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Cardiovascular Research 1999 42(3):567-570; doi:10.1016/S0008-6363(99)00027-9
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Respiratory control in normal and hypertrophic hearts

C. Gibbs

Department of Physiology, Monash University, Clayton, VIC 3168 Australia

Received 10 December 1998; accepted 14 January 1999

See article of Bache et al. [1] (pages 616–626) in this issue.

In a paper in the current issue of Cardiovascular Research, Bache, Zhang, Murakami, Zhang, Cho, Merkle, Gong, Fraser and Ugurbil [1] look at the proposition that the exaggerated depletion of PCr (phosphocreatine) and accumulation of inorganic phosphate (Pi) that occurs in left ventricular hypertrophy (LVH) is the result of impaired myocyte oxygenation. Using 31P- and 1H-NMR spectroscopy to determine myocardial high energy phosphate (HEP) levels and myoglobin desaturation the authors show convincingly that even at high work loads the exaggerated depletion of PCr and the increase in Pi are not the result of inadequate cellular oxygenation. They also conclude that the greater loss of PCr in the hypertrophic hearts during high work rates is probably caused by alterations in the regulation of myocardial oxidative phosphorylation. Their conclusion of adequate cellular oxygenation would receive support from earlier in situ studies of mitochrondrial function in volume- and pressure-overloaded rat hearts, where the authors [2] reported alterations in mitochondrial function despite adequate cytosol oxygenation.

The main energy consuming reactions occur in the cytosol where ATP (adenosine triphosphate) is utilized primarily by the actomyosin ATPase and various ion pumps during contraction and relaxation while resynthesis of ATP occurs within the mitochondria through the process of oxidative phosphorylation. The heart has limited capacity for glycolytic synthesis of ATP and is highly dependent on oxidative phosphorylation, as is evidenced by the fact that mitochondria account for 20–40% of cell volume depending on species. Oxidation of carbohydrates through the tricarboxylic acid cycle (TCA) or fats through the β-oxidation pathway provides intramitochondrial NADH (reduced nicotinamide adenine dinucleotide) and FADH which are the direct source of reducing equivalents (electrons) for the electron transport chain. According to the chemiosmotic hypothesis, the flow of electrons along the electron transport chain results in the translocation of protons across the mitochondrial inner membrane establishing a proton electrochemical gradient ({Delta}FormulaH). The ATP synthase of the mitochondrial inner membrane then couples the movement of protons down their electrochemical gradient to the resynthesis of ATP. {Delta}FormulaH represents the most direct measure of the state of energization of the mitochondria and has two components, a membrane potential ({Delta}{psi}) and a pH gradient ({Delta}pH) according to the relationship {Delta}FormulaH={Delta}{psi}–2.303RT{Delta}pH. Under physiological conditions {Delta}{psi} is the dominant term in this relationship having a value of around –150 mV, whereas {Delta}pH contributes about –60 mV to {Delta}µH (corresponding to {Delta}pH of about 1 pH unit). Despite much effort and considerable knowledge about oxidative phosphorylation there remains uncertainty regarding the regulatory mechanisms that match ATP production to ATP usage.

There are currently five main models of respiratory control and there are several useful reviews [3–5]. These include the so-called classical model (1) where kinetic regulation of metabolism occurs as a result of changes in free [ADP] (adenosine diphosphate) and [Pi] [6–8] or as a result of changes in the [ATP]/[ADP] which can affect the adenine translocase [9] (2) thermodynamic regulation by changes in the free energy of ATP hydrolysis [10–12] (3) regulation by the supply of reducing equivalents (NADH, FADH) for the electron transport chain [13] (4) regulation by changes in intracellular free Ca2+, which are sensed by the mitochondria and either increase intramitochondrial dehydrogenase activity directly or, in some workers view, modulate ATP synthase activity [14,15] (5) modulation by intracellular pO2 [16,17]. It should be apparent that options (1) and (2) and options (3) and (4) are not necessarily independent regulatory factors.

In respect of option (5) i.e. intracellular pO2 being a regulator of respiration, the studies of Gayeski and Honig [18] and a more recent 1H NMR study of perfused rat hearts by Kreutzer and Jue [19] suggest that there is normally a large capillary to cell O2 gradient and a fairly shallow cytosol to mitochondrial gradient. Kreutzer and Jue have shown that ATP signal intensity only starts to fall when cellular O2 drops below 1.0 mmHg and at this time pHi had not changed but lactate production had commenced much earlier, at cellular pO2’s between 4–5 mmHg. Because the contractile response and lactate formation rate changed before the critical pO2 was reached Kreutzer et al suggested that [NADH] rather than free [ADP]i is the key cellular signal of tissue hypoxia. These data together with the evidence in the paper by Bache et al [1] suggest that in blood perfused preparations the critical pO2 is not reached even at high work loads. The safety margin is not great however and in isolated preparations or in saline perfused hearts at even moderate energy flux levels there is the likelihood of cellular hypoxia with resultant changes in NADH levels.

In models (1) and (2) ATP and its hydrolytic products play a central role in respiratory control. In particular there has been a focus on free [ADP]i. However 31P-NMR analysis of whole hearts has often failed to demonstrate changes in the concentration of the adenine nucleotide sufficient to account for the increases in oxygen consumption that are observed when work is increased [12,20]. Balaban [4] in an excellent review says that ‘the ATP/ADP.Pi model (i.e. model (2)) is based on the concept that the majority of the mitochondrial respiratory chain (ie NADH to cytochrome c) is in near equilibrium with the cytosolic phosphorylation potential. This means that shifts in any of the equilibrium constituents (ie NADH, ATP, ADP and Pi and cytochrome c) result in alterations in cytochrome aa3 redox state which ultimately controls respiration through an irreversible step in the reduction of molecular oxygen. Balaban [4] goes on to review some of the experimental data that he believes is not consistent with the classical or thermodynamic model and in relation to the thermodynamic model he states that there is no consistent evidence that either the adenylate translocase or the ATP synthase reaction is actually at or near equilibrium in the heart.

To an outside observer it does appear as if some of the problems arise because workers are comparing results obtained on isolated mitochondria in various set energy states (states 1 to 5) to those obtained in the much more complex intracellular environment found in various physiological preparations some of which, particularly at high work rates, may be inadequately oxygenated. It is interesting that in NMR studies on in situ blood perfused rat hearts Headrick et al. [21] report a mean phosphorylation potential of 700,000 M–1, a mean {Delta}GATP of –63.9 kJ/mol and a mean free cytosolic [ADP] of 18 µm whereas when the same measurements were made on isolated buffer perfused hearts they reported a mean phosphorylation potential of 76,000 M–1, a mean {Delta}GATP=–59.9 kJ/mol and a mean free [ADP] of 65 µM. It needs to be pointed out the latter values are in good agreement with most isolated perfused rat heart literature. These authors concluded that the in situ heart is more highly energized and operates at a different set-point which is in line with the multiple control analysis idea [7]. The authors conclude that since the free cytosolic [ADP] at 18 µM is less than the apparent Km for respiration (about 25 µM), and since increased work rates (two levels of epinephrine infusion) caused three and five fold increase in free ADP, the classical model could predict their experimental data and their [PCr]/[ATP] changes were consistent with the in vivo rat data of Bittl, Balschi and Ingwall [8] and both sets of data are in line with the in situ dog heart data

In recent years there has been increasing experimental support for a regulatory role for Ca2+ in myocardial oxidative phosphorylation. There is experimental evidence that three enzymes of the TCA cycle catalyzing non-equilibrium reactions (ie pyruvate dehydrogenase, isocitrate dehydrogenase and {alpha}-ketoglutarate dehydrogenase) are stimulated by mitochondrial matrix calcium ([Ca2+]m) within the normal physiological range [5]. It is proposed that an increase in [Ca2+]m stimulates these matrix Ca2+-sensitive dehydrogenases leading to increased intramitochondrial [NADH], elevated {Delta}{psi} and hence increased ATP synthesis. Since an increase in cardiac work is often associated with an increase in cytosolic [Ca2+], it is argued that [Ca2+]i serves as the common intermediate, linking cytosolic ATP usage and mitochondrial ATP production.

It is central to this model that variations in cytosolic [Ca2+] evoke parallel changes in mitochondrial matrix [Ca2+]. Under normal conditions there is both an electrical and concentration gradient favouring mitochondrial Ca2+ uptake, and while it is technically difficult to measure [Ca2+]m there is evidence that variations in cytosolic [Ca2+] do lead to changes in [Ca2+]m of sufficient magnitude to regulate these dehydrogenases [22]. Furthermore, recent work by Chacon et al. [23] has demonstrated that, in rabbit myocytes, both the frequency and amplitude of changes in [Ca2+]m might reflect those that are occurring in the cytosol.

Further experimental support that Ca2+ activation of mitochondrial dehydrogenases is important in the control of oxidative phosphorylation is provided by measurements of NADH during work rate transitions in cardiac muscle. According to model (4) an increase in [Ca2+]m should, through stimulation of the dehydrogenases, increase the mitochondrial NADH/NAD+ ratio. Consistent with this view, increases in mitochondrial NADH/NAD+ have been reported to accompany increased myocardial O2 consumption (mVO2) when heart rate was increased, both in vitro and in vivo [24–26].

