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ABSTRACT |
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Abstract
Letter
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The following is the abstract of the article discussed in the subsequent letter:
Portman, Michael A., Yun Xiao, Ying Song,
and Xue-Han Ning. Expression of adenine nucleotide translocator
parallels maturation of respiratory control in heart in vivo.
Am. J. Physiol. 273 (Heart Circ. Physiol. 42):
H1977-H1983, 1997.
Changes in the relationship between myocardial
high-energy phosphates and oxygen consumption in vivo occur during
development, implying that the mode of respiratory control undergoes
maturation. We hypothesized that these maturational changes in sheep
heart are paralleled by alterations in the adenine nucleotide
translocator (ANT), which are in turn related to changes in the
expression of this gene. Increases in myocardial oxygen consumption
(M
O2) were induced by
epinephrine infusion in newborn (0-32 h,
n = 6) and mature sheep (30-32
days, n = 6), and high-energy
phosphates were monitored with 31P
nuclear magnetic resonance. Western blot analyses for the
ANT1 and the 
-subunit of
F1-adenosinetriphosphatase
(ATPase) were performed in these hearts and additional
(n = 9 total per group) as well as in
fetal hearts (130-132 days of gestation,
n = 5). Northern blot analyses were
performed to assess for changes in steady-state RNA transcripts for
these two genes. Kinetic analyses for the
31P spectra data revealed that the
ADP-MO2 relationship for
the newborns conformed to a Michaelis-Menten model but that the mature data did not conform to first- or second-order kinetic control of
respiration through ANT. Maturation from fetal to mature was accompanied by a 2.5-fold increase in ANT protein (by Western blot),
with no detectable change in
-F1-ATPase. Northern blot data
show that steady-state mRNA levels for ANT and
-F1-ATPase increased
~2.5-fold from fetal to mature. These data indicate that
1) respiratory control pattern in
the newborn is consistent with a kinetic type regulation through ANT,
2) maturational decreases in control
through ANT are paralleled by specific increases in ANT content, and
3) regulation of these changes in
ANT may be related to increases in steady-state transcript levels for
its gene.
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LETTER |
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Abstract
Letter
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Do kinetics of ADP stimulation of mitochondria really change during myocardial maturation?
To the Editor: Portman et al. (6) recently correlated expression of the adenine nucleotide translocator (ANT) with a maturational change in mitochondrial respiratory control in cardiac muscle. Here we point out that, contrary to what is claimed, the evidence presented does not prove that myocardial maturation involves a change in the mechanism of respiratory control or even the kinetics of ADP stimulation of mitochondrial respiration.Schönfeld et al. (7) previously showed a maturational increase in ANT expression in rat heart. Portman et al. (6) do not distinguish or separate mRNA transcription, expressed protein amounts, or kinetic properties of the expressed protein. It is the latter two properties that are relevant to the hypothesis that maturation of ANT leads to altered respiratory control. Consequently, the specific increase of ANT in mitochondria claimed (6) must be distinguished from both an increase in total mitochondrial density, which was shown by Veerkamp et al. (8) in the rat, as well as changes in H+-ATPase. The fact that F1-ATPase Western blots seem to support the authors' conclusion may also be correlative, and thus an independent evaluation of mitochondrial density (i.e., citrate synthase activity) should have been a necessary control experiment to include.
The apparent maturational change of respiratory control reported (6)
may be a more complex matter and requires rigorous quantitative
analyses, as we will demonstrate. Much work has already moved our
understanding beyond the dull and simplistic Michaelis-Menten picture
of ADP respiratory control that has pervaded the literature, e.g.,
respiratory control by ANT is not complete and depends on the work load
(2), and respiratory control by ADP concentration ([ADP])
exhibits an apparent cooperativity (4). The claim that the mechanism of
respiratory control changes during maturation from feedback control in
the neonatal myocardium to one in which ADP no longer plays a
significant role cannot be established by the data presented (6).
First, the relationship between [ADP] and myocardial oxygen
consumption (MO2) in the
mature myocardium can be adequately explained by any of three tested
sigmoids with Hill coefficients
(nH) of 3, 5, or 7 (see our Fig. 1, curves
A-C), in agreement with apparent cooperative ADP
regulation of respiration (4). This finding raises the important
question: What value for
nH should be
expected in the mature heart? In the absence of any rigorously
established kinetic mechanism for the apparent cooperativity of
[ADP] stimulation of respiration (4), and because of
complicating issues such as possible nested
Pi concentration ([Pi]) stimulation of
respiration raising the apparent overall kinetic order of the
ADP-MO2
relationship,1
this question cannot be answered at present. As such, the implicit conclusion of Portman et al. (6) that
nH values of 1 and 2 are compatible, but 3, 5, or 7 are no longer compatible, with [ADP] feedback control of respiration was preemptive.
