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. 2002 Aug;83(2):587–604. doi: 10.1016/S0006-3495(02)75194-1

Simultaneous measurements of mitochondrial NADH and Ca(2+) during increased work in intact rat heart trabeculae.

Rolf Brandes 1, Donald M Bers 1
PMCID: PMC1302172  PMID: 12124250

Abstract

The main goal of this study is to investigate the role of mitochondrial [Ca(2+)], [Ca(2+)](m), in the possible up-regulation of the NADH production rate during increased workload. Such up-regulation is necessary to support increased flux through the electron transport chain and increased ATP synthesis rates. Intact cardiac trabeculae were loaded with Rhod-2(AM), and [Ca(2+)](m) and mitochondrial [NADH] ([NADH](m)) were simultaneously measured during increased pacing frequency. It was found that 53% of Rhod-2 was localized in mitochondria. Increased pacing frequency caused a fast, followed by a slow rise of the Rhod-2 signal, which could be attributed to an abrupt increase in resting cytosolic [Ca(2+)], and a more gradual rise of [Ca(2+)](m), respectively. When the pacing frequency was increased from 0.25 to 2 Hz, the slow Rhod-2 component and the NADH signal increased by 18 and 11%, respectively. Based on a new calibration method, the 18% increase of the Rhod-2 signal was calculated to correspond to a 43% increase of [Ca(2+)](m). There was also a close temporal relationship between the rise (time constant approximately 25 s) and fall (time constant approximately 65 s) of [Ca(2+)](m) and [NADH](m) when the pacing frequency was increased and decreased, respectively, suggesting that increased workload and [Ca(2+)](c) cause increased [Ca(2+)](m) and consequently up-regulation of the NADH production rate.

