An in vivo, ex vivo, and in vitro exploration of the use of chronic hypoxia/physioxia and ROS/RNS-mediated alteration of physiological function in mitochondrial disease

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are by-products of cellular O2 metabolism and participate in cell signalling and normal physiological homeostasis. Although ROS/RNS are normal and important molecules in cell physiology, production in excess or in absence of sufficient cellular antioxidant capacity can lead to critical cellular damage which has shown to contribute to a plethora of disease pathologies. Experimental evidence has also supported the use of chronic limitation of ambient O2 gas as a means to reduce the amount of ROS/RNS in both animals and cell culture. The purpose of this thesis was to explore the use of a regulated O2 environment to determine if chronic hypoxia and/or physioxia may influence ROS/RNS production in animal and cell culture models of various known mitochondrial diseases. The absence of superoxide dismutase 2 (SOD2) has been proven to be extremely lethal, so I attempted to breed and house entire mouse dams in hypoxia (11% O2) to investigate if limiting O2 might reduce the amount of ROS/RNS-mediated damage and extend the life span of SOD2 knockout (KO) mouse pups. Many attempts to rescue these KO mice failed due to premature death and/or maternal cannibalism, however, the SOD2 heterozygote (SOD2+/-) – which experience halved SOD2 expression compared to wildtype controls – were viable and body mass data was examined. Although no main effect was found of genotype on body mass over time, male, but not female, mice housed in chronic hypoxia gained significantly less weight than their normoxia (20% O2) counterparts. After examining live animals, I focused on measuring the effects of regulating the O2 environment on ROS production in a variety of cellular models of different mitochondrial disease. Some measure of structural and/or functional integrity is compromised in mitochondrial disease, typically leading to exacerbated proton leak, subsequent superoxide/hydrogen peroxide (H2O2) formation, and, unsurprisingly, a significant potential for cellular damage. These features of mitochondrial disease are further compounded by the fact that a great deal of published cell culture work does not actively regulate O2 levels and thus cells often experience an environment with O2 levels hyperoxic relative to what is typically experienced in vivo. The purpose of this second study was to investigate whether growing and assaying mito-disease cell lines in a regulated, physiologically-relevant O2 environment would reduce H2O2 output to levels similar to wildtype controls. Measuring H2O2 production from various mito-disease cell lines, almost all cell lines produced more H2O2 when grown in normoxia (18% O2) culture conditions compared to physioxia (5% O2), however, only a few of the mitochondrial disease cell lines tested here produced the expected increase in cellular H2O2 efflux than their wildtype counterparts at 18% O2. As expected, almost all mitochondrial disease cell lines produced H2O2 at a similar level to their respective controls when grown in 5% O2. Furthermore, NADPH Oxidases (NOX) were explored as a potentially significant source of elevated ROS production at 18% O2. However, upon NOX inhibition, no significant measurable changes in H2O2 production were reported in any of the cell lines tested here. Finally, the last study in this thesis explored structural and functional consequences which may accompany halved SOD2 expression in adult SOD2+/- female mice over time. Sarco-endoplasmic reticulum ATP-ase (SERCA) is an enzyme responsible for calcium handling in myocytes and its function is critical for proper muscular relaxation and contraction. The soleus and extensor digitorum longus (EDL) were analyzed to determine whether muscle type (ie. slow-oxidative muscle or fast-glycolytic muscle) would influence the effects of heterozygous SOD2 deletion. Interestingly, the soleus muscle showed significant impairments in SERCA function with a reduction in SERCA’s apparent affinity for calcium, whereas there were no differences between genotypes in the EDL muscle. This corresponded well with the fact that SERCA tyrosine nitration was significantly elevated in the soleus, particularly on SERCA2a. Conversely, there were no signs of elevated SERCA tyrosine nitration on SERCA1a, the predominant SERCA isoform in the EDL. In conclusion, the results from these studies provide some insight to the roles that O2 and ROS generation have on physiological function in vivo and in vitro, though it also prompts further investigation due to mixed results in many cases

SERCA2a tyrosine nitration coincides with impairments in maximal SERCA activity in left ventricles from tafazzin deficient mice

