti-carcinogenic effect against ultra-violet B (UVB) and Benzo(a)pyrene (BaP) induced skin tumor and mitochondrial dysfunction in mice. Particle size, morphology and zeta potential of NAp were determined using dynamic light scattering and atomic force microscopy. Tumor incidence and multiplicity in UVB–BaP induced mice with/without NAp treatment were ascertained and their histolopathological sections and chromosomal aberrations were studied. ROS accumulation and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential were analyzed. Mitochondrial volume changes/swelling, cytochrome c (cyt c) release, mRNA and protein expressions of Apaf-1, bax, bcl-2, cyt c, cleaved caspase-9 and 3 were studied. Results showed that NAp produced better effects than Ap, due to their smaller size, and faster mobility. NAp reduced tissue damage and frequency of chromosomal aberrations, increased ROS accumulation to mediate mitochondrial-apoptosis through modulation of several apoptotic markers and mitochondrial matrix swelling. NAp showed ameliorative potentials in combating skin cancer and therefore has greater prospect of use in therapeutic management of skin cancer.

Relationship Between Mitochondrial Matrix Volume and Cellular Volume in Response to Stress and the Role of ATP-Sensitive Potassium Channel

Cardiac myocytes demonstrate significant swelling and associated reduced contractility in response to stress that is prevented by the ATP-sensitive potassium channel opener, diazoxide (DZX) via an unknown mechanism. One proposed mechanism of cardioprotection is mitochondrial matrix swelling. To establish the relationship between mitochondrial and cellular volume during stress, this study examined the effect of DZX on mitochondrial volume. Isolated mouse mitochondria were exposed to the following solutions: Tyrode, isolation buffer, cardioplegia (CPG)±DZX±ATP-sensitive potassium channel inhibitor, 5-hydroxydecanoate, and metabolic inhibition (MI) ± DZX ± 5-hydroxydecanoate. Mitochondrial volume was measured. DZX resulted in significant mitochondrial swelling (P<0.0001 versus Tyrode). MI and CPG resulted in significant mitochondrial swelling compared with baseline volume. The addition of DZX did not alter the response of mitochondrial volume to CPG (P=0.912) but increased swelling in response to MI (P=0.036). The addition of 5-hydroxydecanoate to MI + DZX or CPG+DZX significantly reduced mitochondrial swelling (P<0.003 MI+DZX versus MI + DZX + 5HD; P<0.001 CPG+DZX versus CPG + DZX + 5HD). Both cellular and mitochondrial volume increased during exposure to MI and CPG. DZX did not alter mitochondrial volume during CPG; however, it was associated with an increase in mitochondrial volume during MI. 5-Hydroxydecanoate reduced mitochondrial volume during exposure to both stresses with DZX, supporting a role for a mitochondrial ATP-sensitive potassium channel in the mechanism of cardioprotection by DZX.

Complex I and F0F1-ATP Synthase Mediate Membrane Depolarization and Matrix Acidification by Isoflurane in Mitochondria

Introduction: Short application of volatile anesthetic isoflurane at reperfusion after ischemia exerts strong protection of heart and cardiac mitochondria against injury. Mild depolarization and acidification of mitochondrial matrix are involved in the protective mechanism, but the molecular basis for these changes is not known. In this study we investigated the electron transport chain, F0F1-ATP synthase and mitochondrial ion channels as potential targets of isoflurane in mitochondria.Methods: We have measured mitochondrial respiration, membrane potential, matrix pH, matrix swelling, and H2O2 release in isolated mitochondria in the presence and absence of isoflurane (0.5 mM). Pyruvate/malate, succinate/rotenone, or ascorbate/TMPD, were used as substrates for complex I, II and IV, respectively. Guanosine-diphosphate (GDP), oligomycin, paxilline and 5-hydroxydecanoic acid (5-HD) were used to probe involvement of uncoupling proteins, F0F1-ATP synthase, mitochondrial ATP- and Ca2+-sensitive K+ channel. Nigericin, a K+/H+ exchanger, was used to manipulate the matrix pH.Results: With pyruvate/malate as substrates, isoflurane inhibited mitochondrial respiration by 23±4%, depolarized membrane potential by 2.7±0.7% and decreased matrix pH by 11±3%. With complex II and complex IV-linked substrates, respiration was not changed, but isoflurane still decreased matrix pH and depolarized ΔΨm. Depolarization and matrix acidification were only attenuated by oligomycin, but not GDP, paxilline, or 5-HD. Isoflurane did not induce matrix swelling, but decreased H2O2 release in the presence of succinate in an oligomycin and matrix pH sensitive manner.Conclusion: Our results indicate that isoflurane inhibited the electron transport chain at the site of complex I and also modified F0F1-ATP synthase. Both effects lead to an acidification of the mitochondrial matrix which is beneficial at the time of reperfusion. K+ channels and uncoupling proteins are likely not involved in these direct effects of isoflurane on isolated mitochondria.

