Skip to main content
SpringerLink
Log in

Metabolic phenotyping of the diseased rat heart using 13C-substrates and ex vivo perfusion in the working mode

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

The objective of the present study was to compare energy substrate fluxes through metabolic pathways leading to mitochondrial citrate synthesis and release in normal and diseased rat hearts using 13C-substrates and mass isotopomer analysis by gas chromatography-mass spectrometry (GCMS). This study was prompted by our previous finding of a modulated citrate release by perfused rat hearts and by the possibility that a dysregulated myocardial citrate release represents a specific chronic alteration of energy metabolism in cardiac patients. The 15-week-old spontaneously hypertensive rat (SHR) was chosen as our animal model of disease and the Wistar-Kyoto (WKY) rat as its matched control. Ex vivo work-performing hearts were perfused with a semi-recirculating buffer containing physiological concentrations of unlabeled (glucose) and 13C-labeled ([U-13C3](lactate + pyruvate) and/or [1-13C]oleate) substrates. In parallel to the continuous monitoring of indices of the heart's functional and physiological status, the following metabolic parameters were documented: (i) citrate release rates and citric acid cycle intermediate tissue levels, (ii) the contribution of fatty acids as well as pyruvate decarboxylation and carboxylation to citrate synthesis, and (iii) lactate and pyruvate uptake and efflux rates. Working hearts from both rat species showed a similar percent contribution of carbohydrates for citrate synthesis through decarboxylation (70%) and carboxylation (10%). SHR hearts showed the following metabolic alterations: a higher citrate release rate, which was associated with a parallel increase in its tissue level, a lower contribution of oleate β-oxidation to citrate synthesis, and an accelerated efflux rate of unlabeled lactate from glycolysis. These metabolic changes were not explained by differences in myocardial oxygen consumption, cardiac performance or efficiency, nor correlated with indices of tissue necrosis or ischemia. This study demonstrates how the alliance between ex vivo semi-recirculating working perfused rat hearts with 13C-substrates and mass isotopomer analysis by GCMS, can provide an unprecedented insight into the metabolic phenotype of normal and diseased rat hearts. The clinical relevance of metabolic alterations herein documented in the SHR heart is suggested by its resemblance to those reported in cardiac patients. Taken altogether, our results raise the possibility that the increased citrate release of diseased hearts results from an imbalance between citrate synthesis and utilization rates, which becomes more apparent under conditions of substrate abundance.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Thomassen AR, Nielsen TT, Bagger JP, Henningsen P: Myocardial exchanges of glutamate, alanine and citrate in controls and patients with coronary artery disease. Clin Sci 64: 33-40, 1983

    Google Scholar 

  2. Thuesen L, Nielsen TT, Thomassen A, Bagger JP, Henningsen P: Beneficial effect of a low-fat low-calorie diet on myocardial energy metabolism in patients with angina pectoris. Lancet 2: 59-62, 1984

    Google Scholar 

  3. Thomassen AR, Nielsen TT, Bagger JP, Henningsen P: Cardiac metabolic and hemodynamic effects of insulin in patients with coronary artery disease. Diabetes 38: 1175-1180, 1989

    Google Scholar 

  4. Opie LH: Cardiac metabolism—emergence, decline, and resurgence. Part I. Cardiovasc Res 26: 721-733, 1992

    Google Scholar 

  5. Stanley WC, Lopaschuk GD, Hall JL, McCormack JG: Regulation of myocardial carbohydrate metabolism under normal and ischemic conditions. Potential for pharmacological interventions. Cardiovasc Res 33: 243-257, 1997

    Google Scholar 

  6. Taegtmeyer H, Goodwin GW, Doenst T, Frazier OH: Substrate metabolism as a determinant for postischemic functional recovery of the heart. Am J Cardiol 80: 3A-10A, 1997

    Google Scholar 

  7. Lopaschuk GD, Rebeyka IM, Allard MF: Metabolic modulation: A means to mend a broken heart. Circulation 105: 140-142, 2002

    Google Scholar 

  8. Barr RL, Lopaschuk GD: Methodology for measuring in vitro/ex vivo cardiac energy metabolism. J Pharmacol Toxicol Meth 43: 141-152, 2000

    Google Scholar 

  9. Comte B, Vincent G, Bouchard B, Des Rosiers C: Probing the origin of acetyl-CoA and oxaloacetate entering the citric acid cycle from the 13C labeling of citrate released by perfused rat heart. J Biol Chem 272: 26117-26124, 1997