However, this indeed will need to be addressed in future
studies.
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Second, although the results of Portman et al. (6) do demonstrate
different apparent kinetics of [ADP] stimulation in newborn versus mature hearts, it was not adequately tested (let alone proven)
that these observations reflect altered true kinetics, i.e., a change
in nH or
K0.5 ([ADP] at
which respiration is half-maximally stimulated). Their
conclusion was based on a linearized Hill plot of log
v/(Vmax 
v) versus log
[ADP], where v is the
myocardial oxygen consumption rate and
Vmax is the
maximal oxidative phosphorylation rate, for which they used the same
Vmax for both the
newborn and the mature hearts; however, the authors omitted the
detailed outcome of this analysis. When we analyzed the general trend
in the data given in their Tables 1 and 2 (6) using the same approach, we found nH = 1.5 and K0.5 = 0.187 mM for newborn hearts and
nH = 5.6 and
K0.5 = 0.054 mM
for mature hearts. However, as we stated before (4), such analysis
depends critically on the value for Vmax used. The
authors did not make measurements of
Vmax in either group; rather, they assumed
Vmax to be the
same in both groups. However, this assumption is not supported by the
literature (7, 8). On the contrary, in rat myocardium
Vmax was found to
double with maturation (7). Reanalyzing the newborn heart data by assuming that the same holds for sheep heart, we found
nH = 2.1 and
K0.5 = 0.097 mM
(see our Fig. 1, curve D). If
Vmax increases threefold with maturation, the Hill plots for both mature and newborn
hearts correspond to the same kinetics of [ADP] stimulation of respiration
(nH = 3 and
K0.5 = 0.068 mM)
(see our Fig. 1, curve C). Thus, in
the absence of any data on
Vmax for the
newborn hearts, the simplest explanation that only
Vmax, not
nH or
K0.5, changes during maturation (which may well be consistent with a maturational change in mitochondrial density) cannot be excluded.
In light of these considerations, we view the study by Portman et al. (6) as another call for appropriate studies and analyses of the kinetics of ADP translocation to define the mitochondrial mechanisms responsible for cellular ATP homeostasis in the myocardium.
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FOOTNOTES |
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1
Because [ADP] and
[Pi] covary during
contraction (3), the apparent overall kinetic order of the
ADP-MO2 relationship will
reflect [Pi]
stimulation of respiration superimposed on [ADP] stimulation if [Pi] is
within range of its Michaelis-Menten constant value (
1.25 mM; Ref.
1) over the studied range of respiratory rates. In the mature sheep
myocardium, this is indeed the case (5).
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REFERENCES |
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Top
Abstract
Letter
References
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1.
Bygrave, F. L.,
and
A. L. Lehninger.
Properties of an oligomycin-sensitive ADP-ATP exchange reaction in intact beef heart mitochondria.
J. Biol. Chem.
241:
3894-3903,
1966
2.
Groen, A. K.,
R. J. A. Wanders,
H. V. Westerhoff,
R. van der Meer,
and
J. M. Tager.
Quantification of the distribution of various steps to the control of mitochondrial communication.
J. Biol. Chem.
257:
2754-2757,
1979
3.
Jeneson, J. A. L.,
H. V. Westerhoff,
T. R. Brown,
C. J. A. van Echteld,
and
R. Berger.
Quasi-linear relationship between Gibbs free energy of ATP hydrolysis and power output in human forearm muscle.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1474-C1484,
1995
4.
Jeneson, J. A. L.,
R. W. Wiseman,
H. V. Westerhoff,
and
M. J. Kushmerick.
The signal transduction function for oxidative phosphorylation is at least second order in ADP.
J. Biol. Chem.
271:
27995-27998,
1996
5.
Portman, M. A.,
F. W. Heineman,
and
R. S. Balaban.
Developmental changes in the relation between phosphate metabolites and oxygen consumption in the sheep heart in vivo.
J. Clin. Invest.
83:
456-464,
1989.
6.
Portman, M. A.,
Y. Xiao,
Y. Song,
and
X.-H. Ning.
Expression of adenine nucleotide translocator parallels maturation of respiratory control in heart in vivo.
Am. J. Physiol.
273 (Heart Circ. Physiol. 42):
H1977-H1983,
1997
7.
Schönfeld, P.,
L. Schild,
and
R. Bohnensack.
Expression of the ADP/ATP carrier and expansion of the mitochondrial (ATP + ADP) pool contribute to postnatal maturation of the rat heart.
Eur. J. Biochem.
241:
895-900,
1996[Medline].
8.
Veerkamp, J. H., J. F. C. Glatz, and A. J. M. Wagenmakers. Metabolic changes during cardiac
maturation. Bas. Res. Cardiol. 80, Suppl. 2: 111-113, 1985.
