Am J Physiol Heart Circ Physiol 283: H680–H687, 2002.
First published May 2, 2002; 10.1152/ajpheart.00800.2001.
Mitochondrial creatine kinase is critically necessary
for normal myocardial high-energy phosphate metabolism
MATTHIAS SPINDLER,1 REINHARD NIEBLER,1 HELGA REMKES,1
MICHAEL HORN,1 TITUS LANZ,2 AND STEFAN NEUBAUER1
1
Medizinische Universitätsklinik Würzburg, 97070 Würzburg; and 2Physikalisches
Institut der Universität Würzburg, 97080 Würzburg, Germany
Received 12 September 2001; accepted in final form 19 April 2002
creatine kinase; energy metabolism; nuclear magnetic resonance spectroscopy; transgenic mouse
CREATINE KINASE (CK; EC 2.7.2.2) is a key enzyme involved in energy metabolism in tissues with large fluctuations of energetic demand such as the muscle or
brain. CK catalyses the reversible transfer of a highenergy phosphoryl group between ATP and phosphocreatine (PCr). Four different isoenzymes of CK are
known; three are dimers composed of two subunits
(MM-CK, MB-CK, and BB-CK), whereas sarcomeric
mitochondrial CK (ScCKmit) can form both dimers and
Address for reprint requests and other correspondence: M. Spindler,
Dept. of Medicine, Würzburg Univ.; Josef-Schneider Strasse 2, 97080
Würzburg, Germany (E-mail: [email protected]).
H680
octamers (for a review, see Ref. 28). These isoenzymes
are localized in a compartimentalized fashion in
the cell. MM-CK, the most abundant muscle isoform,
is a structural protein of the myofibrillar M band.
ScCKmit, the second most abundant isoform, is found
on the outer surface of the inner mitochondrial membrane, forming a functional compartment with porin
and adenine nucleotide translocase (29). This characteristic spatial distribution has led to the “CK shuttle”
hypothesis, where PCr serves as an energy transfer
molecule for fast and efficient transport of phosphoryl
moieties from the sites of energy generation (mitochondria) to the sites of energy consumption (myofibrils and
ion pumps) (1). On the other hand, the PCr-CK system
has been generally regarded as a high-energy buffer
system that meets increased energetic requirements
during periods of mismatched energy production and
consumption. The physiological importance of the CK
system in heart muscle is underlined by numerous
reports of alterations in a variety of components of the
PCr-CK system found in various animal models of
heart failure as well as in human heart failure (10,
12–14).
Despite several decades of research, however, the
true nature of the fundamental role of CK, especially in
disease states such as myopathies or heart failure,
remains ill defined. Transgenic animals with null mutations of one or more of the genes of the CK family
may shed new light on the functional significance of the
PCr-CK system. For example, skeletal muscle of mice
lacking the M subunit of CK, referred to here as
M-CK⫺/⫺, demonstrate a transient impairment in contractile function (burst activity) (25). However, concentrations of high-energy phosphate metabolites were
unaltered and PCr was still hydrolyzed and resynthesized during contraction. When ScCKmit is ablated in
addition to M-CK (referred to as M/ScCKmit⫺/⫺), leaving only BB-CK activity, skeletal muscle had a 30%
lower PCr-to-ATP ratio (PCr/ATP) and was unable to
hydrolyze PCr (22).
In cardiac muscle, contractile performance of isolated perfused hearts was unchanged for low-to-modThe costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society
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Spindler, Matthias, Reinhard Niebler, Helga Remkes,
Michael Horn, Titus Lanz, and Stefan Neubauer. Mitochondrial creatine kinase is critically necessary for normal
myocardial high-energy phosphate metabolism. Am J Physiol
Heart Circ Physiol 283: H680–H687, 2002.— First published
May 2, 2002; 10.1152/ajpheart.00800.2001.—The individual
functional significance of the various creatine kinase (CK)
isoenzymes for myocardial energy homeostasis is poorly understood. Whereas transgenic hearts lacking the M subunit
of CK (M-CK) show unaltered cardiac energetics and left
ventricular (LV) performance, deletion of M-CK in combination with loss of sarcomeric mitochondrial CK (ScCKmit)
leads to significant alterations in myocardial high-energy
phosphate metabolites. To address the question as to
whether this alteration is due to a decrease in total CK
activity below a critical threshold or due to the specific loss of
ScCKmit, we studied isolated perfused hearts with selective
loss of ScCKmit (ScCKmit⫺/⫺, remaining total CK activity
⬃70%) using 31P NMR spectroscopy at two different workloads. LV performance in ScCKmit⫺/⫺ hearts (n ⫽ 11) was
similar compared with wild-type hearts (n ⫽ 9). Phosphocreatine/ATP, however, was significantly reduced in ScCKmit⫺/⫺ compared with wild-type hearts (1.02 ⫾ 0.05 vs.
