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.