MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS-based metabolomics workflow that uses insoluble protein- derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow.Weapplied this workflow to study heart metabolism by first comparing two different methods of heart removal: The Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|State||Published - Feb 28 2020|
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health Grant HL131782 (to D. A. A. and J. S.) and UL1TR001998 (to D. A. A., S. V., and R. C. S.); American Heart Association Grants 16GRNT27790094 (to D. A. A. and J. S.) and 17SDG33670578 (to S. V.); American Cancer Society Institutional Research Grant 16-182-28 (to R. C. S.); and a St. Baldrick’s Career Development Award (to R. C. S.). This work was also supported by funding from the Uni-versity of Kentucky Markey Cancer Center, the Saha Cardiovascular Research Center, and the Biostatistics and Bioinformatics Shared Resource Facility of the University of Kentucky Markey Cancer Center Grant P30CA177558. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
© 2020 Andres et al.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology