| First Author | Andres DA | Year | 2020 |
| Journal | J Biol Chem | Volume | 295 |
| Issue | 9 | Pages | 2676-2686 |
| PubMed ID | 31980460 | Mgi Jnum | J:286549 |
| Mgi Id | MGI:6401272 | Doi | 10.1074/jbc.RA119.011081 |
| Citation | Andres DA, et al. (2020) Improved workflow for mass spectrometry-based metabolomics analysis of the heart. J Biol Chem 295(9):2676-2686 |
| abstractText | 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. We applied 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 beta-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. |
