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Assessing mitochondrial respiration in isolated hearts using (17)O MRS.

ABSTRACT: The application of (17)O MRI and MRS for the evaluation of cardiac mitochondrial function has been limited because of the challenge of detecting metabolic H(2)(17)O in the vast background of naturally abundant H(2)(17)O. In this study, we have developed a direct (17)O MRS approach to examine the feasibility and sensitivity of detecting metabolically produced H(2)(17)O in isolated rat hearts perfused with (17)O(2)-enriched Krebs-Henseleit buffer containing normal (1.5 mm) and high (2.5 mm) calcium (Ca(2+)) concentrations to induce high workload. Consistent with increased workload at high Ca(2+) concentration, the measured myocardial oxygen consumption rate (MVO(2)) increased by 82%. Dynamic (17)O MRS showed an accelerated increase in the H(2)(17)O signal at high Ca(2+) concentration, suggesting increased mitochondrial production of H(2)(17)O in concordance with the increased workload. A compartment model was developed to describe the kinetics of H(2)(17)O production as a function of MVO(2). The myocardial (17)O(2) consumption rate (MV(17)O(2) was determined by least-squares fitting of the model to the NMR-measured H(2)(17)O concentration. Consistent with the measured MVO(2), the model-determined MV(17)O(2) showed a 92% increase at high Ca(2+) concentration. The increase in metabolic activity at high workload allowed the balance between ATP production and utilization to be maintained, leading to a similar phosphocreatine to ATP ratio. These results demonstrate that dynamic (17)O MRS can provide a valuable tool for the detection of an altered metabolic rate associated with a change in cardiac workload.

SUBMITTER: Lu M

PROVIDER: S-EPMC3522750 | biostudies-other | 2012 Jun

REPOSITORIES: biostudies-other

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Project description:Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence of CVDs increases markedly with age. Due to the high energetic demand, the heart is highly sensitive to mitochondrial dysfunction. The complexity of the cardiac mitochondrial proteome hinders the development of effective strategies that target mitochondrial dysfunction in CVDs. Mammalian mitochondria are composed of over 1000 proteins, most of which can undergo post-translational protein modifications (PTMs). Top-down proteomics is a powerful technique for characterizing and quantifying all protein sequence variations and PTMs. However, there are still knowledge gaps in the study of age-related mitochondrial proteoform changes using this technique. In this study, we used top-down proteomics to identify intact mitochondrial proteoforms in young and old hearts and determined changes in protein abundance and PTMs in cardiac aging.MethodsIntact mitochondria were isolated from the hearts of young (4-month-old) and old (24-25-month-old) mice. The mitochondria were lysed, and mitochondrial lysates were subjected to denaturation, reduction, and alkylation. For quantitative top-down analysis, there were 12 runs in total arising from 3 biological replicates in two conditions, with technical duplicates for each sample. The collected top-down datasets were deconvoluted and quantified, and then the proteoforms were identified.ResultsFrom a total of 12 LC-MS/MS runs, we identified 134 unique mitochondrial proteins in the different sub-mitochondrial compartments (OMM, IMS, IMM, matrix). 823 unique proteoforms in different mass ranges were identified. Compared to cardiac mitochondria of young mice, 7 proteoforms exhibited increased abundance and 13 proteoforms exhibited decreased abundance in cardiac mitochondria of old mice. Our analysis also detected PTMs of mitochondrial proteoforms, including N-terminal acetylation, lysine succinylation, lysine acetylation, oxidation, and phosphorylation.ConclusionBy combining mitochondrial protein enrichment using mitochondrial fractionation with quantitative top-down analysis using ultrahigh-pressure liquid chromatography (UPLC)-MS and label-free quantitation, we successfully identified and quantified intact proteoforms in the complex mitochondrial proteome. Using this approach, we detected age-related changes in abundance and PTMs of mitochondrial proteoforms in the heart.

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Assessing mitochondrial respiration in isolated hearts using (17)O MRS.

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