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.

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation

“…As presented in our earlier research, both types of skeletal muscle exhibit metabolic preference shift to FA oxidation [9]. Interestingly, the Ranolazine treatment resulted in increased glucose use in cardiac metabolism and thus reduced FA β-oxidation [9,27]. It leads also to the abolishment of observed earlier improvement of exercise capacity in strenuous exercise protocol as well as reduction of total adenine nucleotides pool in gastrocnemius and soleus muscles.…”

Rosiglitazone Ameliorates Cardiac and Skeletal Muscle Dysfunction by Correction of Energetics in Huntington’s Disease

“…Analysis of cardiac and skeletal muscle glucose usage was performed within the method described before [ 30 , 38 ]. d -glucose-1,6- 13 C 2 (Sigma-Aldrich, St. Louis, MO, USA) was administrated in the subcutaneous injection of a 1.8 mg/g body weight dose.…”

“…To unravel the source of the noticed skeletal muscle as well as cardiac functionality improvement, we evaluated the in vivo glycolytic and oxidative metabolism of labeled 13 C glucose. Metabolite tracking (4-13 C glutamate and 3-13 C alanine) after 13 C glucose administration was previously extensively studied by our group [38]. Theoretical assumptions, supported by experimental studies, indicate that after 13 C glucose administration, the heart accumulates 3-13 C pyruvate in proportion to the fraction of glycolytic substrate, supplied by exogenous glucose relative to alternative unlabeled substrate sources (e.g., endogenous glycogen) and 4-13 C α-ketoglutarate in proportion to the fraction of tricarboxylic acid (TCA) cycle carbon flux supported by flux through pyruvate dehydrogenase (PDH), relative to other acetyl-CoA sources (e.g., free fatty acids (FFA)).…”

Endosomal v-ATPase as a Sensor Determining Myocardial Substrate Preference

“…In the healthy heart, the contribution of these alternative substrates to total ATP production is limited, with ketone bodies and AAs accounting for 10% and 2%, at most, of total ATP generated, respectively [ 6 ]. However, at any specific time the actual contribution of each of these substrates could be quite different and can be adjusted rapidly, depending on changes in substrate availability governed by specific (patho)physiologic conditions (e.g., changes during the course of a day) [ 2 , 10 , 11 , 12 ].…”