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J. A. L. Jeneson R. W. Wiseman Department of Radiology University of Washington School of Medicine Seattle, WA 98195 | |||||
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M. J. Kushmerick Departments of Radiology and Physiology and Biophysics and Center for Bioengineering University of Washington School of Medicine Seattle, WA 98195 | |||||
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H. V. Westerhoff Department of Microbial Physiology, Faculty of Biology Free University 1081 HV Amsterdam; and E. C. Slater Institute, Biocenter University of Amsterdam 1105 AZ Amsterdam, The Netherlands |
To the Editor: Jeneson and
colleagues write a critique of our recent paper (9), which in part
disputes their model of respiratory control through cooperative
activation of the adenine nucleotide translocator. They say that we
claimed to have proven the hypothesis that myocardial maturation
involves a change in the mechanism of respiratory control and the
kinetics of ADP stimulation of mitochondrial respiration. Actually, we
did not purport to prove that changes in these mechanisms occur (9),
but we did provide data obtained in vivo supporting the stated
hypothesis, which originated in thought from studies performed in
isolated mitochondrial suspensions.
Their letter further states that we "do not distinguish or separate
mRNA transcription, expressed protein amounts, or kinetic properties of
the expressed protein." The outline of the discussion in our paper
does clearly separate these components. Whereas
F1-ATPase protein reaches adult
levels during fetal life, adenine nucleotide translocator protein
continues to accumulate after birth. These differences in protein
expression occur despite coordinated mRNA expression and imply that
observed increases in mitochondrial density or mass in sheep (1) are
due to specific changes in the adenine nucleotide translocator. These
data conform to results obtained by Wells et al. (10) in sheep heart
that demonstrate that tissue levels of respiratory chain components and
mitochondrial ATPase activity attain adult levels or even greater
before birth. Jeneson and colleagues appear to either ignore or be
unaware of these classic studies when they suggest that an arbitrary
"independent evaluation" for a generalized increase in
mitochondrial density should have been performed during our
experiments. Veerkamp's work in rat heart, cited by Jeneson and
colleagues and used to support their suggestion, is not relevant to our
sheep model.
Although Jeneson and colleagues indicate that they believe that
respiratory control is a complex matter, they have reduced modeling of
this regulation to a single system dependent on second-order kinetics
controlled by ADP. The developers of this model ignore the wealth of
data that indicate that a multifaceted system of control exists
including modulation by mitochondrial
Ca2+ concentrations and reducing
equivalent supply to the respiratory chain (3). Extensive review of the
literature indicates that there is no support for cooperative
stimulation of the adenine nucleotide translocator other than their own
modeling, which has not been substantiated by statistical analyses (see
curve constructions in their Fig. 1). This model (4) was based on
trends in data obtained in Balaban's laboratory (see Ref. 5; Portman
coauthor). To develop this model, they assumed that increases in ADP
accompanied the relatively high increases in myocardial oxygen
consumption (M Our published Hill analyses (9) indicate that no such cooperativity
effect can be observed in mature sheep myocardium. Rigorous statistical
analyses in more than 30 mature sheep from published and unpublished
experiments indicate that there is no significant increase in ADP
during wide and variable increases in
M Construction of their curves for newborns is based on erroneous
assumptions that demonstrate inadequate attention to the literature. First, they assume that maximal oxygen consumption rates
(Vmax) in
mature sheep exceed the newborn by a factor of two to three. Gratama et
al. (2) have shown that
Vmax
values in metabolically mature sheep during treadmill exercise reach
~20
µmol · g Second, Jeneson and colleagues assumed that
Pi concentration
([Pi]) in mature sheep
rests near the Michaelis-Menten constant (Km) value of
1.25 mM and stress that Pi must
therefore play an additional role in regulation of oxygen consumption.
This assumption was extracted from our own initial paper (6),
in which we used a similar value to approximate
Pi changes in the newborn lamb that occur with increases in oxygen consumption. In a subsequent publication (7), we corrected this assumption and demonstrated that
[Pi] values are
actually twice this
Km value in
mature sheep heart and nearly three times this value in newborns. Given
that we have detected no changes in
[Pi] with increases in
oxygen consumption in mature sheep and that
[Pi] values remain
two- and threefold greater than the
Km in newborn and
mature sheep, respectively, Jeneson's comments regarding
Pi stimulation are unconvincing.
As reiterated in our paper, maturational accumulation of adenine
nucleotide translocator protein parallels the apparent loss of kinetic
respiratory regulation by ADP. Kinetic regulation in the newborn
emulates a Michaelis-Menten pattern (9). Jeneson and colleagues do not
provide a convincing argument for control via cooperative action of ADP
on the adenine nucleotide translocator. Their theory that changes in
respiratory patterns are due to maturational increases in
Vmax in sheep,
generalized changes in mitochondrial density, and/or
Pi stimulation is not supported by
data prevalent in the literature.
