MiP2005: Session 2

Mitochondrial Physiology Network 10.9: 23-25 (2005) - download pdf

 

Measuring and modelling the effects of altered O2 delivery on muscle metabolism.

Graham J Kemp

Division of Metabolic and Cellular Medicine, Faculty of Medicine, University of Liverpool, Liverpool L69 3GA, UK. - gkemp@liv.ac.uk

    I consider four main issues, based as far as possible on quantitative analysis of published data in terms of models of mitochondrial function and regulation in vivo.

    (A) Methodological issues in measurement of O2 delivery and O2 usage in vivo

    There are several ways to study muscle 'metabolism (by which I mean ATP turnover in the service of force generation) in vivo. First, for oxidative metabolism, there are whole body VO2 measurements, and (less convenient) invasive arteriovenous difference (AVD) measurements of muscle O2 consumption [5]. Second, 31P magnetic resonance spectroscopy (MRS) can measure ATP production by oxidative and nonoxidative means (the latter including glycolysis to lactate and adjustments of transient ATP demand/supply imbalance by changes in phosphocreatine (PCr) concentration) [1,10,11]. There is a tradition of assessing ‘mitochondrial capacity’ (inferred maximal rate of oxidative ATP synthesis in vivo) by analysing relationships between oxidative ATP synthesis rate and the concentrations of metabolites of presumed regulatory relevance (e.g. ADP) or their correlates (e.g. PCr) [11,13,16]. Third, near-infrared spectroscopy (NIRS) and 1H MRS of deoxymyoglobin can be used to report tissue PO2. NIRS estimates of muscle O2 content can be used to measure rates of O2 usage [22], and to make semi-quantitative inferences about vascular O2 supply abnormalities [11,12]. Recently NIRS has been suggested as a measure of O2 AVD, and in combination with VO2 has been used to estimate the kinetics of blood flow [4]. (I argue that this is not a useful calculation, for algebraic reasons, and because in the data analysed [4] blood flow is always tightly coupled to O2 use). Interpretation of NIRS data [12] is still hampered by disagreement about whether the signal is mainly from capillary deoxyhaemoglobin or myocyte deoxymyoglobin; if the latter [21], then like 1H MRS of deoxymyoglobin [19], NIRS usefully reports PO2 in the vicinity of the mitochondrion (see (D) below). Fourth, 13C MRS labelling methods can be used to measure TCA cycle flux in vivo [9], although current estimates of basal oxidative ATP synthesis by this method are substantially too high, for technical reasons. The same is true of a fifth technique, 31P MR saturation transfer, which can be used with 13C MRS to assess mitochondrial coupling in vivo [17].

    (B) Understanding oxidative ATP synthesis rates in relation to metabolite concentrations

    None of these methods tell us all we want to know, but some key measurements are available. I want to ask to what extent we understand these, a question with several aspects. First, can we fit our measurements of rates and metabolite concentrations into a model consistent with known biochemistry and physiology, both classical, and the newer insights of Metabolic Control Analysis (MCA)? Probably yes, to some extent. Secondly, how much explanatory power do such models have? Does e.g. measured [ADP] predict the rate of mitochondrial O2 consumption? In some circumstances, yes e.g. within a single experiment. However, there is substantial variation between published human studies in inferred maximal rates and relationships between ATP synthesis rates and e.g. [ADP]. Some of this is due to disagreement about resting [PCr] measured by 31P MRS (necessary for calculation of [ADP]), and some is no doubt methodological in other ways, but what remains might be interesting physiology. In addition to longstanding disagreements about candidate force-flow relationships, it is argued (in conformity with MCA [3]) that ‘parallel-activation’ or ‘feed-forward’ influences are important in control of mitochondrial ATP synthesis in vivo [14], detectable in 31P MRS experiments as a lack of correlation between fluxes and concentrations, and in particular as flux changes ‘too large’ for the changes in e.g. [ADP] [14]. This conclusion can be avoided if one posits a higher-order force-flow relation [7], although whether this corresponds to mitochondrial behaviour in vitro remains controversial. Recently attention has been focussed [20] on the direct effects of PCr and free Cr on O2 consumption in vitro, independent of [ADP] [23]. Implications for mitochondrial regulation in vivo are linked to mitochondrial creatine kinase [20], although no detailed predictions have been made. While this is in theory a way in which a hyperbolic flux-ADP relationship in vitro could appear in vivo as the sigmoid relationship [7] needed to explain the dynamic–range problem without parallel activation, the required effect is much larger than has been shown in vitro. However, our understanding of these mechanisms remains incomplete.

    (C) Understanding oxidative ATP synthesis rates in relation to glycolysis

    A third question is, given some task-defined ATP demand, can we predict, nontrivially, how ATP generation will be split between oxidative and glycolytic means? One approach is to use the measurements we have (e.g. pH, PCr, ADP) to predict the oxidative ATP synthesis rate, and then infer glycolytic ATP synthesis essentially by difference. As well as the problems already mentioned, this is hampered by disagreement about how much the pH change which accompanies lactate accumulation itself reduces mitochondrial capacity [6, 8]. Assessing this definitively requires agreement on the relevant flux-force relationships, which, as we have seen, is lacking. A related approach, concentrating on possible activators of glycolysis, is hampered by similar disagreement about whether open- or closed-loop influences dominate [2, 15], and if the latter, which metabolites measurable in vivo are potential predictors of flux in vivo

    (D) Modelling the effect of impaired O2 delivery on oxidative metabolism

    This is physiologically and pathologically important. There have been semi-quantitative approaches. In vascular disease [11] and experimental hypoxia [18] the mitochondrial capacity inferred from 31P MRS measurements is reduced at low cell PO2 inferred from NIRS or 1H MRS (see (A) above), apparently consistent with the known dependence of mitochondrial metabolism on the concentration of its substrate O2 [10]. At present, though, a rate of O2 usage cannot be ‘read off’ from cell PO2 and (say) [ADP], any more than it can be from [ADP] and pH in exercise where ((see (B)  and (C) above), although pH falls, PO2 is not ‘limiting’.

