Rosemary A. Schuh

Measuring mitochondrial respiration in intact single muscle fibers

Measurement of mitochondrial function in skeletal muscle is a vital tool for understanding regulation of cellular bioenergetics. Currently, a number of different experimental approaches are employed to quantify mitochondrial function, with each involving either mechanically or chemically induced disruption of cellular membranes. Here, we describe a novel approach that allows for the quantification of substrate-induced mitochondria-driven oxygen consumption in intact single skeletal muscle fibers isolated from adult mice. Specifically, we isolated intact muscle fibers from the flexor digitorum brevis muscle and placed the fibers in culture conditions overnight. We then quantified oxygen consumption rates using a highly sensitive microplate format. Peak oxygen consumption rates were significantly increased by 3.4-fold and 2.9-fold by simultaneous stimulation with the uncoupling agent, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and/or pyruvate or palmitate exposure, respectively. However, when calculating the total oxygen consumed over the entire treatment, palmitate exposure resulted in significantly more oxygen consumption compared with pyruvate. Further, as proof of principle for the procedure, we isolated fibers from the mdx mouse model, which has known mitochondrial deficits. We found significant reductions in initial and peak oxygen consumption of 51% and 61% compared with fibers isolated from the wild-type (WT) animals, respectively. In addition, we determined that fibers isolated from mdx mice exhibited less total oxygen consumption in response to the FCCP + pyruvate stimulation compared with the WT mice. This novel approach allows the user to make mitochondria-specific measures in a nondisrupted muscle fiber that has been isolated from a whole muscle.

Adaptation of microplate-based respirometry for hippocampal slices and analysis of respiratory capacity.

Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O2 consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate‐based measurement of hippocampal slice O2 consumption. As a first evaluation of the technique, we compared whole‐slice bioenergetics with previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O2 consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O2 consumption that was well coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However, stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared with previous measurements in cells or synaptosomes, though enhanced slightly (to ∼150% of basal respiration) by acute addition of the mitochondrial complex I‐linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose‐derived substrate for maximal respiration. The improved throughput of microplate‐based hippocampal respirometry over traditional O2 electrode‐based methods is conducive to neuroprotective drug screening. When coupled with cell type‐specific pharmacology or genetic manipulations, the ability to measure O2 consumption efficiently from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology.

Mitochondrial dysfunction and nicotinamide dinucleotide catabolism as mechanisms of cell death and promising targets for neuroprotection.

Both acute and chronic neurodegenerative diseases are frequently associated with mitochondrial dysfunction as an essential component of mechanisms leading to brain damage. Although loss of mitochondrial functions resulting from prolonged activation of the mitochondrial permeability transition (MPT) pore has been shown to play a significant role in perturbation of cellular bioenergetics and in cell death, the detailed mechanisms are still elusive. Enzymatic reactions linked to glycolysis, the tricarboxylic acid cycle, and mitochondrial respiration are dependent on the reduced or oxidized form of nicotinamide dinucleotide [NAD(H)] as a cofactor. Loss of mitochondrial NAD+ resulting from MPT pore opening, although transient, allows detrimental depletion of mitochondrial and cellular NAD+ pools by activated NAD+ glycohydrolases. Poly(ADP‐ribose) polymerase (PARP) is considered to be a major NAD+ degrading enzyme, particularly under conditions of extensive DNA damage. We propose that CD38, a main cellular NAD+ level regulator, can significantly contribute to NAD+ catabolism. We discuss NAD+ catabolic and NAD+ synthesis pathways and their role in different strategies to prevent cellular NAD+ degradation in brain, particularly following an ischemic insult. These therapeutic approaches are based on utilizing endogenous intermediates of NAD+ metabolism that feed into the NAD+ salvage pathway and also inhibit CD38 activity.

Postnatal Developmental Regulation of Bcl-2 Family Proteins in Brain Mitochondria

Although it has been long recognized that the relative balance of pro‐ and antiapoptotic Bcl‐2 proteins is critical in determining the susceptibility to apoptotic death, only a few studies have examined the level of these proteins specifically at mitochondria during postnatal brain development. In this study, we examined the age‐dependent regulation of Bcl‐2 family proteins using rat brain mitochondria isolated at various postnatal ages and from the adult. The results indicate that a general down‐regulation of most of the proapoptotic Bcl‐2 proteins present in mitochondria occurs during postnatal brain development. The multidomain proapoptotic Bax, Bak, and Bok are all expressed at high levels in mitochondria early postnatally but decline in the adult. Multiple BH3‐only proteins, including direct activators (Bid, Bim, and Puma) and the derepressor BH3‐only protein Bad, are also present in immature brain mitochondria and are down‐regulated in the adult brain. Antiapoptotic Bcl‐2 family members are differentially regulated, with a shift from high Bcl‐2 expression in immature mitochondria to predominant Bcl‐xL expression in the adult. These results support the concept that developmental differences in upstream regulators of the mitochondrial apoptotic pathway are responsible for the increased susceptibility of cells in the immature brain to apoptosis following injury.