A novel paradigm for rapid ABT-737-induced apoptosis involving outer mitochondrial membrane rupture in primary leukemia and lymphoma cells

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.

Direct mechanical communication between mitochondria and nucleus in cardiac cells

Extracellular forces, which deform the cell and thereby its nucleus, are thought to be able to modulate nuclear functions. However, mechanical signals may also arise from within the cell. We have recently shown (Kaasik et al. FASEB J. 2004, 18, 1219) that mitochondrial modulators, which increase the matrix volume, augment force developed by permeabilized ventricular fibers, due to physical compression of the myofibrillar compartment by swollen mitochondria. In the present study we investigated whether mitochondrial modulators that induce matrix swelling were able to affect nuclear shape and volume by imposing mechanical constraints. Nuclei in rat saponin-permeabilized cardiomyocytes were stained with propidium iodide and analyzed by confocal microscopy after image deconvolution and 3D reconstruction. In an artificial medium mimicking the cytosol, 10 μM valinomycin (a potassium ionophore, inducing mitochondrial matrix swelling) decreased nuclear volume by 12 ± 2% (p<0.001). This effect was not related to inhibition of ATP-generating activity as bongkrekic acid (adenine nucleotide translocator blocker), which did not alter matrix volume, had no effect on nuclei. Furthermore, 150 μM diazoxide, a mitochondrial ATP-sensitive potassium channel opener, also reduced nuclear volume by 12 ± 2% (p=0.001) whereas 150 μM 5-hydroxydecanoate, thought to be a specific inhibitor of these channels, completely blocked the effect. Thus, we have shown for the first time that mitochondria are able to induce nuclear deformation, suggesting that mitochondria may mechanically regulate nuclear function.

Role of the mitochondrial ATP-sensitive K+ channels in cardioprotection.

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel was discovered more than a decade ago. Since then, several pharmacological studies have identified agents that target this channel some of which selectively target mitoK(ATP). These and other studies have also suggested that mitoK(ATP) plays a key role in the process of ischemic preconditioning (IPC) and prevention of apoptosis. The mechanism by which mitoK(ATP) exerts its protective effects is unclear, however, changes in mitochondrial Ca(2+) uptake and levels of reactive oxygen species, and mitochondrial matrix swelling are believed to be involved. Despite major advances, several important issues regarding mitoK(ATP) remain unanswered. These questions include, but are not limited to: the molecular structure of mitoK(ATP), the downstream and upstream mechanisms that leads to IPC and cell death, and the pharmacological profile of the channel. This review attempts to provide an up-to-date overview of the role of mitoK(ATP) in cardioprotection.

Role of the mitochondrial ATP-sensitive K+ channels in cardioprotection.

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel was discovered more than a decade ago. Since then, several pharmacological studies have identified agents that target this channel some of which selectively target mitoK(ATP). These and other studies have also suggested that mitoK(ATP) plays a key role in the process of ischemic preconditioning (IPC) and prevention of apoptosis. The mechanism by which mitoK(ATP) exerts its protective effects is unclear, however, changes in mitochondrial Ca(2+) uptake and levels of reactive oxygen species, and mitochondrial matrix swelling are believed to be involved. Despite major advances, several important issues regarding mitoK(ATP) remain unanswered. These questions include, but are not limited to: the molecular structure of mitoK(ATP), the downstream and upstream mechanisms that leads to IPC and cell death, and the pharmacological profile of the channel. This review attempts to provide an up-to-date overview of the role of mitoK(ATP) in cardioprotection.

Effects of CCCP-induced mitochondrial uncoupling and cyclosporin A on cell volume, cell injury and preconditioning protection of isolated rabbit cardiomyocytes