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Selected References

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  1. Ashruf J. F., Coremans J. M., Bruining H. A., Ince C. Increase of cardiac work is associated with decrease of mitochondrial NADH. Am J Physiol. 1995 Sep;269(3 Pt 2):H856–H862. doi: 10.1152/ajpheart.1995.269.3.H856. [DOI] [PubMed] [Google Scholar]
  2. Backx P. H., Ter Keurs H. E. Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt. Am J Physiol. 1993 Apr;264(4 Pt 2):H1098–H1110. doi: 10.1152/ajpheart.1993.264.4.H1098. [DOI] [PubMed] [Google Scholar]
  3. Baker A. J., Brandes R., Schreur J. H., Camacho S. A., Weiner M. W. Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator indo-1. Biophys J. 1994 Oct;67(4):1646–1654. doi: 10.1016/S0006-3495(94)80637-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bassani J. W., Bassani R. A., Bers D. M. Ca2+ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes. J Physiol. 1993 Jan;460:603–621. doi: 10.1113/jphysiol.1993.sp019489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brandes R., Bers D. M. Analysis of the mechanisms of mitochondrial NADH regulation in cardiac trabeculae. Biophys J. 1999 Sep;77(3):1666–1682. doi: 10.1016/S0006-3495(99)77014-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brandes R., Bers D. M. Increased work in cardiac trabeculae causes decreased mitochondrial NADH fluorescence followed by slow recovery. Biophys J. 1996 Aug;71(2):1024–1035. doi: 10.1016/S0006-3495(96)79303-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brandes R., Bers D. M. Intracellular Ca2+ increases the mitochondrial NADH concentration during elevated work in intact cardiac muscle. Circ Res. 1997 Jan;80(1):82–87. doi: 10.1161/01.res.80.1.82. [DOI] [PubMed] [Google Scholar]
  8. Brandes R., Figueredo V. M., Camacho S. A., Baker A. J., Weiner M. W. Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts. Biophys J. 1993 Nov;65(5):1983–1993. doi: 10.1016/S0006-3495(93)81275-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brandes R., Figueredo V. M., Camacho S. A., Baker A. J., Weiner M. W. Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry. Biophys J. 1993 Nov;65(5):1973–1982. doi: 10.1016/S0006-3495(93)81274-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brandes R., Figueredo V. M., Camacho S. A., Massie B. M., Weiner M. W. Suppression of motion artifacts in fluorescence spectroscopy of perfused hearts. Am J Physiol. 1992 Sep;263(3 Pt 2):H972–H980. doi: 10.1152/ajpheart.1992.263.3.H972. [DOI] [PubMed] [Google Scholar]
  11. Brandes R., Figueredo V. M., Camacho S. A., Weiner M. W. Compensation for changes in tissue light absorption in fluorometry of hypoxic perfused rat hearts. Am J Physiol. 1994 Jun;266(6 Pt 2):H2554–H2567. doi: 10.1152/ajpheart.1994.266.6.H2554. [DOI] [PubMed] [Google Scholar]
  12. Brandes R., Maier L. S., Bers D. M. Regulation of mitochondrial [NADH] by cytosolic [Ca2+] and work in trabeculae from hypertrophic and normal rat hearts. Circ Res. 1998 Jun 15;82(11):1189–1198. doi: 10.1161/01.res.82.11.1189. [DOI] [PubMed] [Google Scholar]
  13. Buntinas L., Gunter K. K., Sparagna G. C., Gunter T. E. The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria. Biochim Biophys Acta. 2001 Apr 2;1504(2-3):248–261. doi: 10.1016/s0005-2728(00)00254-1. [DOI] [PubMed] [Google Scholar]
  14. Cox D. A., Matlib M. A. A role for the mitochondrial Na(+)-Ca2+ exchanger in the regulation of oxidative phosphorylation in isolated heart mitochondria. J Biol Chem. 1993 Jan 15;268(2):938–947. [PubMed] [Google Scholar]
  15. Del Nido P. J., Glynn P., Buenaventura P., Salama G., Koretsky A. P. Fluorescence measurement of calcium transients in perfused rabbit heart using rhod 2. Am J Physiol. 1998 Feb;274(2 Pt 2):H728–H741. doi: 10.1152/ajpheart.1998.274.2.H728. [DOI] [PubMed] [Google Scholar]
  16. Di Lisa F., Gambassi G., Spurgeon H., Hansford R. G. Intramitochondrial free calcium in cardiac myocytes in relation to dehydrogenase activation. Cardiovasc Res. 1993 Oct;27(10):1840–1844. doi: 10.1093/cvr/27.10.1840. [DOI] [PubMed] [Google Scholar]
  17. Eng J., Lynch R. M., Balaban R. S. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes. Biophys J. 1989 Apr;55(4):621–630. doi: 10.1016/S0006-3495(89)82859-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Griffiths E. J., Lin H., Suleiman M. S. NADH fluorescence in isolated guinea-pig and rat cardiomyocytes exposed to low or high stimulation rates and effect of metabolic inhibition with cyanide. Biochem Pharmacol. 1998 Jul 15;56(2):173–179. doi: 10.1016/s0006-2952(98)00016-1. [DOI] [PubMed] [Google Scholar]
  19. Griffiths E. J., Stern M. D., Silverman H. S. Measurement of mitochondrial calcium in single living cardiomyocytes by selective removal of cytosolic indo 1. Am J Physiol. 1997 Jul;273(1 Pt 1):C37–C44. doi: 10.1152/ajpcell.1997.273.1.C37. [DOI] [PubMed] [Google Scholar]
  20. Griffiths E. J., Wei S. K., Haigney M. C., Ocampo C. J., Stern M. D., Silverman H. S. Inhibition of mitochondrial calcium efflux by clonazepam in intact single rat cardiomyocytes and effects on NADH production. Cell Calcium. 1997 Apr;21(4):321–329. doi: 10.1016/s0143-4160(97)90120-2. [DOI] [PubMed] [Google Scholar]
  21. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  22. Gunter T. E., Gunter K. K., Sheu S. S., Gavin C. E. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol. 1994 Aug;267(2 Pt 1):C313–C339. doi: 10.1152/ajpcell.1994.267.2.C313. [DOI] [PubMed] [Google Scholar]
