high-resolution-fluorespirometry-to-assess-dynamic-changes

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these ...

University of Washington

Defects in neuromuscular innervation contribute significantly to the age-related decline in muscle mass and function (sarcopenia). Our previous studies demonstrated that denervation induces muscle mitochondrial hydroperoxide production (HO and lipid hydroperoxides (LOOHs)). Here we define the relative contribution of mitochondrial electron transport chain (ETC) derived HO versus cytosolic phospholipase A (cPLA) derived LOOHs in neurogenic muscle atrophy. We show that denervation increases muscle cPLA protein content, activity, and metabolites downstream of cPLA including LOOHs. Increased scavenging of mitochondrial HO does not protect against denervation atrophy, suggesting ETC generated HO is not a critical player. In contrast, inhibition of cPLA in vivo mitigates LOOH production and muscle atrophy and maintains individual muscle fiber size while decreasing oxidative damage. Overall, we show that loss of innervation in several muscle atrophy models including aging induces generation of LOOHs produced by arachidonic acid metabolism in the cPLA pathway contributing to loss of muscle mass.

Targeting cPLA derived lipid hydroperoxides as a potential intervention for sarcopenia.

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment,  heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.

Elamipretide Improves ADP Sensitivity in Aged Mitochondria by Increasing Uptake through the Adenine Nucleotide Translocator (ANT).

In vivo control over metabolism is at the cutting edge of biomedical research. The particulars of mitochondrial function are especially important to understand in vivo to progress metabolic therapies that will be relevant for diseases of aging. Understanding the differences between how mitochondria function in vitro versus in vivo will be a necessary challenge to overcome to achieve mitochondrial medicine. In this article we outline how discoveries in invertebrate models will be informative for understanding the basic biology of mitochondria to streamline translation to mammals and eventually to humans. Further, we highlight examples of how what is known about mitochondria in vitro is translatable to in vivo models and, in some cases, to human diseases.

From mitochondria to cells to humans: Targeting bioenergetics in aging and disease.

Genomic, transcriptomic and proteomic approaches have been used to gain insight into molecular underpinnings of aging in laboratory animals and in humans. However, protein function in biological systems is under complex regulation and includes factors besides abundance levels, such as modifications, localization, conformation and protein-protein interactions. By making use of quantitative chemical cross-linking technologies, we show that changes in the muscle mitochondrial interactome contribute to mitochondrial functional decline in aging in female mice. Specifically, we identify age-related changes in protein cross-links relating to assembly of electron transport system complexes I and IV, activity of glutamate dehydrogenase, and coenzyme-A binding in fatty acid β-oxidation and tricarboxylic acid cycle enzymes. These changes show a remarkable correlation with complex I respiration differences within the same young-old animal pairs. Each observed cross-link can serve as a protein conformational or protein-protein interaction probe in future studies, which will provide further molecular insights into commonly observed age-related phenotypic differences. Therefore, this data set could become a valuable resource for additional in-depth molecular studies that are needed to better understand complex age-related molecular changes.

Skeletal muscle mitochondrial interactome remodeling is linked to functional decline in aged female mice.

It is unclear whether mitochondrial dysfunction and redox stress contribute to impaired age-related muscle regenerative capacity. Here we characterized a novel compound, BI4500, that inhibits the release of reactive oxygen species (ROS) from the quinone site in mitochondrial complex I (site I). We tested the hypothesis that ROS release from site I contributes to impaired regenerative capacity in aging muscle. Electron transfer system site-specific ROS production was measured in adult and aged mouse isolated muscle mitochondria and permeabilized gastrocnemius fibers. BI4500 inhibited ROS production from site I in a concentration-dependent manner (IC = ∼985 nM) by inhibiting ROS release without impairing complex I-linked respiration. In vivo BI4500 treatment decreased ROS production from site I. Muscle injury and sham injury were induced using barium chloride or vehicle injection to the tibialis anterior (TA) muscle in adult and aged male mice. On the same day as injury, mice began a daily gavage of 30 mg/kg BI4500 (BI) or placebo (PLA). Muscle regeneration (H&E, Sirius Red, Pax7) was measured at 5 and 35 days after injury. Muscle injury increased centrally nucleated fibers (CNFs) and fibrosis with no treatment or age effect. There was a significant age by treatment interaction for CNFs at 5- and 35-days post injury with significantly more CNFs in BI adults compared to PLA adults. Muscle fiber cross-sectional area (CSA) recovered significantly more in adult BI mice (-89 ± 365 μm) compared to old PLA (-599 ± 153 μm) and old BI (-535 ± 222 μm, mean ± SD). In situ TA force recovery was measured 35 days after injury and was not significantly different by age or treatment. Inhibition of site I ROS partially improves muscle regeneration in adult but not old muscle demonstrating a role for CI ROS in the response to muscle injury. Site I ROS does not contribute to impaired regenerative capacity in aging.

A novel mitochondrial complex I ROS inhibitor partially improves muscle regeneration in adult but not old mice.

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH (flavin adenine dinucleotide), O (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein).

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

Gavin Pharaoh

Department of Radiology

University of Washington

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells