5-Hydroxymethylfurfural reduces skeletal muscle superoxide production and modifies force production in rats exposed to hypobaric hypoxia.

Decreased blood-tissue oxygenation at high altitude (HA) increases mitochondrial oxidant production and reduces exercise capacity. 5-Hydroxymethylfurfural (5-HMF) is an antioxidant that increases hemoglobin's binding affinity for oxygen. For these reasons, we hypothesized that 5-HMF would improve muscle performance in rats exposed to a simulated HA of ~5500 m. A secondary objective was to measure mitochondrial activity and dynamic regulation of fission and fusion because they are linked processes impacted by HA. Fisher 344 rats received 5-HMF (40 mg/kg/day) or vehicle during exposure to sea level or HA for 72 h. Right ankle plantarflexor muscle function was measured pre- and post-exposure. Post-exposure measurements included arterial blood gas and complete blood count, flexor digitorum brevis myofiber superoxide production and mitochondrial membrane potential (ΔΨm), and mitochondrial dynamic regulation in the soleus muscle. HA reduced blood oxygenation, increased superoxide levels and lowered ΔΨm, responses that were accompanied by decreased peak isometric torque and force production at frequencies >75 Hz. 5-HMF increased isometric force production and lowered oxidant production at sea level. In HA exposed animals, 5-HMF prevented a decline in isometric force production at 75-125 Hz, prevented an increase in superoxide levels, further decreased ΔΨm, and increased mitochondrial fusion 2 protein expression. These results suggest that 5-HMF may prevent a decrease in hypoxic force production during submaximal isometric contractions by an antioxidant mechanism.

Mitochondrial Fission as a Therapeutic Target for Metabolic Diseases: Insights into Antioxidant Strategies.

Mitochondrial fission is a crucial process in maintaining metabolic homeostasis in normal physiology and under conditions of stress. Its dysregulation has been associated with several metabolic diseases, including, but not limited to, obesity, type 2 diabetes (T2DM), and cardiovascular diseases. Reactive oxygen species (ROS) serve a vital role in the genesis of these conditions, and mitochondria are both the main sites of ROS production and the primary targets of ROS. In this review, we explore the physiological and pathological roles of mitochondrial fission, its regulation by dynamin-related protein 1 (Drp1), and the interplay between ROS and mitochondria in health and metabolic diseases. We also discuss the potential therapeutic strategies of targeting mitochondrial fission through antioxidant treatments for ROS-induced conditions, including the effects of lifestyle interventions, dietary supplements, and chemicals, such as mitochondrial division inhibitor-1 (Mdivi-1) and other mitochondrial fission inhibitors, as well as certain commonly used drugs for metabolic diseases. This review highlights the importance of understanding the role of mitochondrial fission in health and metabolic diseases, and the potential of targeting mitochondrial fission as a therapeutic approach to protecting against these conditions.

Dose-Dependent Effects of Radiation on Mitochondrial Morphology and Clonogenic Cell Survival in Human Microvascular Endothelial Cells.

To better understand radiation-induced organ dysfunction at both high and low doses, it is critical to understand how endothelial cells (ECs) respond to radiation. The impact of irradiation (IR) on ECs varies depending on the dose administered. High doses can directly damage ECs, leading to EC impairment. In contrast, the effects of low doses on ECs are subtle but more complex. Low doses in this study refer to radiation exposure levels that are below those that cause immediate and necrotic damage. Mitochondria are the primary cellular components affected by IR, and this study explored their role in determining the effect of radiation on microvascular endothelial cells. Human dermal microvascular ECs (HMEC-1) were exposed to varying IR doses ranging from 0.1 Gy to 8 Gy (~0.4 Gy/min) in the AFRRI 60-Cobalt facility. Results indicated that high doses led to a dose-dependent reduction in cell survival, which can be attributed to factors such as DNA damage, oxidative stress, cell senescence, and mitochondrial dysfunction. However, low doses induced a small but significant increase in cell survival, and this was achieved without detectable DNA damage, oxidative stress, cell senescence, or mitochondrial dysfunction in HMEC-1. Moreover, the mitochondrial morphology was assessed, revealing that all doses increased the percentage of elongated mitochondria, with low doses (0.25 Gy and 0.5 Gy) having a greater effect than high doses. However, only high doses caused an increase in mitochondrial fragmentation/swelling. The study further revealed that low doses induced mitochondrial elongation, likely via an increase in mitochondrial fusion protein 1 (Mfn1), while high doses caused mitochondrial fragmentation via a decrease in optic atrophy protein 1 (Opa1). In conclusion, the study suggests, for the first time, that changes in mitochondrial morphology are likely involved in the mechanism for the radiation dose-dependent effect on the survival of microvascular endothelial cells. This research, by delineating the specific mechanisms through which radiation affects endothelial cells, offers invaluable insights into the potential impact of radiation exposure on cardiovascular health.

Acute heat stress-indued apoptosis in mouse skeletal muscle is not associated with alteration of glutamine homeostasis.

We previously demonstrated that exposing mice to heat causes functional and ultrastructural mitochondrial alterations and apoptosis in skeletal muscle. Emerging evidence indicates that glutamine (Gln) deprivation may increase cell susceptibility to apoptosis whereas Gln supplementation may protect cells against heat stress. In this study, we investigated the effect of short-term Gln treatment on heat-induced changes in mouse skeletal muscle. Male mice received vehicle, low-dose Gln (100 mg/kg/d) or high-dose Gln (300 mg/kg/d) through daily gavage for 10 days before a heat exposure test. During heat exposure, mice displayed a hyperthermic response and no significant differences in peak core body temperature were noted across the three groups. Neither heat exposure nor pretreatment with low-dose or high-dose Gln significantly affected Gln concentrations in plasma and gastrocnemius muscles. Heat-exposed mice had significantly higher caspase 3/7 levels in gastrocnemius muscle compared to unexposed controls. Heat exposure significantly increased ROS production and mitochondrial fragmentation and decreased mitochondrial membrane potential in flexor digitorum brevis muscle. These changes were not affected by low- or high-dose Gln pretreatment. Together, acute heat stress did not disrupt Gln homeostasis in mouse skeletal muscle and Gln supplementation did not protect mouse skeletal muscle against heat-induced injury. The results of this study do not support a role of Gln in heat-induced skeletal muscle apoptosis.

L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through nitric oxide-mediated Drp1 inhibition in mouse C2C12 myoblasts.

Severe heat exposure causes mitochondrial fragmentation and dysfunction, which contribute to the pathogenesis of heat-related illness. L-citrulline is a naturally occurring amino acid and has been suggested to influence heat shock responses. This study aimed to test whether L-citrulline supplementation would preserve mitochondrial integrity and attenuate heat-induced skeletal muscle injury, and elucidate the underlying mechanisms. At 37°C, L-citrulline (2 mM) increased mitochondrial elongation in mouse C2C12 myoblasts, a process associated with a reduction in mitochondrial fission protein Drp1 levels. Mechanistic studies revealed that L-citrulline increased cellular nitric oxide (NO) levels, but not S-nitrosylation of Drp1. L-citrulline caused a decrease in phosphorylation of Drp1 at Ser 616 and an increase in phosphorylation of Drp1 at Ser 637, which resulted in a reduced mitochondrial localization of Drp1. L-NAME, a non-selective NO synthase inhibitor, abolished the increase in L-citrulline-induced NO levels and inhibited Drp1 phosphorylation changes and mitochondrial elongation, which indicates involvement of a NO-dependent pathway. Under 43°C heat stress conditions, L-citrulline prevented translocation of Drp1 to mitochondria, mitochondrial fragmentation and decreased membrane potential. Finally, L-citrulline pretreatment inhibited heat-induced reactive oxygen species (ROS) overproduction, caspase 3/7 activation, apoptotic cell death, and improved cell viability. NO inhibitor L-NAME abolished all the above protective effects of L-citrulline under heat stress. Our results suggest that L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through NO-mediated Drp1 inhibition in C2C12 myoblasts. L-citrulline may be an effective treatment for heat-related illnesses and other mitochondrial diseases.

