Epigenetic erosion of H4K20me1 induced by inflammation drives aged stem cell ferroptosis.

Aging is associated with a decline in stem cell functionality and number across the organism. In this study, we aimed to further unravel Muscle Stem Cells (MuSCs) aging by assessing how systemic factors influence MuSC fate decisions through long-term epigenetic landscape remodelling. As aging is intricately linked to a pro-inflammatory shift, we studied the epigenetic effects of inflammatory signals in MuSCs and measured decreased H4K20me1 levels. This loss disrupts MuSC quiescence, largely through epigenetic silencing of Notch target genes. In the setting of inflammatory signals or aging, the lack of Kmt5a and the subsequent absence of de novoH4K20me1 culminate in cell death by ferroptosis. Aged MuSCs manifest abnormal iron metabolism and reduced Gpx4 levels, resulting in the accumulation of intracellular iron, increased reactive oxygen species, genomic instability, and lipid peroxidation. We showed that ferroptosis is the predominant mode of cell death in aged MuSCs, with remarkably high levels of lipid peroxidation; a phenomenon we also observed in aged hematopoietic stem cells. Implementing preventative strategies to inhibit systemic inflammation prevented aged MuSC ferroptosis, preserving their numbers and regenerative capabilities. This intervention significantly enhanced aged muscle regeneration and strength recovery and extended both lifespan and healthspan in mice. This study delineates a previously underappreciated fate trajectory for stem cell aging, and offers meaningful insights into the treatment of age-related disorders.

Osmo-respiratory compromise in the mosshead sculpin (Clinocottus globiceps): effects of temperature, hypoxia, and re-oxygenation on rates of diffusive water flux and oxygen uptake.

In nature, mosshead sculpins (Clinocottus globiceps) are challenged by fluctuations in temperature and oxygen levels in their environment. However, it is unclear how mosshead sculpins modulate the permeability of their branchial epithelia to water and O2 in response to temperature or hypoxia stress. Acute decrease in temperature from 13 to 6 oC reduced diffusive water flux rate by 22% and MO2 by 51%, whereas acute increase in temperature from 13 to 25 oC increased diffusive water flux rate by 217% and MO2 by 140%, yielding overall Q10 values of 2.08 and 2.47 respectively. Acute reductions in oxygen tension from >95% to 20% or 10% air saturation did not impact diffusive water flux rates, however, MO2 was reduced significantly by 36% and 65% respectively. During 1-h or 3-h recovery periods diffusive water flux rates were depressed while MO2 exhibited overshoots beyond the normoxic control level. Many responses differed from those seen in our parallel earlier study on the tidepool sculpin, a cottid with similar hypoxia tolerance but much smaller gill area that occupies a similar environment. Overall, our data suggest that during temperature stress, diffusive water flux rates and MO2 follow the traditional osmo-respiratory compromise pattern, but during hypoxia and re-oxygenation stress, diffusive water flux rates are decoupled from MO2.

The Caenorhabditis elegans innexin INX-20 regulates nociceptive behavioral sensitivity.

Organisms rely on chemical cues in their environment to indicate the presence or absence of food, reproductive partners, predators, or other harmful stimuli. In the nematode Caenorhabditis elegans, the bilaterally symmetric pair of ASH sensory neurons serves as the primary nociceptors. ASH activation by aversive stimuli leads to backward locomotion and stimulus avoidance. We previously reported a role for guanylyl cyclases in dampening nociceptive sensitivity that requires an innexin-based gap junction network to pass cGMP between neurons. Here, we report that animals lacking function of the gap junction component INX-20 are hypersensitive in their behavioral response to both soluble and volatile chemical stimuli that signal through G protein-coupled receptor pathways in ASH. We find that expressing inx-20 in the ADL and AFD sensory neurons is sufficient to dampen ASH sensitivity, which is supported by new expression analysis of endogenous INX-20 tagged with mCherry via the CRISPR-Cas9 system. Although ADL does not form gap junctions directly with ASH, it does so via gap junctions with the interneuron RMG and the sensory neuron ASK. Ablating either ADL or RMG and ASK also resulted in nociceptive hypersensitivity, suggesting an important role for RMG/ASK downstream of ADL in the ASH modulatory circuit. This work adds to our growing understanding of the repertoire of ways by which ASH activity is regulated via its connectivity to other neurons and identifies a previously unknown role for ADL and RMG in the modulation of aversive behavior.