1.54 ⫾ 0.07, P ⬍ 0.05). In parallel, free [ADP] was higher
(144 ⫾ 11 vs. 67 ⫾ 7 ␮M, P ⬍ 0.01) and free energy release for
ATP hydrolysis (⌬GATP) was lower (⫺55.8 ⫾ 0.5 vs. ⫺58.5 ⫾
0.5 kJ/mol, P ⬍ 0.01) in ScCKmit⫺/⫺ compared with wildtype hearts. These results demonstrate that M- and B-CK
containing isoenzymes are unable to fully substitute for the
loss of ScCKmit. We conclude that ScCKmit, in contrast to
M-CK, is critically necessary to maintain normal high-energy
phosphate metabolite levels in the heart.
MITOCHONDRIAL CK IS NECESSARY FOR NORMAL HIGH-ENERGY PHOSPHATES
METHODS
Animals. ScCKmit⫺/⫺ mice were generated in the laboratory of Dr. Bé Wieringa (University of Nijmegen, Nijmegen,
The Netherlands) by gene targeting as previously reported
(24). Male and female mice of 20–30 wk of age were studied.
There was no difference between WT and ScCKmit⫺/⫺ mice
regarding heart weight or heart weight-to-body weight ratios. The genotype of each mouse was confirmed by measuring the isoenzymes of CK present using a Helena Cardio-Rep
CK isoenzyme analyzer (Helena Diagnostika). The experimental protocol for the present study followed the American
Physiological Society guidelines for the care and use of laboratory animals.
Isolated perfused heart preparation. Hearts of WT and
ScCKmit⫺/⫺ mice were isolated and perfused in the Langendorff preparation in a 10-mm NMR tube as previously
described (21). Retrograde perfusion via the aorta was carried out at a constant coronary perfusion pressure of 75
mmHg at 37°C. Coronary flow was measured by collecting
coronary sinus effluent through a suction tube. Phosphatefree Krebs-Henseleit buffer containing (in mM) 118 NaCl, 5.3
KCl, 2.0 CaCl2, 1.2 MgSO4, 0.5 EDTA, 25 NaHCO3, 10
glucose, and 0.5 pyruvate as substrates was prepared at the
time of the experiment and equilibrated with 95% O2-5%
CO2, yielding a pH of 7.4. All hearts were paced at 7 Hz using
monophasic square wave pulses delivered from a Hugo Sachs
Elektronik stimulator (model 201, Hugo Sachs Elektronik;
Hugstetten, Germany) through salt bridge pacing wires
made of polyethylene-160 tubing filled with 4 M KCl in 2%
agarose.
Measurement of isovolumic contractile performance. A water-filled balloon custom made of polyvinyl chloride film was
connected to a pressure transducer (Statham P23 Db, Gould
Instruments; Glen Burnie, MD) for continuous recording of
LV pressure and heart rate. The size of the balloon was
carefully matched to the size of the ventricle. The balloon was
inflated to set LV end-diastolic pressure (EDP) between 6
and 8 mmHg for all hearts, and the balloon volume was then
held constant. Contractile performance data were collected
on-line at a sampling rate of 200 Hz using a commercially
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available data-acquisition system (MacLab ADInstruments;
Milford, MA). LV developed pressure (LVDP; the difference
between end-systolic pressure and EDP), minimum and maximum values within a beat of the first derivative of LV
pressure (⫺dP/dt and ⫹dP/dt, respectively), and rate-pressure product (RPP; product of LVDP and heart rate) were
calculated off-line.
31
P NMR spectroscopy. 31P NMR spectra were obtained at
121.50 MHz using a superwide-bore NMR spectrometer
(Bruker; Rheinstetten, Germany) equipped with an Aspect
3000 computer. Hearts were placed in a 10-mm NMR tube
and inserted into a custom-made 1H/31P double-tuned probe
situated in a 150-mm bore, 7.05-T superconducting magnet.
To improve homogeneity of the NMR-sensitive volume, the
perfusate level was adjusted so that the heart was submerged in buffer. Spectra were collected at a pulse length of
17.2 ␮s, pulse angle of 60°, repetition time of 1.84 s, and
sweep width of 6,000 Hz. Single spectra were collected for
8-min periods and consisted of 256 consecutive free induction
decays.