    Google Scholar 

  10. Comte B, Vincent G, Bouchard B, Des Rosiers C: A 13C mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts. J Biol Chem 272: 26125-26131, 1997

    Google Scholar 

  11. Laplante A, Vincent G, Poirier M, Des Rosiers C: Effects and metabolism of fumarate in the perfused rat heart. A 13C mass isotopomer study. Am J Physiol 272: E74-E82, 1997

    Google Scholar 

  12. Vincent G, Comte B, Poirier M, Bouchard B, Des Rosiers C: Citrate release by perfused rat hearts: A window on mitochondrial cataplerosis. Am J Physiol 278: E846-E856, 2000

    Google Scholar 

  13. Panchal AR, Comte B, Huang H, Kerwin T, Darvish A, Des Rosiers C, Brunengraber H, Stanley WC: Partitioning of pyruvate between oxidation and anaplerosis in swine hearts. Am J Physiol 279: H2390-H2398, 2000

    Google Scholar 

  14. Panchal AR, Comte B, Huang H, Dudar B, Roth B, Chandler M, Des Rosiers C, Brunengraber H, Stanley WC: Acute hibernation decreases myocardial pyruvate carboxylation and citrate release. Am J Physiol 281: H1613-H1620, 2001

    Google Scholar 

  15. Depré C, Rider MH, Hue L: Mechanisms of control of heart glycolysis. Eur J Biochem 258: 277-290, 1998

    Google Scholar 

  16. Ruderman NB, Saha AK, Vavvas D, Witters LE: Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol 276: E1-E18, 1999

    Google Scholar 

  17. Cheema-Dhadli S, Robinson BH, Halperin ML: Properties of the citrate transporter in rat heart: Implications for regulation of glycolysis by cytosolic citrate. Can J Biochem 54: 561-565, 1976

    Google Scholar 

  18. Sluse FE, Meijer AJ, Tager JM: Anion translocators in rat-heart mitochondria. FEBS Lett 18: 149-153, 1971

    Google Scholar 

  19. Kinney Lapier TL, Rodnick KJ: Changes in cardiac energy metabolism during early development of female SHR. Am J Hypertens 13: 1074-1081, 2000

    Google Scholar 

  20. Haneda T, Ichihara K, Abiko Y, Onodera S: Functional and metabolic responses to ischemia in the perfused heart isolated from normotensive and spontaneously hypertensive rats. Jpn Circ J 50: 607-613, 1986

    Google Scholar 

  21. Barger PM, Kelly DP: Fatty acid utilization in the hypertrophied and failing heart: Molecular regulatory mechanisms. Am J Med Sci 318: 36-42, 1999

    Google Scholar 

  22. Allard MF, Schönekess BO, Henning SL, English DR, Lopaschuk GD: Contribution of oxidative metabolism and glycolysis to ATP production in hypertrophied hearts. Am J Physiol 267: H742-H750, 1994

    Google Scholar 

  23. Christe ME, Rodgers RL: Altered glucose and fatty acid oxidation in hearts of the spontaneously hypertensive rat. J Mol Cell Cardiol 26: 1371-1375, 1994

    Google Scholar 

  24. Doenst T, Goodwin GW, Cedars AM, Wang M, Stepkowski S, Taegtmeyer H: Load-induced changes in vivo alter substrate fluxes and insulin responsiveness of rat heart in vitro. Metabolism 50: 1083-1090, 2001

    Google Scholar 

  25. Hajri T, Ibrahimi A, Coburn CT, Knapp FF Jr, Kurtz T, Pravenec M, Abumrad NA: Defective fatty acid uptake in the spontaneously hypertensive rat is a primary determinant of altered glucose metabolism, hyperinsulinemia, and myocardial hypertrophy. J Biol Chem 276: 23661-23666, 2001

    Google Scholar 

  26. Seymour A-M L, Chatham JC: The effects of hypertrophy and diabetes on cardiac pyruvate dehydrogenase activity. J Mol Cell Cardiol 29: 2771-2778, 1997

    Google Scholar 

  27. Chatham JC, Des Rosiers C, Forder JR: Evidence of separate pathways for lactate uptake and release by the perfused rat heart. Am J Physiol 281: E794-E802, 2001

    Google Scholar 

  28. Gamcsik MP, Forder JR, Millis KK, McGovern KA: A versatile oxygenator and perfusion system for magnetic resonance studies. Biotech Bioeng 49: 348-354, 1996