A reversible mitochondrial complex I thiol switch mediates hypoxic avoidance behavior in C. elegans.

C. elegans react to metabolic distress caused by mismatches in oxygen and energy status via distinct behavioral responses. At the molecular level, these responses are coordinated by under-characterized, redox-sensitive processes, thought to initiate in mitochondria. Complex I of the electron transport chain is a major site of reactive oxygen species (ROS) production and is canonically associated with oxidative damage following hypoxic exposure. Here, we use a combination of optogenetics and CRISPR/Cas9-mediated genome editing to exert spatiotemporal control over ROS production. We demonstrate a photo-locomotory remodeling of avoidance behavior by local ROS production due to the reversible oxidation of a single thiol on the complex I subunit NDUF-2.1. Reversible thiol oxidation at this site is necessary and sufficient for the behavioral response to hypoxia, does not respond to ROS produced at more distal sites, and protects against lethal hypoxic exposure. Molecular modeling suggests that oxidation at this thiol residue alters the ability for NDUF-2.1 to coordinate electron transfer to coenzyme Q by destabilizing the Q-binding pocket, causing decreased complex I activity. Overall, site-specific ROS production regulates behavioral responses and these findings provide a mechanistic target to suppress the detrimental effects of hypoxia.

Iron Dysregulation in Mitochondrial Dysfunction and Alzheimer's Disease.

Alzheimer's disease (AD) is a devastating progressive neurodegenerative disease characterized by neuronal dysfunction, and decreased memory and cognitive function. Iron is critical for neuronal activity, neurotransmitter biosynthesis, and energy homeostasis. Iron accumulation occurs in AD and results in neuronal dysfunction through activation of multifactorial mechanisms. Mitochondria generate energy and iron is a key co-factor required for: (1) ATP production by the electron transport chain, (2) heme protein biosynthesis and (3) iron-sulfur cluster formation. Disruptions in iron homeostasis result in mitochondrial dysfunction and energetic failure. Ferroptosis, a non-apoptotic iron-dependent form of cell death mediated by uncontrolled accumulation of reactive oxygen species and lipid peroxidation, is associated with AD and other neurodegenerative diseases. AD pathogenesis is complex with multiple diverse interacting players including Aß-plaque formation, phosphorylated tau, and redox stress. Unfortunately, clinical trials in AD based on targeting these canonical hallmarks have been largely unsuccessful. Here, we review evidence linking iron dysregulation to AD and the potential for targeting ferroptosis as a therapeutic intervention for AD.

Osmorespiratory Compromise in Zebrafish (Danio rerio): Effects of Hypoxia and Acute Thermal Stress on Oxygen Consumption, Diffusive Water Flux, and Sodium Net Loss Rates.

The traditional thesis of the osmorespiratory compromise is that low branchial water and ion permeability would be traded off for increased O2 permeability at times of elevated O2 demand. However, there is growing evidence of independent regulation of these permeabilities in hypoxia-tolerant fish. Using 0.5-g zebrafish previously maintained under normoxia at 25°C, we investigated responses to acute temperature challenges (15°C or 35°C), acute hypoxia (15 min at 10% or 5% air saturation), as well as longer-term exposures to 10% hypoxia, on O2 consumption (MO2), diffusive water flux, and net sodium loss rates. Exposure to 35°C increased, and 15°C decreased all three rates, with diffusive water flux showing the lowest temperature sensitivity, and Na+ loss the greatest. Acute 10% and 5% hypoxia increased diffusive water flux and net Na+ loss, and it reduced MO2. All these responses reflected the traditional osmorespiratory compromise. However, during prolonged 10% hypoxia, MO2 recovered, diffusive water flux decreased below control levels, and Na+ loss rate remained elevated, even during posthypoxia recovery. Overall, zebrafish do not fit standard patterns previously seen in either hypoxia-tolerant or -intolerant fish but are clearly able to adjust the effective permeabilities of their gills to O2, water, and ions independently during acute temperature and hypoxia exposures.