In the time domain, the amplitudes of the resonances of
ATP, PCr, and Pi, which are proportional to the number of
phosphorus atoms in the respective compound, were determined using the “AMARES”-routine including prior knowledge. Briefly, J-coupling of 16 Hz, same linewidth, and 1:1 or
1:2:1 ratios for the amplitudes within each duplet or triplet of
ATP was used as prior knowledge for the ATP signals. The
calculation of PCr/ATP was based on the fit integrals of PCr
and ␥-ATP. By comparing the peak amplitude of fully relaxed
(recycle time 15 s) and those of partially saturated (recycle
time 1.84 s) spectra, we calculated the correction factors for
saturation for ATP (1.0), PCr (1.3), and Pi (1.05).
To independently confirm that the ATP concentrations
were not different among the two groups, separate wild-type
and ScCKmit⫺/⫺ hearts were analyzed for ATP content via
HPLC as described below (for details, see Biochemical assays). The concentrations of ATP were 10.6 ⫾ 0.4 mM for
wild-type hearts and 9.6 ⫾ 0.4 mM for ScCKmit⫺/⫺ hearts,
respectively (not significantly different). Thus the ATP resonance area in the first spectrum of each heart was set to the
average concentration of ATP measured biochemically for
that group. The ␥-ATP resonance area of each heart was used
as an internal standard to convert resonance areas of PCr
and Pi to their respective concentrations.
Intracellular pH (pHi) was determined by comparing the
chemical shift of the Pi and PCr peaks in each spectrum to
values from a standard curve.
Cytosolic free [ADP] was calculated using the equilibrium
constant of the CK reaction and from values obtained by
NMR spectroscopy and biochemical assays
[ADP] ⫽ ([ATP][free Cr])/([PCr][H ⫹]Keq)
where the equilibrium constant (Keq) is 1.66 ⫻ 109 M⫺1 for a
[Mg2⫹] of 1.0 mM (9).
The free energy stored in the high-energy phosphate bonds
of ATP and released by ⌬GATP is a negative number. For
purposes of clarity, all values of ⌬GATP are expressed as their
absolute values ( 兩⌬GATP兩 ). Thus increases in 兩⌬GATP兩 (in
kJ/mol) indicate an increase in free energy release
兩⌬GATP兩 ⫽ 兩⌬G° ⫹ RT ln ([ADP][Pi]/[ATP])兩
where ⌬G° (⫺30.5 kJ/mol) is the value of ⌬GATP under
standard conditions of molarity, temperature, pH, and
[Mg2⫹]; R is the gas constant (8.3 J/mol 䡠 K), and T is the
temperature (in K) (4).
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erate workloads in M-CK⫺/⫺ and M/ScCKmit⫺/⫺ mice
(20, 26). Whereas hearts of M-CK⫺/⫺ mice (30% remaining CK activity, consisting of ScCKmit and BBCK) showed no difference in PCr/ATP compared with
wild-type hearts (26), M/ScCKmit⫺/⫺ hearts (with only
3% remaining CK activity) had a 25% lower PCr/ATP.
Thus impaired myocardial energetics have been demonstrated for hearts of M/ScCKmit⫺/⫺ mice only (19,
20). At present, it is unclear whether these reduced
PCr/ATP are predominantly caused by the specific loss
of ScCKmit or by the overall decrease of total CK
activity below a certain threshold necessary to maintain “normal” energetics. This question can only be
directly addressed by studying hearts with an isolated
ablation of ScCKmit (referred to as ScCKmit⫺/⫺).
The purpose of the present work was, therefore, to
define left ventricular (LV) performance, CK activity,
isoenzyme distribution, and high-energy phosphate
metabolite concentrations under two different workloads in ScCKmit⫺/⫺ hearts. 31P NMR spectroscopy
was used to measure [ATP], [PCr], [Pi], [ADP], free
energy for ATP hydrolysis (⌬GATP) , and pH in isolated
beating hearts of mutant and wild-type mice.