REPLY
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Abstract
Letter
References
O2) induced by
phenylephrine infusion in canines. In fact, no statistical change in
ADP occurred despite large increases in oxygen consumption during those
experiments (5). The investigators performing these experiments
attributed the small changes in Pi to the high pressures generated, which potentially resulted in subendocardial ischemia (5).
O2 (6, 8, 9). Nevertheless,
Jeneson and colleagues insist that the data fit the various sigmoid
plots shown in their Fig. 1. They use grouped data from our recent
paper to construct these plots but neglect to provide the standard
error bars or the published results of statistical analyses, which
clearly indicate the lack of significant ADP change in the mature sheep
(their Fig. 1, curves A and
B).
1 · min1.
Jeneson and colleagues would have us believe that
Vmax in newborns is one-third to one-half this rate. Our own published experiments in
anesthetized newborn lambs, in which
MO2 should be well below maximum, have elicited rates greater than the
Vmax postulated by Jeneson (6, 9). Furthermore, detailed studies in isolated sheep
mitochondria indicate that oxidative capacity normalized per tissue
weight does not increase after the late fetal stage (10).
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REFERENCES |
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Top
Abstract
Letter
References
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1.
Brook, W. H.,
S. Connell,
J. Cannata,
J. E. Maloney,
and
A. M. Walker.
Ultrastructure of the myocardium during development from early fetal life to adult life in sheep.
J. Anat.
137:
729-741,
1983.
2.
Gratama, J. W. C.,
J. J. Meuzelaar,
M. Dalinghaus,
J. H. Koers,
A. M. Gerding,
M. T. M. Monchen,
F. C. A. M. te Nijenhuis,
W. G. Zijlstra,
and
J. R. G. Kuipers.
Myocardial blood flow and O2 in lambs with an aortopulmonary shunt during strenuous exercise.
Am J. Physiol.
264 (Heart Circ. Physiol. 33):
H938-H945,
1993
3.
Heineman, F. W.,
and
R. S. Balaban.
Control of mitochondrial respiration in the heart in vivo.
Annu. Rev. Physiol.
52:
523-542,
1990[Medline].
4.
Jeneson, J. A. L.,
R. W. Wiseman,
H. V. Westerhoff,
and
M. J. Kushmerick.
The signal transduction function for oxidative phosphorylation is at least second order in ADP.
J. Biol. Chem.
271:
27995-27998,
1996.
5.
Katz, L. A.,
J. A. Swain,
M. A. Portman,
and
R. S. Balaban.
Relation between phosphate metabolites and oxygen consumption of heart in vivo.
Am. J. Physiol.
256 (Heart Circ. Physiol. 25):
H265-H274,
1989
6.
Portman, M. A.,
F. W. Heineman,
and
R. S. Balaban.
Developmental changes in the relation between phosphate metabolites and oxygen consumption in the sheep heart in vivo.
J. Clin. Invest.
83:
456-464,
1989.
7.
Portman, M. A.,
and
X.-H. Ning.
Developmental adaptations in cytosolic phosphate content and pH regulation in the sheep heart in vivo.
J. Clin. Invest.
86:
1823-1828,
1990.
8.
Portman, M. A.,
and
X.-H. Ning.
Maturational changes in respiratory control through creatine kinase in heart in vivo.
Am. J. Physiol.
263 (Cell Physiol. 32):
C453-C460,
1992
9.
Portman, M. A., Y. Xiao, Y. Song, and X.-H. Ning.
Expression of adenine nucleotide translocator parallels maturation
of respiratory control in heart in vivo. Am.
J. Physiol. (Heart Circ.
Physiol. 42): H1977-H1983, 1997.
10.
Wells, R. J.,
W. F. Friedman,
and
B. E. Sobel.
Increased oxidative metabolism in the fetal and newborn lamb heart in vivo.
Am. J. Physiol
226:
1488-1493,
1972.
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Michael A. Portman Yun Xiao Ying Song Xue-Han Ning Division of Cardiology, Department of Pediatrics University of Washington School of Medicine and Children's Hospital and Regional Medical Center Seattle, WA 98195-6320 |
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)
were normalized as in Ref. 
], Vmax = 1/2(Vmax)mature
(
), or Vmax = 
(Vmax)mature
(
). Curves C,
D, and
E are fitted Hill functions
(C:
nH = 3.1, K0.5 = 0.068 mM;
D:
nH = 2.1, K0.5 = 0.097 mM;
E:
nH = 1.6, K0.5 = 0.196 mM).
v, myocardial oxygen consumption rate.
Nonlinear least-squares curve fitting was performed using Fig.P
software (Elsevier Biosoft, Cambridge, UK).