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is imperative for normal cardiac function regulating both muscle relaxation and contractility. SERCA2a is the predominant isoform in cardiac muscles and is inhibited by phospholamban (PLN). Under conditions of oxidative stress, SERCA2a may also be impaired by tyrosine nitration. Tafazzin (Taz) is a mitochondrial specific transacylase that regulates mature cardiolipin (CL) formation, and its absence leads to mitochondrial dysfunction and excessive production of reactive oxygen/nitrogen species (ROS/RNS). In the present study, we examined SERCA function, SERCA2a tyrosine nitration, and PLN expression/phosphorylation in left ventricles (LV) obtained from young (3-5 months) and old (10-12 months) wild-type (WT) and Taz knockdown (TazKD) male mice. These mice are a mouse model for Barth syndrome, which is characterized by mitochondrial dysfunction, excessive ROS/RNS production, and dilated cardiomyopathy (DCM). Here, we show that maximal SERCA activity was impaired in both young and old TazKD LV, a result that correlated with elevated SERCA2a tyrosine nitration. In addition PLN protein was decreased, and its phosphorylation was increased in TazKD LV compared with control, which suggests that PLN may not contribute to the impairments in SERCA function. These changes in expression and phosphorylation of PLN may be an adaptive response aimed to improve SERCA function in TazKD mice. Nonetheless, we demonstrate for the first time that SERCA function is impaired in LVs obtained from young and old TazKD mice likely due to elevated ROS/RNS production. Future studies should determine whether improving SERCA function can improve cardiac contractility and pathology in TazKD miceBrock University Library Open Access Publishing Fun

Characterizing SERCA Function in Murine Skeletal Muscles after 35–37 Days of Spaceflight

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35–37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA.Brock University Unrestricted Gran

Heterozygous SOD2 deletion selectively impairs SERCA function in the soleus of female mice.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) restores intracellular Ca2+ ([Ca2+ ]i ) to resting levels after muscle contraction, ultimately eliciting relaxation. SERCA pumps are highly susceptible to tyrosine (T)-nitration, impairing their ability to take up Ca2+ resulting in reduced muscle function and increased [Ca2+ ]i and cellular damage. The mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), converts superoxide radicals into less reactive H2 O2 . Heterozygous deletion of SOD2 (Sod2+/- ) in mice increases mitochondrial oxidative stress; however, the consequences of reduced SOD2 expression in skeletal and cardiac muscle, specifically the effect on SERCA pumps, has yet to be investigated. We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from 6 to 7 month-old wild-type (WT) and Sod2+/- female C57BL/6J mice. Ca2+ -dependent SERCA activity assays were performed to assess SERCA function. Western blotting was conducted to examine the protein content of SERCA, phospholamban, and sarcolipin; and immunoprecipitation experiments were done to assess SERCA2a- and SERCA1a-specific T-nitration. Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus or LV but reduced SERCA2a in the EDL compared with WT, though this was not statistically significant. Soleus muscles from Sod2+/- mice showed a significant reduction in SERCA's apparent affinity for Ca2+ when compared to WT, corresponding with significantly elevated SERCA2a T-nitration in the soleus. No effect was seen in the EDL or the LV. This is the first study to investigate the effects of SOD2 deficiency on muscle SERCA function and shows that it selectively impairs SERCA function in the soleus.The Brock Library Open Access Publishing Fun

Inclusive ideologies and passive performances: Exploring masculinities and attitudes toward gay peers among boys in an elite youth football academy

Drawing on data generated from semi-structured, one-to-one interviews in 2012, this article focuses on the attitudinal disposition toward homosexuality of 12 English academy level football players aged 14-15. Results highlight the presence of progressive attitudes toward homosexuality even though some of the youth feel they lack the agency to contest homophobia when espoused in their schools. Using a blend of two dominating masculinities theories we use these interviews to highlight that boys in this setting are best understood as a complex imbrication of inclusivity yet socially-passive acceptance. We suggest that boys of this age are now primed for learning agency to contest the social marginalisation of others

Toward countering muscle and bone loss with spaceflight: GSK3 as a potential target

International audienceWe examined the effects of 30 days of spaceflight on glycogen synthase kinase 3 (GSK3) content and inhibitory serine phosphorylation in murine muscle and bone samples from four separate missions (BION-M1, rodent research [RR]1, RR9, and RR18). Spaceflight reduced GSK3b content across all missions, whereas its serine phosphorylation was elevated with RR18 and BION-M1. The reduction in GSK3b was linked to the reduction in type IIA fibers commonly observed with spaceflight as these fibers are particularly enriched with GSK3. We then tested the effects of inhibiting GSK3 before this fiber type shift, and we demonstrate that muscle-specific Gsk3 knockdown increased muscle mass, preserved muscle strength, and promoted the oxidative fiber type with Earth-based hindlimb unloading. In bone, GSK3 activation was enhanced after spaceflight; and strikingly, muscle-specific Gsk3 deletion increased bone mineral density in response to hindlimb unloading. Thus, future studies should test the effects of GSK3 inhibition during spaceflight