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H682
MITOCHONDRIAL CK IS NECESSARY FOR NORMAL HIGH-ENERGY PHOSPHATES
Table 1. Heart weight, body weight, and biochemical data of wild-type and ScCKmit⫺/⫺ mice
Wild type
ScCKmit⫺/⫺
Body Weight,
g
LV Weight,
mg
Heart Weight/
Body
Weight, mg/g
Protein, mg
protein/
mg wet wt
Creatine,
mM
Citrate Synthase,
IU/mg protein
LDH,
IU/mg protein
Na⫹-Creatine
Cotransporter,
AU of OD
29.2 ⫾ 0.8
28.4 ⫾ 1.1
111 ⫾ 3
101 ⫾ 6
4.9 ⫾ 0.1
5.0 ⫾ 0.2
0.147 ⫾ 0.005
0.141 ⫾ 0.006
18.3 ⫾ 1.2
18.0 ⫾ 1.2
1.13 ⫾ 0.11
1.16 ⫾ 0.06
0.48 ⫾ 0.01
0.51 ⫾ 0.02
27.4 ⫾ 3.3
24.3 ⫾ 3.9
Value are means ⫾ SE; n ⫽ 9 wild-type and 11 sarcometric mitochondrial creatine kinase (CK)-deficient (ScCKmit⫺/⫺) mice. LV, left
ventricular; LDH, lactate dehydrogenase; AU, arbitrary units; OD, optical density.
for ScCKmit⫺/⫺ vs. 58.2 ⫾ 1.4 mM/s for wild type).
Maximal activities of CK isoenzymes and isoenzyme
distribution relative to total CK activity are summarized in Table 2. In contrast to what has been described
for skeletal muscle of ScCKmit⫺/⫺ mice, there was no
compensatory increase in absolute MM-CK activity in
heart tissue.
Furthermore, there was no difference in citrate synthase, a marker of mitochondrial density, and in total
LDH activities (see Table 1) or LDH isoenzyme distribution, an index of the ratio of oxidative to glycolytic
capacity (data not shown).
Intracellular noncollagen protein concentration,
measured as Lowry protein, was 0.147 ⫾ 0.005 and
0.141 ⫾ 0.006 mg protein/mg wet wt for wild-type and
ScCKmit⫺/⫺ hearts, respectively.
Cardiac function and high-energy phosphate metabolism under baseline conditions. When EDP was set to
⬃8 mmHg and isolated hearts were paced at 420 beats/
min, LVDP was not different between wild-type and
ScCKmit⫺/⫺ hearts (see Table 3). Likewise, no difference was observable with regard to RPP, ⫹dP/dt, ⫺dP/
dt, and coronary flow/heart weight. As described previously for isolated hearts with isolated knockout of
M-CK or combined knockout of M-CK and ScCKmit
(20), contractile performance was stable with variations, i.e., in RPP of ⬍5% during a 30-min baseline
perfusion period.
Representative 31P NMR spectra from a wild-type
and a ScCKmit⫺/⫺ heart during baseline perfusion are
shown in Fig. 1. As visible in these spectra, [PCr]
was ⬃35% lower in the ScCKmit⫺/⫺ heart (10.2 ⫾ 0.5
mM) compared with the wild-type heart (15.5 ⫾ 0.7
mM). [ATP], [Pi], and pHi were not different between
both groups. Accordingly, PCr/ATP were lower in
RESULTS
Total CK, mM/s
MM-CK, mM/s
MB-CK, mM/s
BB-CK, mM/s
ScCKmit, mM/s
MM-CK, %total
MB-CK, %total
BB-CK, %total
ScCKmit, %total
General characteristics and CK system of wild-type
and ScCKmit⫺/⫺ mice. Body weight, LV weight, and
LV weight-to-body weight ratios were similar in both
groups, ruling out gross cardiac hypertrophy (Table 1).
Coronary flow was also unchanged in ScCKmit⫺/⫺ compared with wild-type hearts (data not shown).
Total CK activity in hearts of ScCKmit⫺/⫺ mice was
30% less than that of wild-type tissue (39.8 ⫾ 3.1 mM/s
AJP-Heart Circ Physiol • VOL
Table 2. CK isoenzyme distribution and isoenzyme
activities in hearts of wild-type and ScCKmit⫺/⫺ mice
Wild Type (n ⫽ 10)
ScCKmit⫺/⫺ (n ⫽ 10)
58.2 ⫾ 1.4
33.6 ⫾ 0.8
1.6 ⫾ 0.1
0.5 ⫾ 0.1
22.6 ⫾ 1.2
57.8 ⫾ 1.2
2.8 ⫾ 0.2
0.8 ⫾ 0.2
38.6 ⫾ 1.4
39.8 ⫾ 3.1*
37.1 ⫾ 2.9
2.1 ⫾ 0.3
0.6 ⫾ 0.1
ND
93.2 ⫾ 0.4*
5.3 ⫾ 0.3*
1.5 ⫾ 0.2*
ND
Values are means ⫾ SE; n ⫽ no. of hearts. ND, not detectable.
