An official website of the United States government
Mitochondria are hypothesized to display a biphasic response to reactive oxygen species (ROS) exposure. In this study, we evaluated the time course changes in mitochondrial performance and oxidative stress in house mice following X-irradiation. Forty-eight mice were equally divided among six groups, including a nonirradiated control and five experimental groups that varied in time between X-ray exposure and euthanasia (1 h and 1, 4, 7, and 10 days after X-irradiation). We measured parameters associated with mitochondrial respiratory function and ROS emission from isolated liver and skeletal muscle mitochondria and levels of oxidative damage and antioxidants in liver, skeletal muscle, and heart tissues. Mitochondrial function dropped initially after X-irradiation but recovered quickly and was elevated 10 days after the exposure. Hydrogen peroxide production, lipid peroxidation, and protein carbonylation showed inverse U-shaped curves, with levels returning to control or lower than control, 10 days after X-irradiation. Enzymatic antioxidants and markers for mitochondrial biogenesis exhibited a tissue-specific response after irradiation. These data provide the first chronological description of the mitohormetic response after a mild dose of irradiation and highlight the protective response that cells display to ROS exposure. This study also provides valuable information and application for future mitochondrial and oxidative stress studies in numerous physiological settings.
more »
« less
Transient absorption spectroscopy and imaging of redox in muscle mitochondria
Mitochondrial redox is an important indicator of cell metabolism and health, with implications in cancer, diabetes, aging, neurodegenerative diseases, and mitochondrial disease. The most common method to observe redox of individual cells and mitochondria is through fluorescence of NADH and FAD+, endogenous cofactors serve as electron transport inputs to the mitochondrial respiratory chain. Yet this leaves out redox within the respiratory chain itself. To a degree, the missing information can be filled in by exogenous fluorophores, but at the risk of disturbed mitochondrial permeability and respiration. Here we show that variations in respiratory chain redox can be detected up by visible-wavelength transient absorption microscopy (TAM). In TAM, the selection of pump and probe wavelengths can provide multiphoton imaging contrast between non-fluorescent molecules. Here, we applied TAM with a pump at 520nm and probe at 450nm, 490nm, and 620nm to elicit redox contrast from mitochondrial respiratory chain hemeproteins. Experiments were performed with reduced and oxidized preparations of isolated mitochondria and whole muscle fibers, using mitochondrial fuels (malate, pyruvate, and succinate) to set up physiologically relevant oxidation levels. TAM images of muscle fibers were analyzed with multivariate curve resolution (MCR), revealing that the response at 620nm probe provides the best redox contrast and the most consistent response between whole cells and isolated mitochondria.
more »
« less
- Award ID(s):
- 1943595
- PAR ID:
- 10369252
- Publisher / Repository:
- Optical Society of America
- Date Published:
- Journal Name:
- Biomedical Optics Express
- Volume:
- 13
- Issue:
- 4
- ISSN:
- 2156-7085
- Format(s):
- Medium: X Size: Article No. 2103
- Size(s):
- Article No. 2103
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca 2+ , mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca 2+ overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca 2+ -sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.more » « less
-
Mitochondria play a central role in muscle metabolism and function. A unique family of iron–sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3–NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions.more » « less
-
Alzheimer’s disease (AD) includes the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aβ in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aβ1-40 and Aβ1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aβ1-40 and Aβ1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aβ1-42 had more adverse effects than Aβ1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aβ1-42. Mitochondrial dysfunction induced by Aβ was associated with apoptosis of the cells. The effects of Aβ1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aβ1-40 and Aβ1-42 significantly increased Ca2+ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aβ on cell survival and mitochondria function in cardiac cells.more » « less
-
Coenzyme Q (CoQ) is an essential redox-active lipid that plays a major role in the electron transport chain, driving mitochondrial ATP synthesis. In Saccharomyces cerevisiae (yeast), CoQ biosynthesis occurs exclusively in the mitochondrial matrix via a large protein-lipid complex, the CoQ synthome, comprised of CoQ itself, late-stage CoQ-intermediates, and the polypeptides Coq3-Coq9 and Coq11. Coq11 is suggested to act as a negative modulator of CoQ synthome assembly and CoQ synthesis, as its deletion enhances Coq polypeptide content, produces an enlarged CoQ synthome, and restores respiration in mutants lacking the CoQ chaperone polypeptide, Coq10. The CoQ synthome resides in specific niches within the inner mitochondrial membrane, termed CoQ domains, that are often located adjacent to the endoplasmic reticulum-mitochondria encounter structure (ERMES). Loss of ERMES destabilizes the CoQ synthome and renders CoQ biosynthesis less efficient. Here we show that deletion of COQ11 suppresses the respiratory deficient phenotype of select ERMES mutants, results in repair and reorganization of the CoQ synthome, and enhances mitochondrial CoQ domains. Given that ER-mitochondrial contact sites coordinate CoQ biosynthesis, we used a Split-MAM (Mitochondrial Associated Membrane) artificial tether consisting of an ER-mitochondrial contact site reporter, to evaluate the effects of artificial membrane tethers on CoQ biosynthesis in both wild-type and ERMES mutant yeast strains. Overall, this work identifies the deletion of COQ11 as a novel suppressor of phenotypes associated with ERMES deletion mutants and indicates that ER-mitochondria tethers influence CoQ content and turnover, highlighting the role of membrane contact sites in regulating mitochondrial respiratory homeostasis.more » « less
