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Methods in Molecular Biology (Clifton,... 2022The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant...
The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant inducers of mitochondrial swelling is matrix calcium (Ca). Respiring mitochondria will intake available Ca into their matrix until a threshold is reached which triggers the opening of the mitochondrial permeability transition pore (MPTP). Upon opening of the pore, mitochondrial membrane potential dissipates and the mitochondria begin to swell, rendering them dysfunctional. The total amount of Ca taken up by a mitochondrion prior to the engagement of the MPTP is referred to as mitochondrial Ca retention capacity (CRC). The CRC/swelling assay is a useful tool for observing the dose-dependent event of mitochondrial dysfunction in real-time. In this technique, isolated mitochondria are treated with specific boluses of Ca until they reach CRC and undergo swelling. A fluorometer is utilized to detect an increase in transmitted light passing through the sample as the mitochondria lose cristae density, and simultaneously measures calcium uptake by way of a Ca-specific membrane impermeable fluorescent dye. Here we provide a detailed protocol describing the mitochondrial CRC/swelling assay and we discuss how varying amounts of mitochondria and Ca added to the system affect the dose-dependency of the assay. We also report how to validate the assay by using MPTP and calcium uptake inhibitors and troubleshooting common mistakes that occur with this approach.
Topics: Calcium; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Permeability
PubMed: 35771440
DOI: 10.1007/978-1-0716-2309-1_9 -
Progress in Biophysics and Molecular... Aug 2022Existing theoretical approaches were considered that allow modelling of mitochondrial swelling (MS) dynamics. Simple phenomenological kinetic models were reviewed.... (Review)
Review
Existing theoretical approaches were considered that allow modelling of mitochondrial swelling (MS) dynamics. Simple phenomenological kinetic models were reviewed. Simple and extended biophysical and bioenergetic models that ignore mechanical properties of inner mitochondrial membrane (IMM), and similar models that include these mechanical properties were also reviewed. Limitations of these models we considered, as regards correct modelling of MS dynamics. It was found that simple phenomenological kinetic models have significant limitations, due to dependence of the kinetic parameter values estimated by fitting of the experimental data on the experimental conditions. Additionally, such simple models provide no understanding of the detailed mechanisms behind the MS dynamics, nor of the dynamics of various system parameters during MS. Thus, biophysical and bioenergetic models ignoring IMM mechanical properties can't be used to model the transition between reversible and irreversible MS. However, simple and extended biophysical models that include IMM mechanical properties allow modelling the transition to irreversible swelling. These latter models are still limited due to significantly simplified description of biochemistry, compared to those of bioenergetic models. Finally, a strategy of model development is proposed, towards correct interpretation of the mitochondrial life cycle, including the effects of MS dynamics.
Topics: Energy Metabolism; Kinetics; Mitochondria; Mitochondrial Membranes; Mitochondrial Swelling
PubMed: 35447196
DOI: 10.1016/j.pbiomolbio.2022.04.004 -
Toxics Jun 2015The mitochondrial dysfunction has a critical role in several disorders including chemotherapy-induced peripheral neuropathies (CIPN). This is due to a related... (Review)
Review
The mitochondrial dysfunction has a critical role in several disorders including chemotherapy-induced peripheral neuropathies (CIPN). This is due to a related dysregulation of pathways involving calcium signalling, reactive oxygen species and apoptosis. Vincristine is able to affect calcium movement through the Dorsal Root Ganglia (DRG) neuronal mitochondrial membrane, altering its homeostasis and leading to abnormal neuronal excitability. Paclitaxel induces the opening of the mitochondrial permeability transition pore in axons followed by mitochondrial membrane potential loss, increased reactive oxygen species generation, ATP level reduction, calcium release and mitochondrial swelling. Cisplatin and oxaliplatin form adducts with mitochondrial DNA producing inhibition of replication, disruption of transcription and morphological abnormalities within mitochondria in DRG neurons, leading to a gradual energy failure. Bortezomib is able to modify mitochondrial calcium homeostasis and mitochondrial respiratory chain. Moreover, the expression of a certain number of genes, including those controlling mitochondrial functions, was altered in patients with bortezomib-induced peripheral neuropathy.