* P ⬍ 0.05, ScCKmit⫺/⫺ vs. wild-type hearts.
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Biochemical assays. In one group of hearts (6 wild type and
7 ScCKmit⫺/⫺), concentrations of the primary nucleotides
and nucleosides were measured using HPLC (Pharmacia).
After 16 min of baseline perfusion, these hearts were freeze
clamped, the tissue was homogenized in 0.4 N perchloric acid
at 0°C, and aliquots of the homogenate were removed for
protein determination. The homogenates were neutralized
with saturated KOH and centrifugated for 5 min. Aliquots of
the supernatant were applied to a HPLC column (Supelcosil
LC-18, 4.6 ⫻ 250 mm, Supelco). Nucleosides and nucleotides
were eluted at 30°C isocratically using 0.2 M phosphate
buffer (pH 6.0) at a flow rate of 0.8 ml/min. The column
effluent was analyzed at 205 nm for ATP, and amounts were
calculated using external standards. In these hearts, the
measured [ATP] were 22.4 ⫾ 1.7 and 24.3 ⫾ 1.1 nmol
ATP/mg protein in wild-type and ScCKmit⫺/⫺ hearts, respectively. With the use of measured values for protein concentration and the literature value for the ratio of intracellular
volume to total cell volume of 0.48 (16), these values were
converted to [ATP] of 10.6 ⫾ 0.4 and 9.6 ⫾ 0.4 mM for
wild-type and ScCKmit⫺/⫺ hearts, respectively. With the use
of other aliquots, total creatine content was measured using
the method of Kammermeier (8). Noncollagen protein was
measured by the method of Lowry with bovine serum albumin as the standard (11). Total lactate dehydrogenase (LDH)
activity, LDH isoforms, and citrate synthase activity were
measured as previously described (14). Creatine transporter
protein was quantified by Western blot as previously published by our group (15).
Total CK activity and the proportion of this activity attributable to each isoenzyme of CK was measured using methods
previously described. CK activities were measured in international units per milligram of protein and converted to
micromolars per second using the measured concentrations
of cardiac protein. All values are expressed as micromolars
per second at 37°C. The percentage of total CK activity
attributable to each isoenzyme was measured using a Helena
Cardio-Rep CK isoenzyme analyzer (17).
Statistical analysis. All data are expressed as means ⫾ SE.
Paired and unpaired Student’s t-tests as appropriate were
used to compare ScCKmit⫺/⫺ and wild-type hearts at baseline and high workload. Statistical analyses were performed
with the use of Statview (Brainpower; Calabasas, CA), and
values of P ⬍ 0.05 were considered statistically significant.
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MITOCHONDRIAL CK IS NECESSARY FOR NORMAL HIGH-ENERGY PHOSPHATES
Table 3. Isovolumic LV contractile performance at baseline, during increased work, and during recovery of
wild-type and ScCKmit⫺/⫺ hearts
Baseline
Recovery
7⫾1
8⫾1
12 ⫾ 2
11 ⫾ 1
13 ⫾ 3
12 ⫾ 4
81 ⫾ 4
80 ⫾ 2
78 ⫾ 5
89 ⫾ 4
62 ⫾ 3
68 ⫾ 3
34,800 ⫾ 1,900
34,400 ⫾ 1,200
47,000 ⫾ 3,000
53,200 ⫾ 2,300
25,900 ⫾ 1,400
28,100 ⫾ 1,300
3,100 ⫾ 100
3,100 ⫾ 100
3,400 ⫾ 200
3,600 ⫾ 200
2,300 ⫾ 100
2,400 ⫾ 200
2,400 ⫾ 200
2,500 ⫾ 100
3,200 ⫾ 300
3,400 ⫾ 300
1,600 ⫾ 100
1,700 ⫾ 200
Values are means ⫾ SE. EDP, end-diastolic pressure; LVDP, LV developed pressure; RPP, rate-pressure product; ⫹dP/dt and ⫺dP/dt,
maximum and minimum values within a beat of the first derivative of LV pressure. There were no significant differences between the two
groups of hearts at baseline (heart rate ⫽ 420 beats/min, [Ca2⫹] ⫽ 2.0 mM), during increased work (heart rate ⫽ 600 beats/min, [Ca2⫹] ⫽
4.0 mM), or during recovery (heart rate ⫽ 420 beats/min, [Ca2⫹] ⫽ 2.0 mM) for any measure of cardiac function.