Interactive effects of temperature and hypoxia on diffusive water flux and oxygen uptake rate in the tidepool sculpin, Oligocottus maculosus.

The osmorespiratory compromise hypothesis posits that respiratory epithelial characteristics and physiological regulatory mechanisms which promote gas permeability also increase permeability to ions and water. The hypothesis therefore predicts that physiological responses which increase effective gas permeability will result in increased effective ion and water permeabilities. Though analyses of water and gas effective permeabilities using high temperature have generally supported the hypothesis, water permeability responses to hypoxia remain equivocal and the combination of high temperature and hypoxia untested. We measured diffusive water flux (DWF) and oxygen uptake rate (Mo2) in response to acute temperature change, hypoxia, and the combination of high temperature and hypoxia in a hypoxia-tolerant intertidal fish, the tidepool sculpin (Oligocottus maculosus). In support of the osmorespiratory compromise hypothesis, Mo2 and DWF increased with temperature. In contrast, DWF decreased with hypoxia at a constant temperature, a result consistent with previously observed decoupling of water and gas effective permeabilities during hypoxia exposure in some hypoxia tolerant fishes. However, DWF levels during simultaneous high temperature and hypoxia exposure were not different from fish exposed to high temperature in normoxia, possibly suggesting a failure of the mechanism responsible for down-regulating DWF in hypoxia. These results, together with time-course analysis of hypoxia exposure and normoxic recovery, suggest that tidepool sculpins actively downregulate effective water permeability in hypoxia but the mechanism fails with multi-stressor exposure. Future investigations of the mechanistic basis of the regulation of gill permeability will be key to understanding the role of this regulatory ability in the persistence of this species in the dynamic intertidal environment.

Ionoregulatory aspects of the hypoxia-induced osmorespiratory compromise in the euryhaline Atlantic killifish (Fundulus heteroclitus): the effects of salinity.

The osmorespiratory compromise is a physiological trade-off between the characteristics of the gill that promote respiratory gas exchange and those that limit passive flux of ions and water with the environment. In hypoxia, changes in gill blood flow patterns and functional surface area that increase gas transfer can promote an exacerbation in ion and water flux. Our goal was to determine whether the osmorespiratory compromise is flexible, depending on environmental salinity (fresh, isosmotic and sea water) and oxygen levels (hypoxia) in euryhaline killifish, Fundulus heteroclitus Plasma ion concentrations were minimally affected by hypoxia, indicating a maintenance of osmoregulatory homeostasis. In freshwater killifish, hypoxia exposure reduced branchial Na+/K+-ATPase and NEM-sensitive ATPase activities, as well as diffusive water flux rates. Unidirectional Na+ influx and Na+ efflux decreased during hypoxia in freshwater, but net Na+ flux remained unchanged. Net loss rates of Cl-, K+ and ammonia were also attenuated in hypoxia, suggesting both transcellular and paracellular reductions in permeability. These reductions appeared to be regulated phenomena as fluxes were restored immediately in normoxia. Na+ flux rates increased during hypoxia in 11 ppt, but decreased in 35 ppt, the latter suggesting a similar response to hypoxia to that in freshwater. In summary, freshwater and seawater killifish experience a reduction in gill permeability, as seen in other hypoxia-tolerant species. Fish acclimated to isosmotic salinity increased Na+ influx and efflux rates, as well as paracellular permeability in hypoxia, responses in accord with the predictions of the classic osmorespiratory compromise.