Sex-dependent mitochondrial respiratory impairment and oxidative stress in a rat model of neonatal hypoxic-ischemic encephalopathy

Increased male susceptibility to long‐term cognitive deficits is well described in clinical and experimental studies of neonatal hypoxic‐ischemic encephalopathy. While cell death signaling pathways are known to be sexually dimorphic, a sex‐dependent pathophysiological mechanism preceding the majority of secondary cell death has yet to be described. Mitochondrial dysfunction contributes to cell death following cerebral hypoxic‐ischemia (HI). Several lines of evidence suggest that there are sex differences in the mitochondrial metabolism of adult mammals. Therefore, this study tested the hypothesis that brain mitochondrial respiratory impairment and associated oxidative stress is more severe in males than females following HI. Maximal brain mitochondrial respiration during oxidative phosphorylation was two‐fold more impaired in males following HI. The endogenous antioxidant glutathione was 30% higher in the brain of sham females compared to males. Females also exhibited increased glutathione peroxidase (GPx) activity following HI injury. Conversely, males displayed a reduction in mitochondrial GPx4 protein levels and mitochondrial GPx activity. Moreover, a 3–4‐fold increase in oxidative protein carbonylation was observed in the cortex, perirhinal cortex, and hippocampus of injured males, but not females. These data provide the first evidence for sex‐dependent mitochondrial respiratory dysfunction and oxidative damage, which may contribute to the relative male susceptibility to adverse long‐term outcomes following HI.

Ectopic lipid deposition and the metabolic profile of skeletal muscle in ovariectomized mice

Disruptions of ovarian function in women are associated with increased risk of metabolic disease due to dysregulation of peripheral glucose homeostasis in skeletal muscle. Our previous evidence suggests that alterations in skeletal muscle lipid metabolism coupled with altered mitochondrial function may also develop. The objective of this study was to use an integrative metabolic approach to identify potential areas of dysfunction that develop in skeletal muscle from ovariectomized (OVX) female mice compared with age-matched ovary-intact adult female mice (sham). The OVX mice exhibited significant increases in body weight, visceral, and inguinal fat mass compared with sham mice. OVX mice also had significant increases in skeletal muscle intramyocellular lipids (IMCL) compared with the sham animals, which corresponded to significant increases in the protein content of the fatty acid transporters CD36/FAT and FABPpm. A targeted metabolic profiling approach identified significantly lower levels of specific acyl carnitine species and various amino acids in skeletal muscle from OVX mice compared with the sham animals, suggesting a potential dysfunction in lipid and amino acid metabolism, respectively. Basal and maximal mitochondrial oxygen consumption rates were significantly impaired in skeletal muscle fibers from OVX mice compared with sham animals. Collectively, these data indicate that loss of ovarian function results in increased IMCL storage that is coupled with alterations in mitochondrial function and changes in the skeletal muscle metabolic profile.

Calcium-dependent dephosphorylation of brain mitochondrial calcium/cAMP response element binding protein (CREB)

Calcium‐mediated signaling regulates nuclear gene transcription by calcium/cAMP response element binding protein (CREB) via calcium‐dependent kinases and phosphatases. This study tested the hypothesis that CREB is also present in mitochondria and subject to dynamic calcium‐dependent modulation of its phosphorylation state. Antibodies to CREB and phosphorylated CREB (pCREB) were used to demonstrate the presence of both forms in isolated mitochondria and mitoplasts from rat brain. When energized mitochondria were exposed to increasing concentrations of Ca2+ in the physiological range, pCREB was lost while total CREB remained constant. In the presence of Ru360, an inhibitor of the mitochondrial Ca2+ uptake uniporter, calcium‐dependent loss of pCREB levels was attenuated, suggesting that intramitochondrial calcium plays an important role in pCREB dephosphorylation. pCREB dephosphorylation was not, however, inhibited by the phosphatase inhibitors okadaic acid and Tacrolimus. In the absence of Ca2+, CREB phosphorylation was elevated by the addition of ATP to the mitochondrial suspension. Exposure of mitochondria to the pore‐forming molecule alamethicin that causes osmotic swelling and release of intermembrane proteins enriched mitochondrial pCREB immunoreactivity. These results further suggest that mitochondrial CREB is located in the matrix or inner membrane and that a kinase and a calcium‐dependent phosphatase regulate its phosphorylation state.

Mitochondrial Dysfunction and NAD + Metabolism Alterations in the Pathophysiology of Acute Brain Injury

Mitochondrial dysfunction is commonly believed to be one of the major players in mechanisms of brain injury. For several decades, pathologic mitochondrial calcium overload and associated opening of the mitochondrial permeability transition (MPT) pore were considered a detrimental factor causing mitochondrial damage and bioenergetics failure. Mitochondrial and cellular bioenergetic metabolism depends on the enzymatic reactions that require NAD+ or its reduced form NADH as cofactors. Recently, it was shown that NAD+ also has an important function as a substrate for several NAD+ glycohydrolases whose overactivation can contribute to cell death mechanisms. Furthermore, downstream metabolites of NAD+ catabolism can also adversely affect cell viability. In contrast to the negative effects of NAD+-catabolizing enzymes, enzymes that constitute the NAD+ biosynthesis pathway possess neuroprotective properties. In the first part of this review, we discuss the role of MPT in acute brain injury and its role in mitochondrial NAD+ metabolism. Next, we focus on individual NAD+ glycohydrolases, both cytosolic and mitochondrial, and their role in NAD+ catabolism and brain damage. Finally, we discuss the potential effects of downstream products of NAD+ degradation and associated enzymes as well as the role of NAD+ resynthesis enzymes as potential therapeutic targets.

BRCA1 is a Novel Regulator of Metabolic Function in Skeletal Muscle.

Breast cancer type 1 (BRCA1) susceptibility protein is expressed across multiple tissues including skeletal muscle. The overall objective of this investigation was to define a functional role for BRCA1 in skeletal muscle using a translational approach. For the first time in both mice and humans, we identified the presence of multiple isoforms of BRCA1 in skeletal muscle. In response to an acute bout of exercise, we found increases in the interaction between the native forms of BRCA1 and the phosphorylated form of acetyl-CoA carboxylase. Decreasing BRCA1 content using a shRNA approach in cultured primary human myotubes resulted in decreased oxygen consumption by the mitochondria and increased reactive oxygen species production. The decreased BRCA1 content also resulted in increased storage of intracellular lipid and reduced insulin signaling. These results indicate that BRCA1 plays a critical role in the regulation of metabolic function in skeletal muscle. Collectively, these data reveal BRCA1 as a novel target to consider in our understanding of metabolic function and risk for development of metabolic-based diseases.

Interaction of mitochondrial respiratory inhibitors and excitotoxins potentiates cell death in hippocampal slice cultures.

The broad‐spectrum insecticide rotenone, an inhibitor of complex I of the mitochondrial electron transport chain (ETC), gives rise to oxidative stress and bioenergetic failure. Pesticides including rotenone have been implicated in human neurodegenerative diseases, including Parkinsons disease. Another intensively investigated hypothesis of neurodegenerative disease involves the toxic action of the excitatory neurotransmitter glutamate. In the present study, we determined whether concomitant exposure of rotenone plus tetraethylammonium chloride (TEA) or the specific glutamate receptor agonists N‐methyl‐D‐aspartate (NMDA) or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid (AMPA) would cause greater cell death in organotypic hippocampal slice cultures than when given separately. Low, sublethal rotenone (100 nM), TEA (0.5–2.0 mM), NMDA (1.0–10 μM), and AMPA (1.0–10 μM) alone resulted in little cell death as determined by propidium iodide fluorescence. However, cell death was significantly to dramatically potentiated when the hippocampal slices were coincubated with comparable concentrations of rotenone plus TEA, NMDA, or AMPA. Similarly, in the presence of 10 μM NMDA, ETC inhibitors blocking other mitochondrial complexes also potentiated cell death. Immunohistochemical analysis using glial fibrillary acidic protein antibody determined that the cell death was preferentially neuronal. These results demonstrate that two different classes of toxicants can interact, resulting in potentiation of neurotoxicity, and further suggest that a combinatorial therapeutic approach may be required to ameliorate the potentiated cell death.