ScCKmit⫺/⫺ (1.02 ⫾ 0.05) versus wild-type hearts
(1.54 ⫾ 0.07) (Fig. 2). Total creatine concentration,
measured biochemically with HPLC in the same
hearts, revealed similar values for ScCKmit⫺/⫺ and
wild-type hearts (Table 1). Furthermore, myocardial
Na⫹-creatine cotransporter, the protein responsible for
regulating the myocardial creatine concentration, was
also similar in both groups.
When intracellular free [ADP] was calculated from
the equilibrium equation for the CK reaction, significantly higher [ADP] were found for ScCKmit⫺/⫺ (144 ⫾
11 ␮M) compared with wild-type hearts (67 ⫾ 7 ␮M)
(Fig. 2). Because of this doubling of free [ADP], calculated ⌬GATP was significantly lower in ScCKmit⫺/⫺
compared with wild-type hearts (⫺55.8 ⫾ 0.5 vs.
⫺58.5 ⫾ 0.5 kJ/mol, respectively) (Fig. 2).
Cardiac high-energy phosphate metabolism and cardiac function during increased work and during recovery. When workload was increased by doubling of the
extracellular [Ca2⫹] and increasing pacing frequency
from 420 to 600 beats/min, EDP increased slightly and
LVDP essentially remained constant, leading to an
increase in RPP on average of 40–55% (see Table 3).
There were no differences in relative and absolute
increases in RPP between both groups during increased work.
When perfusate [Ca2⫹] and pacing frequency was
returned to baseline levels, LVDP and RPP fell below
values observed during baseline perfusion. On average, RPP returned to 75 ⫾ 4% of baseline for wildtype hearts and 79 ⫾ 3% for ScCKmit⫺/⫺hearts after
16 min of recovery (see Table 3). There were no
significant differences between isolated wild-type and
ScCKmit⫺/⫺ hearts with regard to EDP, LVDP, ⫹dP/
dt, and ⫺dP/dt during high workload and during recovery from increased cardiac work.
Increasing workload by 40–55% lead to the expected
changes of high- and low-energy phosphate concentrations. [Pi] more than doubled, whereas [PCr] decreased
5.7 mM (by 37%) in wild-type hearts and 2.4 mM (by
24%) in ScCKmit⫺/⫺ hearts. During recovery, a similar
amount of PCr was resynthetized in both groups of
hearts, and [PCr] after 10 min of recovery reached
almost the baseline level. In parallel to this resynthesis
of PCr, [Pi] decreased in both groups to baseline levels.
Increasing RPP caused [ATP] to decrease similarly
in both groups, by 1.5 mM in wild-type hearts and by
0.9 mM in ScCKmit⫺/⫺ hearts. The total cardiac phos-
Fig. 1. Representative 31P NMR spectra from a wild-type (left) and a sarcomeric mitochondrial creatine kinase
(CK)-deficient (ScCKmit⫺/⫺; right)
mouse heart. Each spectrum is the average of 256 consecutive scans collected over a 8-min period. The major
resonances are assigned (from left to
right) as Pi, phosphocreatine (PCr),
and ␥-, ␣-, and ␤-phosphates of ATP.
Note that ScCKmit⫺/⫺ hearts have
similar ATP areas as wild-type hearts
but smaller PCr areas. PPM, parts per
million.
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EDP, mmHg
Wild type
ScCKmit⫺/⫺
LVDP, mmHg
Wild type
ScCKmit⫺/⫺
RPP, mmHg 䡠 beats 䡠 min⫺1
Wild type
ScCKmit⫺/⫺
⫹dP/dt, mmHg/s
Wild type
ScCKmit⫺/⫺
⫺dP/dt, mmHg/s
Wild type
ScCKmit⫺/⫺
Increased Work
H684
MITOCHONDRIAL CK IS NECESSARY FOR NORMAL HIGH-ENERGY PHOSPHATES
phate pool ([Pi] ⫹ [PCr] ⫹ 3 ⫻ [ATP]) decreased by
⬃15% from baseline to recovery in the two groups of
hearts (data not shown).
Free [ADP] increased by 82 ␮M in wild-type hearts
and by 42 ␮M in ScCKmit⫺/⫺ hearts in response to
increased workload (P ⬍ 0.05). In absolute terms, free
[ADP] at high workload showed a trend for higher
concentrations in ScCKmit⫺/⫺ compared with wildtype hearts. The ⌬GATP decreased significantly as
workload was increased in both groups, 4.9 kJ/mol for
wild-type hearts and 2.9 kJ/mol for ScCKmit⫺/⫺ hearts
(Fig. 2). However, in contrast to baseline conditions, no
differences were detectable in ⌬GATP during increased
workload and after recovery between wild-type and
ScCKmit⫺/⫺ hearts.