Protective effects of dietary curcumin and astaxanthin against heat-induced ROS production and skeletal muscle injury in male and female C57BL/6J mice.

AIMS: This study aimed to: 1) investigate sex differences in heat-induced mitochondrial dysfunction, ROS production, and skeletal muscle injury in mice; 2) evaluate whether curcumin and astaxanthin, alone or together, would prevent those heat-induced changes. MAIN METHODS: Male and female C57BL/6J mice were treated with curcumin and astaxanthin for 10 days, then exposed to 39.5 °C heat for up to 3 h. Heat-induced hyperthermia, changes in mitochondrial morphology and function, and oxidative damage to skeletal muscle were evaluated. KEY FINDINGS: Although female mice had a slightly higher basal core body temperature (Tc) than male mice, peak Tc during heat exposure was significantly lower in females than in males. Heat increased ROS levels in skeletal muscle in both sexes; interestingly, the increases in ROS were greater in females than in males. Despite the above-mentioned differences, heat induced similar levels of mitochondrial fragmentation and membrane potential depolarization, caspase 3/7 activation, and injury in male and female skeletal muscle. Individual treatment of curcumin or astaxanthin did not affect basal and peak Tc but prevented heat-induced mitochondrial dysfunction, ROS increases, and apoptosis in a dose-dependent manner. Moreover, a low-dose combination of curcumin and astaxanthin, which individually showed no effect, reduced the heat-induced oxidative damage to skeletal muscle. SIGNIFICANCE: Both male and female mice can develop mitochondrial dysfunction and oxidative stress in skeletal muscle when exposed to heat stress. High doses of either curcumin or astaxanthin limit heat-induced skeletal muscle injury, but a low-dose combination of these ingredients may increase their efficacy.

Astaxanthin Protects Against Heat-induced Mitochondrial Alterations in Mouse Hypothalamus.

The hypothalamus plays an essential role in regulating whole-body energy and temperature homeostasis when adapting to environmental changes. We previously reported that heat exposure causes mitochondrial dysfunction and apoptosis in mouse skeletal muscle, and pretreatment with astaxanthin (AST), an antioxidant, prevents this effect. How the hypothalamus responds to heat stress remains largely unexplored. In this study, we investigated the effects of heat exposure on hypothalamic mitochondria in mice with and without AST pretreatment. During heat exposure, both vehicle and AST-treated mice had a hyperthermic response though no significant differences in peak core body temperature were noted between the two groups. Heat exposure induced mitochondrial fission in the hypothalamus, as manifested by increased mitochondrial fragmentation and expression of both total and phosphorylated dynamin-related protein 1. In addition, transmission electron microscopy revealed damaged and degraded mitochondria in the hypothalamus of heat-exposed mice. Heat induced apoptosis and mitophagy were further confirmed by increased formation of reactive oxygen species, activation of caspase 3/7 and expression of LC3 proteins. Moreover, heat exposure increased the expression of PINK1 and Parkin in mouse hypothalamus. In contrast, pretreatment with AST reduced these effects. These results demonstrate that heat stress-induced hypothalamic apoptosis is associated with altered mitochondrial dynamics favoring fission and mitophagy. AST protects the hypothalamus against heat-induced injury by preserving redox homeostasis and mitochondrial integrity.

Mouse liver is more resistant than skeletal muscle to heat-induced apoptosis.

During passive heat stress, shifting of blood flow from the hepato-splanchnic to peripheral regions produces less favorable physiological conditions in the liver than in the skeletal muscle. We were wondering if the two organs differ in susceptibility to heat injury and thus examined the effects of heat shock exposure on apoptotic and heat stress-related markers in the gastrocnemius muscle and liver of mice. During heat exposure, mice had a peak core body temperature of 41.1 ± 0.7 °C. Heat-exposed mice showed higher levels of reactive oxygen species (ROS), cleaved caspases, fragmented DNA, and Drp1 protein expression in the gastrocnemius muscles than control mice. These changes were not observed in the livers of heat-exposed mice. Furthermore, the levels of glucocorticoid receptor, HSP70, and HSF1 proteins were significantly elevated in the gastrocnemius muscles of heat-exposed mice compared with that of control mice. The livers of heat-exposed mice also revealed increased expression of HSP70 but no changes in the other proteins. These results demonstrate that heat exposure induces significantly lower levels of the stress response and apoptosis in the liver than in the skeletal muscle of mice. The liver tissue resistance against heat stress is associated with low levels of heat-induced ROS production and mitochondrial fission protein expression.