1.  Blei ML, Conley KE, Kushmerick MJ (1993) Separate measures of ATP utilization and recovery in human skeletal muscle [correction in J Physiol 1994;475:548]. J. Physiol. 465: 203-222.

2.  Conley KE, Kushmerick MJ, Jubrias SA (1998) Glycolysis is independent of oxygenation state in stimulated human skeletal muscle in vivo. J. Physiol. 511: 935-945.

3.  Fell DA, Thomas S (1995) Physiological control of metabolic flux: the requirement for multisite modulation. Biochem. J. 311: 35-39.

4.  Ferreira L, Townsend D, Lutjemeier B, Barstow T (2005) Muscle capillary blood flow kinetics estimated from pulmonary O2 uptake and near-infrared spectroscopy. J. Appl. Physiol. 98: 1820-1828.

5.  Grassi B, Poole D, Richardson RS, Knight D, Erickson B, Wagner PD (1996) Muscle O2 uptake kinetics in humans: implications for metabolic control. J. Appl. Physiol. 80: 988-998.

6.  Harkema S, Meyer RA (1997) Effect of acidosis on control of respiration in skeletal muscle. Am. J. Physiol. 272: C491-500.

7.  Jeneson JAL, Wiseman RW, Westerhoff HV, Kushmerick MJ (1996) The signal transduction function for oxidative phosphorylation is at least second order in ADP. J. Biol. Chem. 271: 27995-28.

8.  Jubrias SA, Crowther G, Shankland E, Gronka R, Conley KE (2003) Acidosis inhibits oxidative phosphorylation in contracting human skeletal muscle in vivo. J. Physiol. 533: 589-599.

9.  Jucker B, Dufour S, Ren J, Cao X, Previs S, Underhill B, Cadman K, Shulman GI (2000) Assessment of mitochondrial energy coupling in vivo by 13C/31P NMR [correction in PNAS USA (2001) 98, 3624]. Proc. Natl. Acad. Sci. USA 97: 6880-6884.

10.       Kemp GJ (2004) Mitochondrial dysfunction in chronic ischemia and peripheral vascular disease. Mitochondrion 4: 629-640.

11.       Kemp GJ, Roberts N, Bimson WE, Bakran A, Frostick SP (2002) Muscle oxygenation and ATP turnover when blood flow is impaired by vascular disease. Spectroscopy Int. J. 16: 317-334.

12.       Kemp GJ, Roberts N, Bimson WE, Bakran A, Harris P, Gilling-Smith G, Brennan J, Rankin A, Frostick SP (2001) Mitochondrial function and oxygen supply in normal and in chronically ischaemic muscle: a combined 31P magnetic resonance spectroscopy and near infra-red spectroscopy study in vivo. J. Vasc. Surg. 34: 1103-1110.

13.       Kemp GJ, Thompson CH, Taylor DJ, Hands L, Rajagopalan B, Radda GK (1993) Quantitative analysis by 31P MRS of abnormal mitochondrial oxidation in skeletal muscle during recovery from exercise. NMR Biomed. 6: 302-310.

14.       Korzeniewski B (1998) Regulation of ATP supply during muscle contraction: theoretical studies. Biochem. J. 330: 1189-1195.

15.       Lambeth MJ, Kushmerick MJ (2002) A computational model for glycogenolysis in skeletal muscle. Ann. Biomed. Eng. 30: 808-827.

16.       McCully K, Fielding R, Evans W, Leigh JJ, Posner J (1993) Relationships between in vivo and in vitro measurements of metabolism in young and old human calf muscles. J. Appl. Physiol. 75: 813-819.

17.       Petersen KF, Befroy D, Dufour S, Dziura J, Ariyan C, Rothman DL, DiPietro L, Cline GL, Shulman GI (2003) Mitochondrial dysfunction in the elderly: possible role in insulin resistance. Science 300: 1140-1142.

18.       Richardson RS, Leigh JS, Wagner PD, Noyszewski E (1999) Cellular PO2 as a determinant of maximal mitochondrial O2 consumption in trained human skeletal muscle. J. Appl. Physiol. 87: 325-331.

19.       Richardson RS, Noyszewski EA, Kendrick KF, Leigh JS, Wagner PD (1995) Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport. J. Clin. Invest. 96: 1916-1926.

20.       Smith SA, Montain SJ, Zientara GP, Fielding RA (2004) Use of phosphocreatine kinetics to determine the influence of creatine on muscle mitochondrial respiration: an in vivo 31P-MRS study of oral creatine ingestion. J. Appl. Physiol. 96: 2288-2292.

21.       Tran T, Sailasuta N, Kreutzer U, Hurd R, Chung Y, Mole P, Kuno S, Jue T (1999) Comparative analysis of NMR and NIRS measurements of intracellular PO2 in human skeletal muscle. Am. J. Physiol. 276: R1682-R1690.

22.       Van Beekvelt M, Colier W, Wevers R, Van Engelen B (2001) Performance of near-infrared spectroscopy in measuring local O2 consumption and blood flow in skeletal muscle. J. Appl. Physiol. 90: 511-519.

23.       Walsh B, Tonkonogi M, Soderlund K, Hultman E, Saks V, Sahlin K (2001) The role of phosphorylcreatine and creatine in the regulation of mitochondrial respiration in human skeletal muscle. J.  Physiol. 537: 971-978.


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