Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

Fisher‐Wellman, Kelsey H.; Davidson, Michael T.; Narowski, Tara M.; Lin, Chien-Te; Koves, Timothy R.; Muoio, Deborah M.

doi:10.1016/j.celrep.2018.08.091

Cited by 104 publications

(224 citation statements)

References 47 publications

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“…One potential mechanism for the observed effect on energetics is that 3OHB provides a substrate for generating additional acetyl-CoA (32,33), leading to increased production of NADH to drive energy transfer and ATP production (32,33). We sought to discern the energetic value of 3OHB using a recently developed bioenergetics assay platform in which comprehensive assessment of mitochondrial respiratory flux is conducted on freshly isolated mouse heart mitochondria (34). The platform features a modified version of the creatine kinase (CK) energetic clamp technique (25,35,36), which permits dynamic control of the extramitochondrial concentration ratio of ATP/ADP -and, thus, the energy of ATP hydrolysis (ΔG ATP ) -within a near-physiologic range.…”

Section: Resultsmentioning

confidence: 99%

“…The bioenergetic effects of 3OHB oxidation were evaluated in freshly isolated mouse heart mitochondria using a recently developed assay platform that permits comprehensive assessment of respiratory fluxes and determinants of energy transfer (34). Importantly, this approach provides for a highly tractable model system wherein substrate supply and energetic backpressure (ΔG ATP ) can be precisely controlled.…”

Section: Discussionmentioning

confidence: 99%

“…Rates of NADH production were determined as previously described (34). More detail can be found in Supplemental Methods.…”

Section: Bdh1 Activitymentioning

confidence: 99%

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The failing heart utilizes 3-hydroxybutyrate as a metabolic stress defense

et al. 2019

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The failing heart utilizes 3-hydroxybutyrate as a metabolic stress defense

et al. 2019

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“…In this assay, the free energy of ATP hydrolysis (ΔG′ ATP, depicted throughout the manuscript as ΔG ATP ) can be calculated based on known amounts of creatine (Cr), phosphocreatine (PCr) and ATP in combination with excess amounts of creatine kinase (CK) and the equilibrium constant for the CK reaction (i.e., K CK ). Calculation of ΔG′ ATP was performed according to the following formula using the previously published online resource (https://dmpio.github.io/ bioenergetic-calculators/ck_clamp/) 20 :…”

Section: Chemical and Reagentsmentioning

confidence: 99%

“…Following substrate additions, sequential additions of PCr to 6, 9,15 and 21 mM were performed to gradually slow JO 2 back toward baseline. Plotting the calculated ΔG ATP against the corresponding JO 2 reveals a linear force-flow relationship, the slope of which represents the conductance/sensitivity of the entire respiratory system under specified substrate constraints as previously described 20 . For each sample, respiratory conductance values from all substrates were pooled and subsequently correlated with protein expression across the mitochondrial proteome.…”

Section: Chemical and Reagentsmentioning

confidence: 99%

Subcellular proteomics combined with bioenergetic phenotyping reveals protein biomarkers of respiratory insufficiency in the setting of proofreading-deficient mitochondrial polymerase