Glutamine depletion disrupts mitochondrial integrity and impairs C2C12 myoblast proliferation, differentiation, and the heat-shock response.

Glutamine and glucose are both oxidized in the mitochondria to supply the majority of usable energy for processes of cellular function. Low levels of plasma and skeletal muscle glutamine are associated with severe illness. We hypothesized that glutamine deficiency would disrupt mitochondrial integrity and impair cell function. C2C12 mouse myoblasts were cultured in control media supplemented with 5.6 mmol/L glucose and 2 mmol/L glutamine, glutamine depletion (Gln-) or glucose depletion (Glc-) media. We compared mitochondrial morphology and function, as well as cell proliferation, myogenic differentiation, and heat-shock response in these cells. Glc- cells exhibited slightly elongated mitochondrial networks and increased mitochondrial mass, with normal membrane potential (ΔΨm). Mitochondria in Gln- cells became hyperfused and swollen, which were accompanied by severe disruption of cristae and decreases in ΔΨm, mitochondrial mass, the inner mitochondrial membrane remodeling protein OPA1, electron transport chain complex IV protein expression, and markers of mitochondrial biogenesis and bioenergetics. In addition, Gln- increased the autophagy marker LC3B-II on the mitochondrial membrane. Notably, basal mitochondrial respiration was increased in Glc- cells as compared to control cells, whereas maximal respiration remained unchanged. In contrast, basal respiration, maximal respiration and reserve capacity were all decreased in Gln- cells. Consistent with the aforementioned mitochondrial deficits, Gln- cells had lower growth rates and myogenic differentiation, as well as a higher rate of cell death under heat stress conditions than Glc- and control cells. We conclude that glutamine is essential for mitochondrial integrity and function; glutamine depletion impairs myoblast proliferation, differentiation, and the heat-shock response.

IL-18 binding protein (IL-18BP) as a novel radiation countermeasure after radiation exposure in mice.

Recent studies suggested that radiation exposure causes local and systemic inflammatory responses and induces cell and tissue damage. We have reported that IL-18 plays an important role in radiation-induced injury. Here, we demonstrate that IL-18 binding protein (IL-18BP), a natural antagonist of IL-18, was significantly increased (1.7-63 fold) in mouse serum on day 1 after 0.5-10 Gy TBI. However, this high level of IL-18BP was not sufficient to neutralize the active IL-18 in irradiated mice, resulting in a radiation dose-dependent free IL-18 increase in these mice's serum which led to pathological alterations to the irradiated cells and tissues and finally caused animal death. Administration of recombinant human (rh) IL-18BP (1.5 mg/kg) with single (24, 48 or 72 h post-TBI) or double doses (48 h and 5 days post-TBI) subcutaneous (SC) injection increased 30-day survival of CD2F1 mice after 9 Gy TBI 12.5-25% compared with the vehicle control treated group, respectively. Furthermore, the mitigative effects of rhIL-18BP included balancing the ratio of IL-18/IL-18BP and decreasing the free IL-18 levels in irradiated mouse serum and significantly increasing blood cell counts, BM hematopoietic cellularity and stem and progenitor cell clonogenicity in mouse BM. Furthermore, IL-18BP treatment inhibited the IL-18 downstream target interferon (IFN)-γ expression in mouse BM, decreased reactive oxygen species (ROS) level in the irradiated mouse heart tissues, attenuated the stress responsive factor GDF-15 (growth differentiation factor-15) and increased the intestine protector citrulline level in total body irradiated mouse serum, implicating that IL-18BP may protect multiple organs from radiation-induced inflammation and oxidative stress. Our data suggest that IL-18 plays a key role in radiation-induced cell and tissue damage and dysfunction; and for the first time demonstrated that IL-18BP counters IL-18 activation and therefore may mitigate/treat radiation-induced multiple organ injuries and increase animal survival with a wider therapeutic window from 24 h and beyond after lethal doses of radiation exposure.