Reverse translation: effects of acclimation temperature and acute temperature challenges on oxygen consumption, diffusive water flux, net sodium loss rates, Q10 values and mass scaling coefficients in the rainbow trout (Oncorhynchus mykiss).

Our understanding is limited on how fish adjust the effective permeability of their branchial epithelium to ions and water while altering O2 uptake rate (MO2) with acute and chronic changes in temperature. We investigated the effects of acclimation temperature (8 °C, 13 °C and 18 °C) and acute temperature challenges [acute rise (acclimated at 8 °C, measured at 13 °C and 18 °C), acute drop (acclimated at 18 °C, measured at 8 °C and 13 °C) and intermediate (acclimated at 13 °C, measured at 8 °C and 18 °C)] on routine MO2, diffusive water flux, and net sodium loss rates in 24-h fasted rainbow trout (Oncorhynchus mykiss). In the temperature challenge tests, measurements were made during the first hour. In acclimated trout at all temperatures, allometric mass scaling coefficients were much higher for diffusive water flux than for MO2. Furthermore, the diffusive water flux rate was more responsive (overall Q10 = 2.75) compared to MO2 (Q10 = 1.80) over the 8-18 °C range, and for both, Q10 values were greater at 8-13 °C than at 13-18 °C. The net Na+ flux rates were highly sensitive to acclimation temperature with an overall Q10 of 3.01 for 8-18 °C. In contrast, very different patterns occurred in trout subjected to acute temperature challenges. The net Na+ flux rate was temperature-insensitive with a Q10 around 1.0. Both MO2 and diffusive water flux rates exhibited lower Q10 values than for the acclimated rates in response to either acute increases or decreases in temperature. These results fit Pattern 5 of Precht (undercompensation, reverse effect) and more precisely Pattern IIB of Prosser (reverse translation). These inverse compensatory patterns suggest that trout do not alter their rates very much when undergoing acute thermal challenges (diurnal fluctuations, migration through the thermocline). The greater changes seen with acclimation may be adaptive to long-term seasonal changes in temperature. We discuss the roles of aquaporins, spontaneous activity, and recent feeding in these responses.

Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O2•-) and singlet oxygen (1O2) quantum yields with irradiation at 561 nm. The formation of the O2•--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O2•- quantum yield (ΦO2•-) of SuperNova was determined to be 1.5 × 10-3, KillerRed was 0.97 × 10-3, and mCherry 1.2 × 10-3. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 µM O2•-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O2 quantum yield (Φ1O2) for SuperNova to be 22.0 × 10-3, KillerRed 7.6 × 10-3, and mCherry 5.7 × 10-3. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.

Physiologic Implications of Reactive Oxygen Species Production by Mitochondrial Complex I Reverse Electron Transport.

Mitochondrial reactive oxygen species (ROS) can be either detrimental or beneficial depending on the amount, duration, and location of their production. Mitochondrial complex I is a component of the electron transport chain and transfers electrons from NADH to ubiquinone. Complex I is also a source of ROS production. Under certain thermodynamic conditions, electron transfer can reverse direction and reduce oxygen at complex I to generate ROS. Conditions that favor this reverse electron transport (RET) include highly reduced ubiquinone pools, high mitochondrial membrane potential, and accumulated metabolic substrates. Historically, complex I RET was associated with pathological conditions, causing oxidative stress. However, recent evidence suggests that ROS generation by complex I RET contributes to signaling events in cells and organisms. Collectively, these studies demonstrate that the impact of complex I RET, either beneficial or detrimental, can be determined by the timing and quantity of ROS production. In this article we review the role of site-specific ROS production at complex I in the contexts of pathology and physiologic signaling.

The osmorespiratory compromise in rainbow trout (Oncorhynchus mykiss): The effects of fish size, hypoxia, temperature and strenuous exercise on gill diffusive water fluxes and sodium net loss rates.

