Non-targeted metabolomics analysis of cardiac Muscle Ring Finger-1 (MuRF1), MuRF2, and MuRF3 in vivo reveals novel and redundant metabolic changes
- 356 Downloads
The muscle-specific ubiquitin ligases MuRF1, MuRF2, MuRF3 have been reported to have overlapping substrate specificities, interacting with each other as well as proteins involved in metabolism and cardiac function. In the heart, all three MuRF family proteins have proven critical to cardiac responses to ischemia and heart failure. The non-targeted metabolomics analysis of MuRF1−/−, MuRF2−/−, and MuRF3−/− hearts was initiated to investigate the hypothesis that MuRF1, MuRF2, and MuRF3 have a similarly altered metabolome, representing alterations in overlapping metabolic processes. Ventricular tissue was flash frozen and quantitatively analyzed by GC/MS using a library built upon the Fiehn GC/MS Metabolomics RTL Library. Non-targeted metabolomic analysis identified significant differences (via VIP statistical analysis) in taurine, myoinositol, and stearic acid for the three MuRF−/− phenotypes relative to their matched controls. Moreover, pathway enrichment analysis demonstrated that MuRF1−/− had significant changes in metabolite(s) involved in taurine metabolism and primary acid biosynthesis while MuRF2−/− had changes associated with ascorbic acid/aldarate metabolism (via VIP and t test analysis vs. sibling-matched wildtype controls). By identifying the functional metabolic consequences of MuRF1, MuRF2, and MuRF3 in the intact heart, non-targeted metabolomics analysis discovered common pathways functionally affected by cardiac MuRF family proteins in vivo. These novel metabolomics findings will aid in guiding the molecular studies delineating the mechanisms that MuRF family proteins regulate metabolic pathways. Understanding these mechanism is an important key to understanding MuRF family proteins’ protective effects on the heart during cardiac disease.
KeywordsCardiac Ubiquitin ligase Metabolomics Muscle Ring Finger-1 (MuRF1) MuRF2 MuRF3
Muscle ring finger-1(2,3)
Variable importance in projection
Principal components analysis
Partial least squares discriminant analysis
The authors would like to thank Tim Koves for his guidance and valuable discussion and suggestions for harvesting and preparing heart samples for metabolomics analysis. This work was supported by the National Institutes of Health (R01HL104129 to M.W.), a Jefferson-Pilot Corporation Fellowship (to M.W.), and the Fondation Leducq (to M.W.).
Conflict of interest
The authors report no conflicts of interest.
- Guaiquil, V. H., Golde, D. W., Beckles, D. L., Mascareno, E. J., & Siddiqui, M. A. (2004). Vitamin C inhibits hypoxia-induced damage and apoptotic signaling pathways in cardiomyocytes and ischemic hearts. Free Radical Biology and Medicine, 37, 1419–1429. doi: 10.1016/j.freeradbiomed.2004.06.041.CrossRefPubMedGoogle Scholar
- Halket, J. M., Przyborowska, A., Stein, S. E., Mallard, W. G., Down, S., & Chalmers, R. A. (1999). Deconvolution gas chromatography/mass spectrometry of urinary organic acids–potential for pattern recognition and automated identification of metabolic disorders. Rapid Communications in Mass Spectrometry, 13, 279–284. doi: 10.1002/(SICI)1097-0231(19990228)13:4<279:AID-RCM478>3.0.CO;2-I.CrossRefPubMedGoogle Scholar
- Huang, J., et al. (2001). Dehydroascorbic acid, a blood-brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke. Proceedings of the National Academy of Sciences United States of America, 98, 11720–11724. doi: 10.1073/pnas.171325998.CrossRefGoogle Scholar
- Kim, O. Y., Jung, Y. S., Cho, Y., Chung, J. H., Hwang, G. S., & Shin, M. J. (2013). Altered heart and kidney phospholipid fatty acid composition are associated with cardiac hypertrophy in hypertensive rats. Clinical Biochemistry, 46, 1111–1117. doi: 10.1016/j.clinbiochem.2013.04.008.CrossRefPubMedGoogle Scholar
- Lopaschuk, G. D., Wambolt, R. B., & Barr, R. L. (1993). An imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. Journal of Pharmacology and Experimental Therapeutics, 264, 135–144.PubMedGoogle Scholar
- Mallard, W. G., Reed J. (1997). Automated Mass Spectral Deconvolution and Identification System: AMDIS User Guide. National Institute of Standards and Technology, US Department of Commerce iv, 58. http://chemdata.nist.gov/mass-spc/amdis/docs/amdis.pdf.
- Rupert, B. E., Segar, J. L., Schutte, B. C., & Scholz, T. D. (2000). Metabolic adaptation of the hypertrophied heart: Role of the malate/aspartate and alpha-glycerophosphate shuttles. Journal of Molecular and Cellular Cardiology, 32, 2287–2297. doi: 10.1006/jmcc.2000.1257.CrossRefPubMedGoogle Scholar
- Shekhawat, P. S., Matern, D., & Strauss, A. W. (2005). Fetal fatty acid oxidation disorders, their effect on maternal health and neonatal outcome: Impact of expanded newborn screening on their diagnosis and management. Pediatric Research, 57, 78R–86R. doi: 10.1203/01.PDR.0000159631.63843.3E.CrossRefPubMedCentralPubMedGoogle Scholar
- Styczynski, M. P., Moxley, J. F., Tong, L. V., Walther, J. L., Jensen, K. L., & Stephanopoulos, G. N. (2007). Systematic identification of conserved metabolites in GC/MS data for metabolomics and biomarker discovery. Analytical Chemistry, 79, 966–973. doi: 10.1021/ac0614846.CrossRefPubMedGoogle Scholar
- Ueki, I., et al. (2011). Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide. American Journal of Physiology-Endocrinology and Metabolism, 301, E668–E684. doi: 10.1152/ajpendo.00151.2011.CrossRefPubMedCentralPubMedGoogle Scholar
- Willis, M. S., et al. (2014). Muscle ring finger 1 and muscle ring finger 2 are necessary but functionally redundant during developmental cardiac growth and regulate E2F1-mediated gene expression in vivo. Cell Biochemistry and Function, 32, 39–50. doi: 10.1002/cbf.2969.CrossRefPubMedCentralPubMedGoogle Scholar