Skeletal muscle mitochondrial fragmentation and impaired bioenergetics from nutrient overload are prevented by carbon monoxide.

Nutrient excess increases skeletal muscle oxidant production and mitochondrial fragmentation that may result in impaired mitochondrial function, a hallmark of skeletal muscle insulin resistance. This led us to explore whether an endogenous gas molecule, carbon monoxide (CO), which is thought to prevent weight gain and metabolic dysfunction in mice consuming high-fat diets, alters mitochondrial morphology and respiration in C2C12 myoblasts exposed to high glucose (15.6 mM) and high fat (250 µM BSA-palmitate) (HGHF). Also, skeletal muscle mitochondrial morphology, distribution, respiration, and energy expenditure were examined in obese resistant (OR) and obese prone (OP) rats that consumed a high-fat and high-sucrose diet for 10 wk with or without intermittent low-dose inhaled CO and/or exercise training. In cells exposed to HGHF, superoxide production, mitochondrial membrane potential (ΔΨm), mitochondrial fission regulatory protein dynamin-related protein 1 (Drp1) and mitochondrial fragmentation increased, while mitochondrial respiratory capacity was reduced. CO decreased HGHF-induced superoxide production, Drp1 protein levels and mitochondrial fragmentation, maintained ΔΨm, and increased mitochondrial respiratory capacity. In comparison with lean OR rats, OP rats had smaller skeletal muscle mitochondria that contained disorganized cristae, a normal mitochondrial distribution, but reduced citrate synthase protein expression, normal respiratory responses, and a lower energy expenditure. The combination of inhaled CO and exercise produced the greatest effect on mitochondrial morphology, increasing ADP-stimulated respiration in the presence of pyruvate, and preventing a decline in resting energy expenditure. These data support a therapeutic role for CO and exercise in preserving mitochondrial morphology and respiration during metabolic overload.

Curcumin Ameliorates Heat-Induced Injury through NADPH Oxidase-Dependent Redox Signaling and Mitochondrial Preservation in C2C12 Myoblasts and Mouse Skeletal Muscle.

BACKGROUND: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the mitochondrial electron transport chain are the primary sources of reactive oxygen species (ROS). Previous studies have shown that severe heat exposure damages mitochondria and causes excessive mitochondrial ROS production that contributes to the pathogenesis of heat-related illnesses. OBJECTIVES: We tested whether the antioxidant curcumin could protect against heat-induced mitochondrial dysfunction and skeletal muscle injury, and characterized the possible mechanism. METHODS: Mouse C2C12 myoblasts and rat flexor digitorum brevis (FDB) myofibers were treated with 5 µM curcumin; adult male C57BL/6J mice received daily curcumin (15, 50, or 100 mg/kg body weight) by gavage for 10 consecutive days. We compared ROS levels and mitochondrial morphology and function between treatment and nontreatment groups under unheated or heat conditions, and investigated the upstream mechanism and the downstream effect of curcumin-regulated ROS production. RESULTS: In C2C12 myoblasts, curcumin prevented heat-induced mitochondrial fragmentation, ROS overproduction, and apoptosis (all P < 0.05). Curcumin treatment for 2 and 4 h at 37°C induced increases in ROS levels by 42% and 59% (dihydroethidium-derived fluorescence), accompanied by increases in NADPH oxidase protein expression by 24% and 32%, respectively (all P < 0.01). In curcumin-treated cells, chemical inhibition and genetic knockdown of NADPH oxidase restored ROS to levels similar to those of controls, indicating NADPH oxidase mediates curcumin-stimulated ROS production. Moreover, curcumin induced ROS-dependent shifting of the mitochondrial fission-fusion balance toward fusion, and increases in mitochondrial mass by 143% and membrane potential by 30% (both P < 0.01). In rat FDB myofibers and mouse gastrocnemius muscles, curcumin preserved mitochondrial morphology and function during heat stress, and prevented heat-induced mitochondrial ROS overproduction and tissue injury (all P < 0.05). CONCLUSIONS: Curcumin regulates ROS hormesis favoring mitochondrial fusion/elongation, biogenesis, and improved function in rodent skeletal muscle. Curcumin may be an effective therapeutic target for heat-related illness and other mitochondrial diseases.

Involvement of p53 in the Responses of Cardiac Muscle Cells to Heat Shock Exposure and Heat Acclimation.

Intense heat stress induces damage to the heart, whereas mild to moderate heat stress protects the heart against subsequent ischemic injury. The mechanisms underlying the detrimental and beneficial effects of heat stress remain unclear. In this study, we investigated the role of p53 in the responses of cardiac muscle cells to acute heat exposure and heat acclimation (HA). Heat exposure increased the levels of caspase and annexin, and levels of cytosolic, nuclear, and mitochondrial p53 protein in H9c2 cells. Pifithrin-α or pifithrin-µ reduced heat-induced apoptotic response in these cells. HA reduced localization of p53 in the mitochondria and improved cell viability during heat exposure. The effects of heat exposure and HA on p53 were further verified in vivo in mouse heart tissue. These results suggest that p53 plays a role in heat-induced apoptosis in cardiac muscle cells. The protective effect of HA against heat injury likely involves a p53-dependent mechanism.

Carbon Monoxide and Exercise Prevents Diet-Induced Obesity and Metabolic Dysregulation Without Affecting Bone.

OBJECTIVE: Carbon monoxide (CO) may counteract obesity and metabolic dysfunction in rodents consuming high-fat diets, but the skeletal effects are not understood. This study investigated whether low-dose inhaled CO (250 ppm) with or without moderate intensity aerobic exercise (3 h/wk) would limit diet-induced obesity and metabolic dysregulation and preserve bone health. METHODS: Obesity-resistant (OR) rats served as controls, and obesity-prone (OP) rats were randomized to sedentary, sedentary plus CO, exercise, or CO plus exercise. For 10 weeks, OP rats consumed a high-fat, high-sucrose diet, whereas OR rats consumed a low-fat control diet. Measurements included indicators of obesity and metabolism, bone turnover markers, femoral geometry and microarchitecture, bone mechanical properties, and tibial morphometry. RESULTS: A high-fat, high-sucrose diet led to obesity, hyperinsulinemia, and hyperleptinemia, without impacting bone. CO alone led only to a modest reduction in weight gain. Exercise attenuated weight gain and improved the metabolic profile; however, bone fragility increased. Combined CO and exercise led to body mass reduction and a metabolic state similar to control OR rats and prevented the exercise-induced increase in bone fragility. CONCLUSIONS: CO and aerobic exercise training prevent obesity and metabolic sequelae of nutrient excess while stabilizing bone physiology.

Genetic association of FKBP5 with PTSD in US service members deployed to Iraq and Afghanistan.

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder with a prevalence of more than 7% in the US population and 12% in the military. An interaction of childhood trauma with FKBP5 (a glucocorticoid-regulated immunophilin) has been reported to be associated with PTSD in the general population. However, there are few reports on the association of FKBP5 with PTSD, particularly in important high-risk population such as the military. Here, we examined the association between four single-nucleotide polymorphisms (SNPs; rs3800373, rs9296158, rs1360780, rs9470080) covering the FKBP5 gene and probable PTSD in US service members deployed to Iraq and Afghanistan, a high-risk military population (n = 3890) (Hines et al., 2014). We found that probable PTSD subjects were significantly more likely to carry the A-allele of rs3800373, G-allele of rs9296158, C-allele of rs1360780, and C-allele of rs9470080. Furthermore, the four SNPs were in one block of strong pairwise linkage disequilibrium (r = 0.91-0.96). Within the block there were two major haplotypes of CATT and AGCC (rs3800373-rs9296158-rs1360780-rs9470080) that account for 99% of haplotype diversity. The distribution of the AGCC haplotype was significantly higher in probable PTSD subjects compared to non-PTSD (p<.05). The diplotype-based analysis indicated that the AGCC carriers tended to be probable PTSD. In this study, we demonstrated the association between FKBP5 and probable PTSD in US service members deployed to Iraq and Afghanistan, indicating that FKBP5 might be a risk factor for PTSD.

Updates in PTSD Animal Models Characterization.

Post-traumatic stress disorder (PTSD) is a chronic, debilitating mental disorder afflicting more than 7% of the US population and 12% of military service members. Since the Afghanistan and Iraq wars, thousands of US service members have returned home with PTSD. Despite recent progress, the molecular mechanisms underlying the pathology of PTSD are poorly understood. To promote research on PTSD (especially its molecular mechanisms) and to set a molecular basis for discovering novel medications for this disorder, well-validated animal models are needed. However, to develop PTSD animal models is a challenging process, due to predisposing factors such as physiological, behavioral, emotional, and cognitive changes that emerge after trauma. Currently, there is no well-validated animal model of PTSD, although several stress paradigms mimic the behavioral symptoms and neurological alterations seen in PTSD. In this chapter, we will provide an overview of animal models of PTSD including learned helplessness, footshock, restraint stress, inescapable tail shock, single-prolonged stress, underwater trauma, social isolation, social defeat, early-life stress, and predator-based stress. We emphasize rodent models because they reproduce some of the behavioral and biotical phenotypes seen in PTSD. We will also present data showing that homologous biological measures are increasingly incorporated in studies to assess markers of risk and therapeutic response in these models. Therefore, PTSD animal models may be refined in hopes of capitalizing on the understanding of the molecular mechanisms and delivering tools in order to develop new and more efficacious treatments for PTSD.

Association between leukocyte telomere length and hostility in US army service members.

Hostility is a common form of emotionally charged anger which can lead to maladaptive and unhealthy behaviors. Significant association between shortened telomeres and greater levels of hostility has been observed in civilian populations, but has not yet been comprehensively studied in military populations. Our study investigates the relationship between hostility, post-traumatic stress disorder (PTSD), and leukocyte telomere length (LTL) in a sample of United States Army Special Operations personnel (n = 474) who deployed to Iraq and/or Afghanistan as part of combat operations. Hostility was measured with five items from the Brief Symptom Inventory (BSI). PTSD was determined using the PTSD Checklist (PCL) total score. The LTL was assessed using quantitative polymerase chain reaction methods and regression analyses were conducted to determine the association of hostility and telomere length. PTSD subjects reported higher hostility scores compared with those without PTSD. Among the participants with PTSD, those with medium or high level of hostility had shorter LTL than those with low level hostility (P < 0.01). Stepwise regression indicated that hostility level and age, but not gender and PTSD, were negatively correlated with LTL. Univariate regression showed that total hostility score was negatively associated with LTL (CI= -0.06 to -0.002, Beta= -0.095, p < 0.039) as well as a significant correlation between LTL and hostility impulses (HI) (CI= -0.108 to -0.009, Beta= -0.106, p < 0.021) and hostility controlling (HC) (CI= -0.071 to -0.002, Beta= -0.095, p < 0.004). Multiple regression analyses revealed that, while HC has no significant association with LTL, HI was still negatively correlated with LTL (p = 0.021). Our data indicates that LTL is associated with HI levels. Prevention and treatment efforts designed to reduce hostility may help mitigate risk for LTL shortening, a process of cellular aging, and thus slow accelerated aged-related health outcomes.

Astaxanthin but not quercetin preserves mitochondrial integrity and function, ameliorates oxidative stress, and reduces heat-induced skeletal muscle injury.