PubMed: 29056658
DOI: 10.3390/toxics3020198 -
International Journal of Molecular... Feb 2022Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In... (Review)
Review
Analysis of the function, structure, and intracellular organization of mitochondria is important for elucidating energy metabolism and intracellular energy transfer. In addition, basic and clinically oriented studies that investigate organ/tissue/cell dysfunction in various human diseases, including myopathies, cardiac/brain ischemia-reperfusion injuries, neurodegenerative diseases, cancer, and aging, require precise estimation of mitochondrial function. It should be noted that the main metabolic and functional characteristics of mitochondria obtained in situ (in permeabilized cells and tissue samples) and in vitro (in isolated organelles) are quite different, thereby compromising interpretations of experimental and clinical data. These differences are explained by the existence of the mitochondrial network, which possesses multiple interactions between the cytoplasm and other subcellular organelles. Metabolic and functional crosstalk between mitochondria and extra-mitochondrial cellular environments plays a crucial role in the regulation of mitochondrial metabolism and physiology. Therefore, it is important to analyze mitochondria in vivo or in situ without their isolation from the natural cellular environment. This review summarizes previous studies and discusses existing approaches and methods for the analysis of mitochondrial function, structure, and intracellular organization in situ.
Topics: Animals; Cell Respiration; Energy Metabolism; Humans; Mitochondria, Heart; Muscle, Skeletal; Myocytes, Cardiac
PubMed: 35216368
DOI: 10.3390/ijms23042252 -
The Journal of Neuroscience : the... Jun 2022Zn is an important contributor to ischemic brain injury, and recent studies support the hypothesis that mitochondria are key sites of its injurious effects. In murine...
Zn is an important contributor to ischemic brain injury, and recent studies support the hypothesis that mitochondria are key sites of its injurious effects. In murine hippocampal slices (both sexes) subjected to oxygen glucose deprivation (OGD), we found that Zn accumulation and its entry into mitochondria precedes and contributes to the induction of acute neuronal death. In addition, if the ischemic episode is short (and sublethal), there is ongoing Zn accumulation in CA1 mitochondria after OGD that may contribute to their delayed dysfunction. Using this slice model of sublethal OGD, we have examined Zn contributions to the progression of changes evoked by OGD and occurring over 4-5 h. We detected progressive mitochondrial depolarization occurring from ∼2 h after ischemia, a large increase in spontaneous synaptic activity between 2 and 3 h, and mitochondrial swelling and fragmentation at 4 h. Blockade of the primary route for Zn entry, the mitochondrial Ca uniporter (with ruthenium red [RR]) or Zn chelation shortly after OGD withdrawal substantially attenuated the mitochondrial depolarization and the changes in synaptic activity. RR also largely reversed the mitochondrial swelling. Finally, using an rat (male) asphyxial cardiac arrest model of transient global ischemia, we found that ∼8 min asphyxia induces considerable injury of CA1 neurons 4 h later that is associated with strong Zn accumulation within many damaged mitochondria. These effects were substantially attenuated by infusion of RR on reperfusion. Our findings highlight mitochondrial Zn accumulation after ischemia as a possible target for neuroprotective therapy. Brain ischemia is a leading cause of mortality and long-term disability that still lacks effective treatment. After transient ischemia, delayed death of neurons occurs in vulnerable brain regions. There is a critical need to understand mechanisms of this delayed neurodegeneration which can be targeted for neuroprotection. We found progressive and long-lasting mitochondrial Zn accumulation to occur in highly vulnerable CA1 neurons after ischemia. Here we demonstrate that this Zn accumulation contributes strongly to deleterious events occurring after ischemia, including mitochondrial dysfunction, swelling, and structural changes. We suggest that this mitochondrial Zn entry may constitute a promising target for development of therapeutic interventions to be delivered after termination of an episode of transient global ischemia.
PubMed: 35623885
DOI: 10.1523/JNEUROSCI.0874-21.2022 -
Biochemical and Biophysical Research... Jul 2020We here used fluorescence imaging to explore the effect of co-overexpression of Mcl-1 and Bak/BH3-only proteins on mitochondrial morphology. The cells co-expressing...
We here used fluorescence imaging to explore the effect of co-overexpression of Mcl-1 and Bak/BH3-only proteins on mitochondrial morphology. The cells co-expressing CFP-Mcl-1 and YFP-Bak/BimL/Puma/tBid showed co-localization of Mcl-1 with Bak/Puma/BimL/tBid and also showed the inhibitory action of Mcl-1 on the Bak-, BimL-, Puma- or tBid-mediated cell death. Co-expression of Mcl-1 and Bak but not BH3-only proteins induced time-dependent mitochondrial swelling. Fluorescence resonance energy transfer (FRET) imaging proved the direct binding of Mcl-1 to Bak, BimL, Puma and tBid, respectively. In addition, Mcl-1 prevented Bak oligomerization by retrotranslocating Bak from mitochondria into cytoplasm. Moreover, Mcl-1-Bak complex exhibited a good co-localization with mitochondria, and co-expression of Mcl-1 and Bak for more than 24 h not only induced mitochondrial swelling but also impaired mitochondrial membrane potential. Collectively, co-expression of Mcl-1 and Bak but not BH3-only proteins significantly induced mitochondrial swelling and subsequent loss of mitochondrial membrane potential.