In the context of the osmorespiratory compromise, hypoxia and temperature have been little studied relative to exercise, and diffusive water flux rates (as assessed by 3H2O efflux) have received almost no attention. We investigated the effects of fish size, hypoxia, exercise and acute temperature increase on diffusive water flux rates and net sodium loss rates in juvenile rainbow trout. Trout weighing 13-50 g were used to determine the effects of fish size under normoxia. Thereafter 25-50 g trout were selected to assess the effects of different hypoxia levels (3.15, 5.25 and 8.40 KPa), time course of hypoxia (1 h 8.40 KPa, 3 h 8.40 KPa, 1 h 8.40 KPa +1 h normoxic recovery, and 1 h 8.40 KPa + 3 h normoxic recovery), strenuous exercise (5 min) and acute temperature challenge (transfer from 8 °C to 13 °C or to 18 °C). Small fish (13 g) had higher diffusive water flux rates than larger fish, turning over >100% of their fractional body water pool per hour against 34% per hour for 50 g fish. Hypoxic exposure exerted a biphasic effect, increasing the diffusive water flux rate at 8.40 KPa and 5.25 KPa, while returning it to control levels at 3.15 KPa. All the levels of hypoxia increased net Na+ loss. One hour hypoxia (8.40 KPa) increased diffusive water flux rate while prolonged 3 h hypoxia (8.40 KPa), and short or prolonged normoxic recovery returned diffusive water flux rates to control levels. All the treatments over the time course of hypoxia and normoxic recovery increased net Na+ loss rates. Strenuous exercise increased both the diffusive water flux and net Na+ loss rates. Acute temperature rise increased diffusive water flux rates, with Q10 values of 4.03 for 8 to 13 °C and 2.16 for 8 to 18 °C, but the net Na+ loss rate did not change. There was no significant correlation between diffusive water flux rate and net Na+ loss rates at different hypoxia levels, over the course of hypoxia and normoxic recovery, or during acute temperature stress. In contrast, we observed a significant correlation between diffusive water flux and net Na+ loss rates following exercise. Overall, diffusive water flux and sodium loss were regulated differently during acute temperature challenge and hypoxia, while following exercise the two parameters were regulated in a similar fashion.

Combined effects of cadmium, temperature and hypoxia-reoxygenation on mitochondrial function in rainbow trout (Oncorhynchus mykiss).

Although aquatic organisms face multiple environmental stressors that may interact to alter adverse outcomes, our knowledge of stressor-stressor interaction on cellular function is limited. We investigated the combined effects of cadmium (Cd), hypoxia-reoxygenation (H-R) and temperature on mitochondrial function. Liver mitochondria from juvenile rainbow trout were exposed to Cd (0-20µM) and H-R (0 and 5min) at 5, 13 and 25°C followed by measurements of mitochondrial Cd load, volume, complex І active (A)↔deactive (D) transition, membrane potential, ROS release and ultrastructural changes. At high temperature Cd exacerbated H-R-imposed reduction of maximal complex I (CI) respiration whereas at low temperature 5 and 10µM stimulated maximal CI respiration post H-R. The basal respiration showed a biphasic response at high temperatures with low Cd concentrations reducing the stimulatory effect of H-R and high concentrations enhancing this effect. At low temperature Cd monotonically enhanced H-R-induced stimulation of basal respiration. Cd and H-R reduced both the P/O ratio and the RCR at all 3 temperatures. Temperature rise alone increased mitochondrial Cd load and toxicity, but combined H-R and temperature exposure reduced mitochondrial Cd load but surprisingly exacerbated the mitochondrial dysfunction. Mitochondrial dysfunction induced by H-R was associated with swelling of the organelle and blocking of conversion of CІ D to A form. However, low amounts of Cd protected against H-R induced swelling and prevented the inhibition of H-R-induced CI D to A transition. Both H-R and Cd dissipated mitochondrial membrane potential Δψm and damaged mitochondrial structure. We observed increased reactive oxygen species (H2O2) release that together with the protection afforded by EGTA, vitamin E and N-acetylcysteine against the Δψm dissipation suggested direct involvement of Cd and oxidative stress. Overall, our findings indicate that mitochondrial sensitivity to Cd toxicity was enhanced by the effects of H-R and temperature, and changes in mitochondrial Cd load did not always explain this effect.