DISCUSSION
The CK system constitutes a highly organized isoenzyme system of central importance for energy maintenance, transfer, and buffering in excitable tissue, evidenced by a large body of work examining the kinetic,
thermodynamic, and functional properties of this enzyme system under various conditions and disease
states (6, 18, 28). However, the functional coupling and
complex interactions of the CK system with other components of the energy generation, transport, and utiliAJP-Heart Circ Physiol • VOL
zation machinery of the cell have challenged the classical view of an enzyme system only necessary to
catalyze the reversible transfer of a phosphoryl group
between PCr and ATP via the following reaction:
PCr ⫹ ADP ⫹ H⫹ 7 ATP ⫹ creatine. Because specific
and selective inhibitors of CK are not available, gene
knockout technology offers a unique opportunity to
dissect the functional significance of the different CK
isoenzymes in muscle.
The present study thus sought to determine LV
function, high-energy phosphate metabolism, and
isoenzyme distribution in hearts exclusively lacking
the ScCKmit isoenzyme (30% decrease in total CK
activity). Specifically, we asked whether lowering CK
activity below a certain critical threshold was responsible for the inability to maintain normal energetics or,
alternatively, whether the specific loss of ScCKmit lead
to the “energetic phenotype” of M/ScCKmit⫺/⫺ hearts.
Our results demonstrate that, first, no compensatory
increase in other CK isoforms or in a variety of other
enzymes involved in cellular energy homeostasis was
observed in ScCKmit⫺/⫺ hearts. Second, deletion of
ScCKmit is not associated with any functional alteration in isolated perfused hearts during different
workloads. Third, and most importantly, hearts from
ScCKmit⫺/⫺ mice demonstrate significant changes in
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Fig. 2. Myocardial energetics measured by 31P NMR spectroscopy from
wild-type and ScCKmit⫺/⫺ hearts during baseline, increased work, and recovery. Values are means ⫾ SE. * P ⬍
0.05, ScCKmit⫺/⫺ vs. wild-type hearts.
⌬GATP, free energy release for ATP hydrolysis (for details, see METHODS).
MITOCHONDRIAL CK IS NECESSARY FOR NORMAL HIGH-ENERGY PHOSPHATES
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“normal” energetics has led to the prediction that
ScCKmit⫺/⫺ hearts, with a remaining total CK activity
of 70%, will have unchanged high-energy phosphates.
The view was supported by the results of skeletal
muscle of ScCKmit⫺/⫺ mice, showing no impairment in
baseline energetics and a normal capacity to hydrolyze
PCr during ischemia (5). For the other CK knockout
strains such as M-CK⫺/⫺ and M/ScCKmit⫺/⫺ mice, the
changes in high-energy phosphate profile in the heart
were correctly “predicted” by the results from skeletal
muscle. This, however, is not the case for ScCKmit⫺/⫺
hearts. This report is therefore the first to describe
significant discrepancies in steady-state high-energy
phosphate concentrations between the heart and skeletal muscle in CK knockout mice. These differences
highlight the unique role of ScCKmit in cardiac compared with skeletal muscle. This may be related to the
fact that cardiac muscle with its higher mitochondrial
density and larger proportion of ScCKmit but lower
total CK activity relies predominantly on aerobic ATP
generation (2, 27).
In agreement with the results from skeletal muscle
showing preserved ability to hydrolyze PCr during
ischemia (23), ScCKmit⫺/⫺ hearts were able to hydrolyze PCr during increased work and, more importantly,
resynthesize PCr during recovery. Because changes in
PCr content reflect the balance between ATP synthesis
and hydrolysis, this result demonstrates that during
recovery the rate of ATP synthesis and therefore PCr
generation exceeds the rate of ATP hydrolysis. Thus we
conclude that not only is oxidative energy production
still workload dependent in ScCKmit⫺/⫺ hearts, but
also that effective high-energy phosphate transport
across the mitochondrial membrane is maintained
without ScCKmit. Whether the remaining CK isoenzymes directly substitute for ScCKmit, relocating into
intermembrane space, or whether other enzyme systems are upregulated cannot directly be answered from
our data.