There is however a considerable body of experimental evidence that is inconsistent with this model of respiratory control. Since mitochondrial [NADH] can be viewed as being a substrate for electron transport, an increase in NADH would be expected to increase {Delta}{psi} and consequently {Delta}µH. There are only a few studies where the relationship between ATP utilization and {Delta}{psi} has been examined. Wan et al. [27] and Doumen et al. [28], using the distribution of the lipophilic cation tetraphenylphosphonium to measure {Delta}{psi}, found that the NADH/NAD ratio decreased when cardiac work was varied over a range that produced a four-fold increase in (mVO2) of perfused rat hearts. These observations have since been confirmed by Panov and Scaduto [29] who found a decrease in {Delta}{psi} when the respiratory activity of isolated mitochondria was stimulated by ADP.

Work from our laboratory in the 1970’s showed that as long as the workload was low to moderate, so that oxygen supply was not limiting, mitochondrial NADH/NAD+ decreases with increasing workload in isolated papillary muscles [30,31]. Since then similar results have been obtained in isolated perfused hearts [32,33] and in myocytes [34]. It is possible that the widely varying literature results for NADH reflects differences in preparation, workload and metabolic substrate utilized. Several investigators are quite critical of many of the NADH fluorescence measurements in the literature believing them to often reflect cellular hypoxia rather than normal respiratory control. The paper by Ashruf et al. [32] has an excellent discussion of the problem.

The observations that increases in cardiac work can occur with a decrease in NADH/NAD+ and a fall in {Delta}{psi} cast some doubt on whether activation of mitochondrial matrix dehydrogenases by Ca2+ is a sufficient stimulus for increasing the rate of ATP synthesis. An alternative explanation for these observations involves modulation of the kinetic properties of the ATP synthase by Ca2+. Harris and Das [14] have shown that in cultured rat myocytes ATP synthase activity increased when quiescent cells were stimulated to contract, and increased even further when the cells were exposed to the β-adrenergic agonist isoproterenol. The increase in activity could be blocked by the mitochondrial Ca2+ uptake inhibitor, ruthenium red, thereby implicating intramitochondrial Ca2+ as a primary effector in the stimulation of ATP synthase activity. Essentially a similar observation was made by Scholz and Balaban [15] who found that in an in vivo dog heart preparation infusion of phenylephrine caused a greater than 2-fold increase in mVO2 and a 30% increase in ATP synthase activity.

In the last few years a series of papers from Brandes, Bers and colleagues have been published that I think have unequivocally progressed our ideas on cardiac respiratory control [35,36]. Using isolated but very small trabeculae where oxygenation is not a factor, they have varied work rate over physiologically relevant ranges while monitoring NADH using fluorescence microscopy and using an improved methodology for correcting motion artifacts. At low pacing rates their data is identical to the earlier work of Chapman [30] in that NADH decreases with each contraction and then recovers slowly. At higher pacing rates NADH initially decreases to an amount dependent upon the new work rate but then with prolonged stimulation the NADH level recovers to a steady state level which is still below the initial level. They concluded that oxidative phosphorylation is not initially stimulated by increased mitochondrial [NADH] (i.e. model (3) prediction) but with prolonged activity [NADH] levels do increase. In a subsequent paper they varied work rate either by altering extracellular calcium or by varying filament overlap [36]. They clearly show that when work rate is increased by varying overlap there is no [NADH] recovery but when cellular calcium rises so does the extent of the secondary recovery phase which they attribute to Ca2+-dependent stimulation of mitochondrial dehydrogenases (model (4) prediction). My own view would be that the early fall is almost certainly caused by an increase in [ADP] as predicted by the classical model [6]. In their most recent paper the fluorescence technique has been extended to trabeculae from pressure overloaded hearts and cytosolic Ca2+ has also been monitored [37]. They find that compared to control hearts the initial fall in [NADH] levels is greater in hypertrophy but that with prolonged stimulation there was recovery of the NADH level to values close to that seen in control animals and they conclude that the Ca2+-dependent recovery mechanism is enhanced in hypertrophy i.e. the drive to dehydrogenase activity is increased.

It seems that we are much closer to understanding respiratory control in the heart but it should be remembered that we are dealing with a system where perhaps only a five fold increase in mVO2 is physiologically possible. In intact skeletal muscle the VO2 changes may be of the order of several hundred fold and this has led some authors to believe that the ADP signal in exercise is nowhere near large enough to be explained by a traditional Michaelis-Menton mechanism and that signal transduction for oxidative phosphorylation is at least second order in ADP [38]. Whether this level of complexity is also needed in cardiac tissue, is I believe an open question at the present time, but it has been speculated that in order to produce the big differences that are seen in myocardial mVO2 per g tissue across species [39] there may well have been an evolutionary need for some type of regulation that produces a higher kinetic gain in smaller mammals.


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