Cell swelling may contribute to acute cell injury subsequent to ischemia/reperfusion. The potential role of mitochondrial uncoupling and the resultant mitochondrial swelling, due to opening of the mitochondrial permeability transition pore (MPTP), were examined in an in vitro ischemically pelleted isolated rabbit cardiomyocyte model using the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) to uncouple mitochondria. Cyclosporin A (CsA) was employed to inhibit MPTP opening. Cell volume was determined by a cell-flotation, density-gradient assay, using bromododecane. Cell viability, subsequent to an osmotic stress, was determined by trypan blue permeability. Ischemic preconditioning (IPC) facilitated volume regulation following an osmotic stress. Ischemic-cell swelling was reduced by IPC. IPC protected ischemically pelleted cells, but CsA had no significant effects on injury or IPC protection. CCCP ischemia accelerated rates of ischemic contracture and injury, and abolished IPC protection. IPC protection was restored by CsA. In CCCP-ischemic-uncoupled cells, subjected to a reduced (170 mOsm) osmotic stress, CsA and IPC afforded independent and additive protection. Chelerythrine and 5-hydroxydecanoate (5-HD) blocked IPC, but did not reduce CsA protection. Electron microscopy confirmed that CCCP ischemia induced mitochondrial matrix swelling that was reduced by CsA. Cardioprotection by IPC and CsA was accompanied by proportional reductions in cell swelling. Morphometric analysis of the electron photomicrographs demonstrated that the mitochondrial volume fractions were significantly reduced in the CsA/CCCP (29.8 ± 2.3%, P < 0.004) and IPC/CsA/CCCP (31.5 ± 1.7%, P < 0.0008) groups as compared to the CCCP-ischemic group (40.5 ± 1.7%) The IPC/CCCP group (39.5 ± 4.2%) was not significantly different from the CCCP-ischemic group. NIM 811, a CsA analogue MPTP blocker with no calcineurin inhibitory activity, afforded protection similar to CsA. The results suggest that CsA protection may, in part, be mediated by reduction of mitochondrial swelling.

Bcl-xL overexpression blocks bax-mediated mitochondrial contact site formation and apoptosis in rod photoreceptors of lead-exposed mice.

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited and disease-, injury-, and chemical-induced retinal degeneration. A clinically relevant mouse model of progressive rod photoreceptor-selective apoptosis was produced by low-level developmental lead exposure and studied in combination with transgenic mice overexpressing Bcl-x(L) only in the photoreceptors. A multiparametric analysis of rod apoptosis and mitochondrial structure-function was performed. Mitochondrial cristae topography and connectivity, matrix volume, and contact sites were examined by using 3D electron tomography. Lead-induced rod-selective apoptosis was accompanied by rod Ca(2+) overload, rhodopsin loss, translocation of Bax from the cytosol to the mitochondria, decreased rod mitochondrial respiration and membrane potential, mitochondrial cytochrome c release, caspase-3 activation, and an increase in the number of mitochondrial contact sites. These effects occurred without mitochondrial matrix swelling, outer membrane rupture, caspase-8 activation, or Bid cleavage. Bcl-x(L) overexpression completely blocked all apoptotic events, except Ca(2+) overload, and maintained normal rod mitochondrial function throughout adulthood. This study presents images of mitochondrial contact sites in an in vivo apoptosis model and shows that Bcl-x(L) overexpression blocks increased contact sites and apoptosis. These findings extend our in vitro retinal studies with Pb(2+) and Ca(2+) and suggest that developmental lead exposure produced rod-selective apoptosis without mitochondrial swelling by translocating cytosolic Bax to the mitochondria, which likely sensitized the Pb(2+) and Ca(2+) overloaded rod mitochondria to release cytochrome c. These results have relevance for therapies in a wide variety of progressive retinal and neuronal degenerations where Ca(2+) overload, lead exposure, andor mitochondrial dysfunction occur.

The permeability transition pore signals apoptosis by directing Bax translocation and multimerization.

Mitochondria are key players of apoptosis and can irreversibly commit the cell to death by releasing cytochrome c (Cyt.c) to the cytosol, where caspases 9 and 3 subsequently get activated. Under conditions of oxidative stress, opening of the mitochondrial permeability transition pore (PTP) represents an early trigger and is crucial in causing Cyt.c release. To account for the latter, current models propose that PTP gating would result, as is the case in vitro, in the rupture of the outer mitochondrial membrane caused by mitochondrial matrix swelling. Using live cell imaging and recombinant fluorescent probes based on the green fluorescent protein (GFP) and its mutants, we report that directed repetitive gating of the PTP triggers a delayed Cyt.c efflux, which is not associated with mitochondrial swelling. Instead, subcellular imaging shows that PTP opening signals the redistribution of the cytosolic protein Bax to the mitochondria, where it secondarily forms clusters that appear to be a prerequisite for Cyt.c release. Fluorescence resonance energy transfer imaging further reveals that Bax clustering coincides with the formation of Bax multimers. We conclude that the PTP is not itself a component of the Cyt.c release machinery, but that it acts indirectly by signaling Bax translocation and multimerization.