It is, however, important to point out the possibity
that complex adaptational processes and subcellular
rearrangements already during fetal development
might have taken place in the CK-deficient hearts,
ensuring an alternative energy transfer and signal
transduction system. This hypothesis has been recently confirmed in M/ScCKmit⫺/⫺ hearts showing significant cytoarchitectural rearrangements and biochemical adaptations (7). It is concluded that the
genetically induced loss of an important component of
the CK system is obviously partially compensated by
an effective rearrangement of subcellular organelles,
explaining the preserved cardiac function of CK-deficient mice under moderate workload. Whether these
compensatory changes and mechanisms enable the
ScCKmit⫺/⫺ hearts to withstand acute or chronic
stress conditions is completely unknown at present and
warrant further examinations.
When cardiac energetics of ScCKmit⫺/⫺ hearts from
this study are compared with M/ScCKmit⫺/⫺ hearts
from previous studies, two differences are apparent:
First, whereas we found ⌬GATP to be significantly re-
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high-energy phosphate metabolism similar to the
changes in M/ScCKmit⫺/⫺ hearts, such as an increase
in free [ADP] and a decrease in ⌬GATP.
Biochemical effects of deletion of ScCKmit. Deletion
of ScCKmit produced the expected 30% decreased in
total CK activity. We did not observe any compensatory increase in the specific activity of the remaining
CK isoenzymes. This is in line with the results of
Saupe et al. (19) demonstrating complete independence of CK isoenzyme regulation in developing and
mature hearts from M-CK⫺/⫺ and M/ScCKmit⫺/⫺
compared with wild-type mice. In contrast, Boehm et
al. (3) found an ⬃30% decrease in MM-CK activity in
ScCKmit⫺/⫺ hearts, leading to an overall 47% decrease
in total CK activity in these hearts. This decrease in
MM-CK activity was only observed in ventricular muscle; in skeletal muscle, a significant increase in
MM-CK activity of ScCKmit⫺/⫺ mice was reported. The
reasons for the discrepancy between these two investigations cannot be elucidated from the currently available data and warrant further study.
In agreement with others (23, 24), we did not detect
changes in total LDH activity and LDH isoenzyme
distribution in hearts from ScCKmit⫺/⫺. Boehm et al.
(3) showed that this distribution, reflecting the oxidative or glycolytic preference of carbohydrate metabolism, was altered in slow twitch skeletal muscle of
ScCKmit⫺/⫺ mice toward a more glycolytic pattern.
This shift towards a more glycolytic energy production
in skeletal muscle was not observed in heart muscle.
Similarly, Steeghs et al. (23) did not find a difference in
the ability of mitochondria of ScCKmit⫺/⫺ hearts to
oxidate pyruvate. Although changes in substrate utilization cannot completely be ruled out by these measurements, it is apparent that more adaptational
changes take place in skeletal muscle than in heart
muscle from ScCKmit⫺/⫺ mice. This is further confirmed by our finding that citrate synthease activity,
a marker of mitochondrial mass, is unaffected in
ScCKmit⫺/⫺ hearts.
Myocardial function and energy metabolism with
varying workload demand. During baseline perfusion
conditions, producing a moderate degree of workload in
wild-type mice, contractile performance was similar in
ScCKmit⫺/⫺ and wild-type hearts. Despite comparable
isovolumic contractile work, hearts from ScCKmit⫺/⫺
mice had a significantly lower (⫺34%) concentration of
PCr. In parallel, Pi showed a trend for higher values in
ScCKmit⫺/⫺ mice; statistical significance, however,
was not reached. When the free cytosolic [ADP] was
calculated, a significant increase was observed in
ScCKmit⫺/⫺ hearts. These results are remarkable for
several reasons: It has previously been shown that
hearts with deletion of M-CK⫺/⫺ (remaining CK activity 28%) were able to maintain a completely normal
high-energy phosphate profile during baseline and increased workload, whereas a combined loss of M-CK
and ScCKmit (leaving only 3% CK activity) led to
reduced PCr, increased ADP, and decreased ⌬GATP
values (20). The assumption that a total CK activity
below a critical threshold is necessary to maintain
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MITOCHONDRIAL CK IS NECESSARY FOR NORMAL HIGH-ENERGY PHOSPHATES
The authors thank Dr. Bé Wieringa and Dr. Klaas Nicolay for
generously providing the founder mice used in this study and Dr.
Ernest Boehm for thoughtful contributions to this manuscript.
This work was supported by Deutsche Forschungsgemeinschaft
Grant Sonder Forschungsbereich 355 “Pathophysiologie der Herzinsuffizienz,” TPA3, and the British Heart Foundation.
Present address of M. Horn: Wallenberg Laboratory, Sahlgrenska
Hospital, Gothenburg University, 41345 Gothenburg, Sweden.
Present address of S. Neubauer: Department of Cardiovascular
Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK
OX3 9DU.
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