    Google Scholar 

  29. Rodgers RL, Christe ME, Tremblay GC, Babson JR, Daniels T: Insulin-like effects of a physiologic concentration of carnitine on cardiac metabolism. Mol Cell Biochem 226: 97-105, 2001

    Google Scholar 

  30. Bergmeyer HU: In: Methods of Enzymatic Analysis, vol. 3, 2nd edn. Verlag Chemie International, Academic Press, New York, 1974

    Google Scholar 

  31. Starnes JW, Wilson DF, Erecinska M: Substrate dependence of metabolic state and coronary flow in perfused rat heart. Am J Physiol 249: H799-806, 1985

    Google Scholar 

  32. Bünger R, Mallet RT, Hartman DA: Pyruvate-enhanced phosphorylation potential and inotropism in normoxic and postischemic isolated working heart. Near-complete prevention of reperfusion contractile failure. Eur J Biochem 180: 221-233, 1989

    Google Scholar 

  33. Goodwin GW, Cohen DM, Taegtmeyer H: [5-3H]glucose overestimates glycolytic flux in isolated working rat heart: Role of pentose phosphate pathway. Am J Physiol 280: E502-E508, 2001

    Google Scholar 

  34. Gibala MJ, Young ME, Taegtmeyer H: Anaplerosis of the citric acid cycle: Role in energy metabolism of heart and skeletal muscle. Acta Physiol Scand 168: 657-665, 2000

    Google Scholar 

  35. Brooks GA: Intra-and extra-cellular lactate shuttles. Med Sci Sports Exerc 32: 790-799, 2000

    Google Scholar 

  36. Weiss JN, Venkatesh N: Metabolic regulation of cardiac ATP-sensitive K+ channels. Cardiovasc Drugs Ther 7: 499-505, 1993

    Google Scholar 

  37. Gardner PR, Fridovich I: Superoxide sensitivity of the Escherichi coli aconitase. J Biol Chem 266: 19328-19333, 1991

    Google Scholar 

  38. Janero DR, Hreniuk D: Suppression of TCA cycle activity in the cardiac muscle cell by hydroperoxide-induced oxidant stress. Am J Physiol 270: C1735-C1742, 1996

    Google Scholar 

  39. Nulton-Persson AC, Szweda LI: Modulation of mitochondrial function by hydrogen peroxide. J Biol Chem 276: 23357-23361, 2001

    Google Scholar 

  40. Dhalla AK, Hill MF, Singal PK: Role of oxidative stress in transition of hypertrophy to heart failure. J Am Coll Cardiol 28: 506-514, 1996

    Google Scholar 

  41. Rupp H, Schulze W, Vetter R: Dietary medium-chain triglycerides can prevent changes in myosin and SR due to CPT-1 inhibition by etomoxir. Am J Physiol 269: R630-R640, 1995

    Google Scholar 

  42. Boomsma F, van den Meiracker AH: Plasma A-and B-type natriuretic peptides: Physiology, methodology and clinical use. Cardiovasc Res 51: 442-449, 2001

    Google Scholar 

  43. H'Doubler PB Jr, Petersen M, Shek W, Auchincloss H, Abbott WM, Orkin RW: Spontaneously hypertensive and Wistar-Kyoto rats are genetically disparate. Lab Anim Sci 41: 471-473, 1991

    Google Scholar 

  44. Kurtz TW, Morris RC Jr: Biological variability in Wistar-Kyoto rats. Hypertension 10: 127-131, 1987

    Google Scholar 

  45. Belke DD, Larsen TS, Lopaschuk, GD, Severson DL: Glucose and fatty acid metabolism in the isolated working mouse heart. Am J Physiol 277: R1210-R1217, 1999

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry, University of Montreal, Montreal, Quebec, Canada, H3C 3J7

    Genevièe Vincent & Maya Khairallah

  2. Department of Nutrition, University of Montreal, Montreal, Quebec, Canada, H3C 3J7

    Bertrand Bouchard

  3. Departments of Biochemistry and Nutrition, University of Montreal, Montreal, Quebec, Canada, H3C 3J7

    Christine Des Rosiers

Authors
  1. Genevièe Vincent
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maya Khairallah
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bertrand Bouchard
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christine Des Rosiers
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vincent, G., Khairallah, M., Bouchard, B. et al. Metabolic phenotyping of the diseased rat heart using 13C-substrates and ex vivo perfusion in the working mode. Mol Cell Biochem 242, 89–99 (2003). https://doi.org/10.1023/A:1021189728877

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1021189728877

Search

Navigation