Heat stress causes mitochondrial dysfunction and increases mitochondrial production of reactive oxygen species (ROS), both of which contribute to heat-induced skeletal muscle injury. In this study, we tested whether either astaxanthin or quercetin, two dietary antioxidants, could ameliorate heat-induced skeletal muscle oxidative injury. In mouse C2C12 myoblasts exposed to 43°C heat stress, astaxanthin inhibited heat-induced ROS production in a concentration-dependent manner (1-20 µM), whereas the ROS levels remained high in cells treated with quercetin over a range of concentrations (2-100 µM). Because mitochondria are both the main source and a primary target of heat-induced ROS, we then tested the effects of astaxanthin and quercetin on mitochondrial integrity and function, under both normal temperature (37°C) and heat stress conditions. Quercetin treatment at 37°C induced mitochondrial fragmentation and decreased membrane potential (ΔΨ m ), accompanied by reduced protein expression of the master regulator of mitochondrial biogenesis peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). It also induced cleavage of mitochondrial inner-membrane fusion protein OPA1. In contrast, astaxanthin at 37°C increased protein expression of PGC-1α and mitochondrial transcription factor A (TFAM), and maintained tubular structure and normal ΔΨm . Under 43°C heat stress conditions, whereas quercetin failed to rescue C2C12 cells from injury, astaxanthin treatment prevented heat-induced mitochondrial fragmentation and depolarization, and apoptotic cell death. We also isolated rat flexor digitorum brevis myofibers and confirmed the data from C2C12 myoblasts that astaxanthin but not quercetin preserves mitochondrial integrity and function and ameliorates heat-induced skeletal muscle injury. These results confirm that mitochondria may be a potential therapeutic target for heat-related illness and suggest that astaxanthin may potentially be an effective preventive strategy.

Curcumin induces concentration-dependent alterations in mitochondrial function through ROS in C2C12 mouse myoblasts.

Curcumin exhibits antioxidant properties in normal cells where the uptake is low, unlike in tumor cells where uptake is high and curcumin increases reactive oxygen species (ROS) production and cell death. Mitochondria are the main source and primary target of cellular ROS. We hypothesized that curcumin would regulate cellular redox status and mitochondrial function, depending on cell sensitivity and/or curcumin concentration in normal cells. We examined the differences between low and high concentrations of curcumin, with specific attention focused on ROS levels, mitochondrial function, and cell viability in mouse C2C12 myoblast under normal and simulated conditions of diabetes. Cells incubated with high concentrations of curcumin (10-50 µM) resulted in decreased cell viability and sustained robust increases in ROS levels. Mechanistic studies showed that increased ROS levels in cells incubated with 20 µM curcumin induced opening of mitochondrial permeability transition pores and subsequent release of cytochrome c, activation of caspases 9 and 3/7, and apoptotic cell death. Low concentrations of curcumin (1-5 µM) did not affect cell viability, but induced a mild increase in ROS levels, which peaked at 2 hr after the treatment. Incubation with 5 µM curcumin also induced ROS-dependent increases in mitochondrial mass and membrane potential. Finally, pretreatment with 5 µM curcumin prevented high glucose-induced oxidative cell injury. Our study suggests that mitochondria respond differentially depending on curcumin concentration-dependent induction of ROS. The end result is either cell protection or death. Curcumin may be an effective therapeutic target for diabetes and other mitochondrial diseases when used in low concentrations.

Testosterone mediates hyperthermic response of mice to heat exposure.

AIMS: Testosterone is implicated as a potential contributing factor to heat-induced injury. We examined effects of testosterone on thermal response of mice to heat exposure. MAIN METHODS: Adult C57BL/6J male mice received gonadectomy or sham surgery subsequent vehicle (Gnx) or testosterone implants (Gnx+T). Body core temperature (Tc) of mice was recorded telemetrically during acute heat exposure. Thermal responses to heat exposure were also examined in age-matched female mice at each stage of the estrous cycle. KEY FINDINGS: Basal Tc was lower in sham male mice than females, but did not differ among sham, Gnx or Gnx+T males. No alterations in expression of uncoupling proteins 2 and 3 in the gastrocnemius muscle were found in either Gnx or Gnx+T mice compared to sham males. During heat exposure, sham male mice had a faster and greater rise in Tc, compared to females. This rapid hyperthermic response to heat was abolished in Gnx males, but not in Gnx+T males. No significant correlation was revealed between peak Tc values and plasma testosterone concentrations in Gnx+T males treated with either low- or high-dose testosterone. No effects of estrous cycle phase on the thermal response to heat exposure were detected in female mice. SIGNIFICANCE: We found that male mice were more susceptible to development of heat-induced hyperthermia than females and this was prevented by castration, not by castration with testosterone replacement. These results suggest that testosterone mediates heat-induced hyperthermia and is a heat stress susceptibility factor.