High Throughput Procedure for Comparative Analysis of In Vivo Cardiac Glucose or Amino Acids Use in Cardiovascular Pathologies and Pharmacological Treatments

Abstract

The heart is characterized by the prominent flexibility of its energy metabolism and is able to use diverse carbon substrates, including carbohydrates and amino acids. Cardiac substrate preference could have a major impact on the progress of cardiac pathologies. However, the majority of methods to investigate changes in substrates’ use in cardiac metabolism in vivo are complex and not suitable for high throughput testing necessary to understand and reverse these pathologies. Thus, this study aimed to develop a simple method that would allow for the analysis of cardiac metabolic substrate use. The developed methods involved the subcutaneous injection of stable ¹³C isotopomers of glucose, valine, or leucine with mass spectrometric analysis for the investigation of its entry into cardiac metabolic pathways that were deducted from ¹³C alanine and glutamate enrichments in heart extracts. The procedures were validated by confirming the known effects of treatments that modify glucose, free fatty acids, and amino acid metabolism. Furthermore, we studied changes in the energy metabolism of CD73 knock-out mice to demonstrate the potential of our methods in experimental research. The methods created allowed for fast estimation of cardiac glucose and amino acid use in mice and had the potential for high-throughput analysis of changes in pathology and after pharmacological treatments.