McLaughlin

Kew

McClung

et al. 2020

Sci Rep

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the mitochondrial mutator mouse is a well-established model of premature aging. in addition to accelerated aging, these mice develop hypertrophic cardiomyopathy at ~13 months of age, presumably due to overt mitochondrial dysfunction. Despite evidence of bioenergetic disruption within heart mitochondria, there is little information about the underlying changes to the mitochondrial proteome that either directly underly or predict respiratory insufficiency in mutator mice. Herein, nLC-MS/MS was used to interrogate the mitochondria-enriched proteome of heart and skeletal muscle of aged mutator mice. the mitochondrial proteome from heart tissue was then correlated with respiratory conductance data to identify protein biomarkers of respiratory insufficiency. The majority of downregulated proteins in mutator mitochondria were subunits of respiratory complexes I and IV, including both nuclear and mitochondrial-encoded proteins. Interestingly, the mitochondrial-encoded complex V subunits, were unchanged or upregulated in mutator mitochondria, suggesting a robustness to mtDNA mutation. Finally, the proteins most strongly correlated with respiratory conductance were PPM1K, NDUFB11, and C15orf61. These results suggest that mitochondrial mutator mice undergo a specific loss of mitochondrial complexes i and iV that limit their respiratory function independent of an upregulation of complex V. Additionally, the role of PPM1K in responding to mitochondrial stress warrants further exploration. The mitochondrial polymerase gamma (Polg) is responsible for both replicating and maintaining the fidelity of the mitochondrial DNA (mtDNA) 1. Disruption of the proofreading capacity of this enzyme complex has been shown to lead to an accumulation of single point mutations and deletions throughout the mitochondrial genome 2-5. The mtDNA mutator mouse is a genetic knock-in model in which mice express a proofreading-deficient Polg harboring a D257A mutation, thus increasing mutational burden of the mtDNA 2,4. Mice homozygous (D257A +/+) for this transgene display a progeroid phenotype characterized by kyphosis, hair loss, hearing loss, pronounced cardiomyopathy, and reduced lifespan compared to wild-type littermates 2,4,6. At the cellular level, increased mtDNA mutational load and/or overt respiratory dysfunction has been associated with increased levels of reactive oxygen species, possibly due to a high amount of electron leak from the respiratory system 7-9. Our lab and others have demonstrated profound bioenergetic limitations in mitochondria isolated from adult D257A +/+ tissues 2,4,10. Recently we found that respiratory conductance, a measure of

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Interventional‐ and amputation‐stage muscle proteomes in the chronically threatened ischemic limb

Ryan

Kim

Scali

et al. 2022

Clinical & Translational Med

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Background Despite improved surgical approaches for chronic limb‐threatening ischemia (CLTI), amputation rates remain high and contributing tissue‐level factors remain unknown. The purpose of this study was twofold: (1) to identify differences between the healthy adult and CLTI limb muscle proteome, and (2) to identify differences in the limb muscle proteome of CLTI patients prior to surgical intervention or at the time of amputation. Methods and results Gastrocnemius muscle was collected from non‐ischemic controls (n = 19) and either pre‐interventional surgery (n = 10) or at amputation outcome (n = 29) CLTI patients. All samples were subjected to isobaric tandem‐mass‐tag‐assisted proteomics. The mitochondrion was the primary classification of downregulated proteins (> 70%) in CLTI limb muscles and paralleled robust functional mitochondrial impairment. Upregulated proteins (> 38%) were largely from the extracellular matrix. Across the two independent sites, 39 proteins were downregulated and 12 upregulated uniformly. Pre‐interventional CLTI muscles revealed a robust upregulation of mitochondrial proteins but modest functional impairments in fatty acid oxidation as compared with controls. Comparison of pre‐intervention and amputation CLTI limb muscles revealed mitochondrial proteome and functional deficits similar to that between amputation and non‐ischemic controls. Interestingly, these observed changes occurred despite 62% of the amputation CLTI patients having undergone a prior surgical intervention. Conclusions The CLTI proteome supports failing mitochondria as a phenotype that is unique to amputation outcomes. The signature of pre‐intervention CLTI muscle reveals stable mitochondrial protein abundance that is insufficient to uniformly prevent functional impairments. Taken together, these findings support the need for future longitudinal investigations aimed to determine whether mitochondrial failure is causally involved in amputation outcomes from CLTI.

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Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

Cited by 104 publications

References 47 publications

The failing heart utilizes 3-hydroxybutyrate as a metabolic stress defense

The failing heart utilizes 3-hydroxybutyrate as a metabolic stress defense

Subcellular proteomics combined with bioenergetic phenotyping reveals protein biomarkers of respiratory insufficiency in the setting of proofreading-deficient mitochondrial polymerase

Interventional‐ and amputation‐stage muscle proteomes in the chronically threatened ischemic limb

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