Bioenergetic and volume regulatory effects of mitoKATP channel modulators protect against hypoxia-reoxygenation-induced mitochondrial dysfunction.

The mitochondrial ATP-sensitive K(+) (mitoKATP) channel plays a significant role in mitochondrial physiology and protects against ischemic reperfusion injury in mammals. Although fish frequently face oxygen fluctuations in their environment, the role of the mitoKATP channel in regulating the responses to oxygen stress is rarely investigated in this class of animals. To elucidate whether and how the mitoKATP channel protects against hypoxia-reoxygenation (H-R)-induced mitochondrial dysfunction in fish, we first determined the mitochondrial bioenergetic effects of two key modulators of the channel, diazoxide and 5-hydroxydecanoate (5-HD), using a wide range of doses. Subsequently, the effects of low and high doses of the modulators on mitochondrial bioenergetics and volume under normoxia and after H-R using buffers with and without magnesium and ATP (Mg-ATP) were tested. In the absence of Mg-ATP (mitoKATP channel open), both low and high doses of diazoxide improved mitochondrial coupling, but only the high dose of 5-HD reversed the post-H-R coupling-enhancing effect of diazoxide. In the presence of Mg-ATP (mitoKATP channel closed), diazoxide at the low dose improved coupling post-H-R, and this effect was abolished by 5-HD at the low dose. Interestingly, both low and high doses of diazoxide reversed H-R-induced swelling under mitoKATP channel open conditions, but this effect was not sensitive to 5-HD. Under mitoKATP channel closed conditions, diazoxide at the low dose protected the mitochondria from H-R-induced swelling and 5-HD at the low dose reversed this effect. In contrast, diazoxide at the high dose failed to reduce the swelling caused by H-R, and the addition of the high dose of 5-HD enhanced mitochondrial swelling. Overall, our study showed that in the presence of Mg-ATP, both opening of mitoKATP channels and bioenergetic effects of diazoxide were protective against H-R in fish mitochondria, while in the absence of Mg-ATP only the bioenergetic effect of diazoxide was protective.

Hypoxia-reoxygenation differentially alters the thermal sensitivity of complex I basal and maximal mitochondrial oxidative capacity.

Hypoxia-reoxygenation (H-R) transitions and temperature fluctuations occur frequently in biological systems and likely interact to alter cell function. To test how H-R modulates mitochondrial function at different temperatures we measured the effects of H-R on isolated fish liver mitochondrial oxidation rates over a wide temperature range (5-25°C). Subsequently, the mechanisms underlying H-R induced mitochondrial responses were examined. H-R inhibited the complex I (CI) maximal (state 3) and stimulated the basal (state 4) mitochondrial oxidation rates with temperature enhancing both effects. As a result, the thermal sensitivity (Q10) for CI maximal respiration was reduced while that for basal respiration was increased by H-R. H-R reduced both the coupling and phosphorylation efficiencies more profoundly at high temperature suggesting that mitochondria were more resistant to H-R at low temperature. The H-R induced mitochondrial impairments were associated with increased reactive oxygen species (ROS) production and proton leak, dissipation of membrane potential, and loss of structural integrity of the organelles. Overall, our study provides insight into the mechanisms of H-R induced mitochondrial morphofunctional disruption and shows that the moderation of effects of H-R on oxidative phosphorylation by temperature depends on the functional state.

Modulation of cadmium-induced mitochondrial dysfunction and volume changes by temperature in rainbow trout (Oncorhynchus mykiss).