  23. Hansford R. G. Dehydrogenase activation by Ca2+ in cells and tissues. J Bioenerg Biomembr. 1991 Dec;23(6):823–854. doi: 10.1007/BF00786004. [DOI] [PubMed] [Google Scholar]
  24. Haworth R. A., Redon D. Calibration of intracellular Ca transients of isolated adult heart cells labelled with fura-2 by acetoxymethyl ester loading. Cell Calcium. 1998 Oct;24(4):263–273. doi: 10.1016/s0143-4160(98)90050-1. [DOI] [PubMed] [Google Scholar]
  25. Hove-Madsen L., Bers D. M. Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties. Biophys J. 1992 Jul;63(1):89–97. doi: 10.1016/S0006-3495(92)81597-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hunter D. R., Haworth R. A., Berkoff H. A. Cellular manganese uptake by the isolated perfused rat heart: a probe for the sarcolemma calcium channel. J Mol Cell Cardiol. 1981 Sep;13(9):823–832. doi: 10.1016/0022-2828(81)90239-x. [DOI] [PubMed] [Google Scholar]
  27. Hunter D. R., Komai H., Haworth R. A., Jackson M. D., Berkoff H. A. Comparison of Ca2+, Sr2+, and Mn2+ fluxes in mitochondria of the perfused rat heart. Circ Res. 1980 Nov;47(5):721–727. doi: 10.1161/01.res.47.5.721. [DOI] [PubMed] [Google Scholar]
  28. Jacobus W. E., Moreadith R. W., Vandegaer K. M. Mitochondrial respiratory control. Evidence against the regulation of respiration by extramitochondrial phosphorylation potentials or by [ATP]/[ADP] ratios. J Biol Chem. 1982 Mar 10;257(5):2397–2402. [PubMed] [Google Scholar]
  29. Koretsky A. P., Katz L. A., Balaban R. S. Determination of pyridine nucleotide fluorescence from the perfused heart using an internal standard. Am J Physiol. 1987 Oct;253(4 Pt 2):H856–H862. doi: 10.1152/ajpheart.1987.253.4.H856. [DOI] [PubMed] [Google Scholar]
  30. Leyssens A., Nowicky A. V., Patterson L., Crompton M., Duchen M. R. The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes. J Physiol. 1996 Oct 1;496(Pt 1):111–128. doi: 10.1113/jphysiol.1996.sp021669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Maier L. S., Brandes R., Pieske B., Bers D. M. Effects of left ventricular hypertrophy on force and Ca2+ handling in isolated rat myocardium. Am J Physiol. 1998 Apr;274(4 Pt 2):H1361–H1370. doi: 10.1152/ajpheart.1998.274.4.H1361. [DOI] [PubMed] [Google Scholar]
  32. McCormack J. G., Halestrap A. P., Denton R. M. Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev. 1990 Apr;70(2):391–425. doi: 10.1152/physrev.1990.70.2.391. [DOI] [PubMed] [Google Scholar]
  33. Miyata H., Silverman H. S., Sollott S. J., Lakatta E. G., Stern M. D., Hansford R. G. Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes. Am J Physiol. 1991 Oct;261(4 Pt 2):H1123–H1134. doi: 10.1152/ajpheart.1991.261.4.H1123. [DOI] [PubMed] [Google Scholar]
  34. Nuutinen E. M. Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart. Basic Res Cardiol. 1984 Jan-Feb;79(1):49–58. doi: 10.1007/BF01935806. [DOI] [PubMed] [Google Scholar]
  35. Schreur J. H., Figueredo V. M., Miyamae M., Shames D. M., Baker A. J., Camacho S. A. Cytosolic and mitochondrial [Ca2+] in whole hearts using indo-1 acetoxymethyl ester: effects of high extracellular Ca2+. Biophys J. 1996 Jun;70(6):2571–2580. doi: 10.1016/S0006-3495(96)79828-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sparagna G. C., Gunter K. K., Sheu S. S., Gunter T. E. Mitochondrial calcium uptake from physiological-type pulses of calcium. A description of the rapid uptake mode. J Biol Chem. 1995 Nov 17;270(46):27510–27515. doi: 10.1074/jbc.270.46.27510. [DOI] [PubMed] [Google Scholar]
  37. Spurgeon H. A., Stern M. D., Baartz G., Raffaeli S., Hansford R. G., Talo A., Lakatta E. G., Capogrossi M. C. Simultaneous measurement of Ca2+, contraction, and potential in cardiac myocytes. Am J Physiol. 1990 Feb;258(2 Pt 2):H574–H586. doi: 10.1152/ajpheart.1990.258.2.H574. [DOI] [PubMed] [Google Scholar]
  38. Territo P. R., French S. A., Dunleavy M. C., Evans F. J., Balaban R. S. Calcium activation of heart mitochondrial oxidative phosphorylation: rapid kinetics of mVO2, NADH, AND light scattering. J Biol Chem. 2000 Oct 11;276(4):2586–2599. doi: 10.1074/jbc.M002923200. [DOI] [PubMed] [Google Scholar]
  39. Territo P. R., Mootha V. K., French S. A., Balaban R. S. Ca(2+) activation of heart mitochondrial oxidative phosphorylation: role of the F(0)/F(1)-ATPase. Am J Physiol Cell Physiol. 2000 Feb;278(2):C423–C435. doi: 10.1152/ajpcell.2000.278.2.C423. [DOI] [PubMed] [Google Scholar]
  40. Trollinger D. R., Cascio W. E., Lemasters J. J. Mitochondrial calcium transients in adult rabbit cardiac myocytes: inhibition by ruthenium red and artifacts caused by lysosomal loading of Ca(2+)-indicating fluorophores. Biophys J. 2000 Jul;79(1):39–50. doi: 10.1016/S0006-3495(00)76272-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Unitt J. F., McCormack J. G., Reid D., MacLachlan L. K., England P. J. Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using 31P n.m.r. and ruthenium red. Biochem J. 1989 Aug 15;262(1):293–301. doi: 10.1042/bj2620293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wan B., Doumen C., Duszynski J., Salama G., Vary T. C., LaNoue K. F. Effects of cardiac work on electrical potential gradient across mitochondrial membrane in perfused rat hearts. Am J Physiol. 1993 Aug;265(2 Pt 2):H453–H460. doi: 10.1152/ajpheart.1993.265.2.H453. [DOI] [PubMed] [Google Scholar]
  43. White R. L., Wittenberg B. A. Effects of calcium on mitochondrial NAD(P)H in paced rat ventricular myocytes. Biophys J. 1995 Dec;69(6):2790–2799. doi: 10.1016/S0006-3495(95)80152-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

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