Topics: Apoptosis; Gene Expression; HeLa Cells; Humans; Mitochondria; Mitochondrial Swelling; Myeloid Cell Leukemia Sequence 1 Protein; bcl-2 Homologous Antagonist-Killer Protein
PubMed: 32430171
DOI: 10.1016/j.bbrc.2020.04.154 -
Chemico-biological Interactions Nov 2020A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two...
A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.
Topics: Adenosine Triphosphate; Animals; Calcium; Cell Line; Cromakalim; Male; Mice; Mice, Inbred C57BL; Mitochondria; Oxygen Consumption; Potassium Channels; Respiratory Rate
PubMed: 33010220
DOI: 10.1016/j.cbi.2020.109272 -
Circulation Research May 2024During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability...
BACKGROUND
During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined.
METHODS
Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca, ROS, and Fe to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective.
RESULTS
In the absence of Ca, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca synergize to induce mPTP opening. Furthermore, this synergistic form of Ca- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone.
CONCLUSIONS
In the present study, we have investigated the relationship between Ca and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.
Topics: Animals; Lipid Peroxidation; Mitochondrial Permeability Transition Pore; Reactive Oxygen Species; Mice; Mitochondria, Heart; Male; Myocardial Reperfusion Injury; Mice, Inbred C57BL; Mitochondrial Membrane Transport Proteins; Mitochondria, Liver; Calcium; Mitochondrial Swelling
PubMed: 38618716
DOI: 10.1161/CIRCRESAHA.123.323882 -
Bio Systems Dec 2021An extended biophysical model was obtained by upgrading the previously reported one (Khmelinskii and Makarov, 2021). The upgraded model accommodates variations of solute...
An extended biophysical model was obtained by upgrading the previously reported one (Khmelinskii and Makarov, 2021). The upgraded model accommodates variations of solute transport rates through the inner mitochondrial membrane (IMM) within the mitochondrial population, described by a Gaussian distribution. However, the model may be used for any functional form of the distribution. The dynamics of system parameters as predicted by the current model differed from that predicted by the previous model in the same initial conditions (Khmelinskii and Makarov, 2021). The amount of change varied from one parameter to the other, remaining in the 1-38% range. The upgraded model fitted the available experimental data with a better accuracy (R = 0.993) compared to the previous model (R = 0.978) using the same experimental data (Khmelinskii and Makarov, 2021). The fitting procedure also estimated the Gaussian distribution parameters. The new model requires much larger computational resources, but given its higher accuracy, it may be used for better analysis of experimental data and for better prediction of MS dynamics in different initial conditions. Note that activities of individual mitochondria in mitochondrial populations should vary within biological tissues. Thus, the currently upgraded model is a better tool for biological and bio-medical applications. We believe that this model is much better adapted to the analysis of MS dynamics in vivo.
Topics: Animals; Biophysical Phenomena; Humans; Mitochondria; Mitochondrial Membranes; Mitochondrial Swelling; Models, Biological
PubMed: 34627969
DOI: 10.1016/j.biosystems.2021.104559 -
Mitochondrion Jul 2023As the cell's energy factory and metabolic hub, mitochondria are critical for ATP synthesis to maintain cellular function. Mitochondria are highly dynamic organelles... (Review)
Review
As the cell's energy factory and metabolic hub, mitochondria are critical for ATP synthesis to maintain cellular function. Mitochondria are highly dynamic organelles that continuously undergo fusion and fission to alter their size, shape, and position, with mitochondrial fusion and fission being interdependent to maintain the balance of mitochondrial morphological changes. However, in response to metabolic and functional damage, mitochondria can grow in size, resulting in a form of abnormal mitochondrial morphology known as megamitochondria. Megamitochondria are characterized by their considerably larger size, pale matrix, and marginal cristae structure and have been observed in various human diseases. In energy-intensive cells like hepatocytes or cardiomyocytes, the pathological process can lead to the growth of megamitochondria, which can further cause metabolic disorders, cell damage and aggravates the progression of the disease. Nonetheless, megamitochondria can also form in response to short-term environmental stimulation as a compensatory mechanism to support cell survival. However, extended stimulation can reverse the benefits of megamitochondria leading to adverse effects. In this review, we will focus on the findings of the different roles of megamitochondria, and their link to disease development to identify promising clinical therapeutic targets.
Topics: Humans; Mitochondrial Swelling; Mitochondria; Metabolic Diseases; Hepatocytes; Mitochondrial Membranes; Mitochondrial Dynamics
PubMed: 37276954
DOI: 10.1016/j.mito.2023.06.001