We investigated how temperature modulates cadmium (Cd)-induced mitochondrial bioenergetic disturbances, metal accumulation and volume changes in rainbow trout (Oncorhynchus mykiss). In the first set of experiments, rainbow trout liver mitochondrial function and Cd content were measured in the presence of complex I substrates, malate and glutamate, following exposure to Cd (0-100 µM) at three (5, 13 and 25 °C) temperatures. The second set of experiments assessed the effect of temperature on Cd-induced mitochondrial volume changes, including the underlying mechanisms, at 15 and 25 °C. Although temperature stimulated both state 3 and 4 rates of respiration, the coupling efficiency was reduced at temperature extremes due to greater inhibition of state 3 at low temperature and greater stimulation of state 4 at the high temperature. Cadmium exposure reduced the stimulatory effect of temperature on state 3 respiration but increased that on state 4, consequently exacerbating mitochondrial uncoupling. The interaction of Cd and temperature yielded different responses on thermal sensitivity of state 3 and 4 respiration; the Q10 values for state 3 respiration increased at low temperature (5-13 °C) while those for state 4 increased at high temperature (13-25 °C). Importantly, the mitochondria accumulated more Cd at high temperature suggesting that the observed greater impairment of oxidative phosphorylation with temperature was due, at least in part, to a higher metal burden. Cadmium-induced mitochondrial volume changes were characterized by an early phase of contraction followed by swelling, with temperature changing the kinetics and intensifying the effects. Lastly, using specific modulators of mitochondrial ion channels, we demonstrated that the mitochondrial volume changes were associated with Cd uptake via the mitochondrial calcium uniporter (MCU) without significant contribution of the permeability transition pore and/or potassium channels. Overall, it appears that high temperature exacerbates Cd-induced mitochondrial dysfunction and volume changes in part by increasing metal uptake through the MCU.

Effects of hypoxia-cadmium interactions on rainbow trout (Oncorhynchus mykiss) mitochondrial bioenergetics: attenuation of hypoxia-induced proton leak by low doses of cadmium.

The goal of the present study was to elucidate the modulatory effects of cadmium (Cd) on hypoxia/reoxygenation-induced mitochondrial dysfunction in light of the limited understanding of the mechanisms of multiple stressor interactions in aquatic organisms. Rainbow trout (Oncorhynchus mykiss) liver mitochondria were isolated and energized with complex I substrates (malate-glutamate), and exposed to hypoxia (0>PO2<2 Torr) for 0-60 min followed by reoxygenation and measurement of coupled and uncoupled respiration and complex I enzyme activity. Thereafter, 5 min hypoxia was used to probe interactions with Cd (0-20 µmol l(-1)) and to test the hypothesis that deleterious effects of hypoxia/reoxygenation on mitochondria were mediated by reactive oxygen species (ROS). Hypoxia/reoxygenation inhibited state 3 and uncoupler-stimulated (state 3u) respiration while concomitantly stimulating states 4 and 4ol (proton leak) respiration, thus reducing phosphorylation and coupling efficiencies. Low doses of Cd (≤5 µmol l(-1)) reduced, while higher doses enhanced, hypoxia-stimulated proton leak. This was in contrast to the monotonic enhancement by Cd of hypoxia/reoxygenation-induced reductions of state 3 respiration, phosphorylation efficiency and coupling. Mitochondrial complex I activity was inhibited by hypoxia/reoxygenation, hence confirming the impairment of at least one component of the electron transport chain (ETC) in rainbow trout mitochondria. Similar to the effect on state 4 and proton leak, low doses of Cd partially reversed the hypoxia/reoxygenation-induced complex I activity inhibition. The ROS scavenger and sulfhydryl group donor N-acetylcysteine, administrated immediately prior to hypoxia exposure, reduced hypoxia/reoxygenation-stimulated proton leak without rescuing the inhibited state 3 respiration, suggesting that hypoxia/reoxygenation influences distinct aspects of mitochondria via different mechanisms. Our results indicate that hypoxia/reoxygenation impairs the ETC and sensitizes mitochondria to Cd via mechanisms that involve, at least in part, ROS. Moreover, we provide, for the first time in fish, evidence for a hormetic effect of Cd on mitochondrial bioenergetics--the attenuation of hypoxia/reoxygenation-stimulated proton leak and partial rescue of complex I inhibition by low Cd doses.