RESUMO
We critically assess the proposal that succinate-fuelled reverse electron flow (REF) drives mitochondrial matrix superoxide production from Complex I early in reperfusion, thus acting as a key mediator of ischemia/reperfusion (IR) injury. Real-time surface fluorescence measurements of NAD(P)H and flavoprotein redox state suggest that conditions are unfavourable for REF during early reperfusion. Furthermore, rapid loss of succinate accumulated during ischemia can be explained by its efflux rather than oxidation. Moreover, succinate accumulation during ischemia is not attenuated by ischemic preconditioning (IP) despite powerful cardioprotection. In addition, measurement of intracellular reactive oxygen species (ROS) during reperfusion using surface fluorescence and mitochondrial aconitase activity detected major increases in ROS only after mitochondrial permeability transition pore (mPTP) opening was first detected. We conclude that mPTP opening is probably triggered initially by factors other than ROS, including increased mitochondrial [Ca2+]. However, IP only attenuates [Ca2+] increases later in reperfusion, again after initial mPTP opening, implying that IP regulates mPTP opening through additional mechanisms. One such is mitochondria-bound hexokinase 2 (HK2) which dissociates from mitochondria during ischemia in control hearts but not those subject to IP. Indeed, there is a strong correlation between the extent of HK2 loss from mitochondria during ischemia and infarct size on subsequent reperfusion. Mechanisms linking HK2 dissociation to mPTP sensitisation remain to be fully established but several related processes have been implicated including VDAC1 oligomerisation, the stability of contact sites between the inner and outer membranes, cristae morphology, Bcl-2 family members and mitochondrial fission proteins such as Drp1.
Assuntos
Traumatismo por Reperfusão Miocárdica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Succínico/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade MitocondrialRESUMO
ß-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for ß-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in ß-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.
Assuntos
Talassemia beta , Humanos , Talassemia beta/genética , Talassemia beta/terapia , Eritropoese/genética , Células Eritroides , FenótipoRESUMO
We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6-phosphate at pH 6.3. Prior HK2 dissociation from pre- or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre- and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca2+].
Assuntos
Hexoquinase/metabolismo , Mitocôndrias Cardíacas/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Isquemia Miocárdica/enzimologia , Animais , Respiração Celular/efeitos dos fármacos , Dinaminas/metabolismo , Glucose-6-Fosfato/farmacologia , Hemodinâmica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Precondicionamento Isquêmico , Ligantes , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Dinâmica Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Isquemia Miocárdica/patologia , Ligação Proteica/efeitos dos fármacos , Ratos WistarRESUMO
Cardiac mitochondria can take up Ca(2+), competing with Ca(2+) transporters like the sarcoplasmic reticulum (SR) Ca(2+)-ATPase. Rapid mitochondrial [Ca(2+)] transients have been reported to be synchronized with normal cytosolic [Ca(2+)](i) transients. However, most intra-mitochondrial free [Ca(2+)] ([Ca(2+)](mito)) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca(2+)](mito) in single rat myocytes using the ratiometric Ca(2+) indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca(2+)](mito) dependence on [Ca(2+)](i) (with 5 mM EGTA) was sigmoid with [Ca(2+)](mito)<[Ca(2+)](i) for [Ca(2+)](i) below 475 nM. With low [EGTA] (50 microM) and 150 nM [Ca(2+)](i) (+/-15 mM Na(+)) cyclical spontaneous SR Ca(2+) release occurred (5-15/min). Changes in [Ca(2+)](mito) during individual [Ca(2+)](i) transients were small ( approximately 2-10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca(2+) handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca(2+)](mito) and spontaneous Ca(2+) transients (confirming that SR Ca(2+) releases caused the [Ca(2+)](mito) rise). Confocal imaging of local [Ca(2+)](mito) (using rhod-2) showed that [Ca(2+)](mito) rose rapidly with a delay after SR Ca(2+) release (with amplitude up to 10 nM), but declined much more slowly than [Ca(2+)](i) (time constant 2.8+/-0.7 s vs. 0.19+/-0.06 s). Total Ca(2+) uptake for larger [Ca(2+)](mito) transients was approximately 0.5 micromol/L cytosol (assuming 100:1 mitochondrial Ca(2+) buffering), consistent with prior indirect estimates from [Ca(2+)](i) measurements, and corresponds to approximately 1% of the SR Ca(2+) uptake during a normal Ca(2+) transient. Thus small phasic [Ca(2+)](mito) transients and gradually integrating [Ca(2+)](mito) signals occur during repeating [Ca(2+)](i) transients.
Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Anestésicos Locais/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cafeína/farmacologia , Inibidores Enzimáticos/farmacologia , Cinética , Masculino , Microscopia de Fluorescência , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Sprague-Dawley , Tetracaína/farmacologia , Tapsigargina/farmacologiaRESUMO
BACKGROUND: Pluripotent stem cells are attractive progenitor cells for the generation of erythroid cells in vitro as have expansive proliferative potential. However, although embryonic (ESC) and induced pluripotent (iPSC) stem cells can be induced to undergo erythroid differentiation, the majority of cells fail to enucleate and the molecular basis of this defect is unknown. One protein that has been associated with the initial phase of erythroid cell enucleation is the intermediate filament vimentin, with loss of vimentin potentially required for the process to proceed. METHODS: In this study, we used our established erythroid culture system along with western blot, PCR and interegation of comparative proteomic data sets to analyse the temporal expression profile of vimentin in erythroid cells differentiated from adult peripheral blood stem cells, iPSC and ESC throughout erythropoiesis. Confocal microscopy was also used to examine the intracellular localisation of vimentin. RESULTS: We show that expression of vimentin is turned off early during normal adult erythroid cell differentiation, with vimentin protein lost by the polychromatic erythroblast stage, just prior to enucleation. In contrast, in erythroid cells differentiated from iPSC and ESC, expression of vimentin persists, with high levels of both mRNA and protein even in orthochromatic erythroblasts. In the vimentin-positive iPSC orthochromatic erythroblasts, F-actin was localized around the cell periphery; however, in those rare cells captured undergoing enucleation, vimentin was absent and F-actin was re-localized to the enucleosome as found in normal adult orthrochromatic erythroblasts. CONCLUSION: As both embryonic and adult erythroid cells loose vimentin and enucleate, retention of vimentin by iPSC and ESC erythroid cells indicates an intrinsic defect. By analogy with avian erythrocytes which naturally retain vimentin and remain nucleated, retention in iPSC- and ESC-derived erythroid cells may impede enucleation. Our data also provide the first evidence that dysregulation of processes in these cells occurs from the early stages of differentiation, facilitating targeting of future studies.
Assuntos
Eritropoese/fisiologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteômica/métodos , Vimentina/metabolismo , Diferenciação Celular , Células Cultivadas , Células Eritroides , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Development of in vitro culture systems for the generation of red blood cells is a goal of scientists globally with the aim of producing clinical grade products for transfusion. Although mature reticulocytes can be efficiently generated by such systems, the numbers produced fall short of that required for therapeutics, due to limited proliferative capacity of the erythroblasts. To overcome this hurdle, approaches are required to increase the expansion potential of such culture systems. The OP9 mouse stromal cell line is known to promote haematopoietic differentiation of pluripotent stem cells, however an effect of OP9 cells on erythropoiesis has not been explored. In this study, we show not only OP9 co-culture, but factors secreted by OP9 cells in isolation increase the proliferative potential of adult erythroid cells by delaying differentiation and hence maintaining self-renewing cells for an extended duration. The number of reticulocytes obtained was increased by approximately 3.5-fold, bringing it closer to that required for a therapeutic product. To identify the factors responsible, we analysed the OP9 cell secretome using comparative proteomics, identifying 18 candidate proteins. These data reveal the potential to increase erythroid cell numbers from in vitro culture systems without the need for genetic manipulation or co-culture.