Creatine and Creatinine Metabolism - Physiological Reviews

1150 MARKUS WYSS AND RIMA KADDURAH-DAOUK Volume 80
31 P-NMR saturation transfer, MM- and Mi-CK activity, mitochondrial
adenylate kinase activity, total Cr content, as
well as in Cr- and AMP-stimulated mitochondrial respiration
(48, 69, 420, 461, 462, 509, 622, 699, 771, 835, 879,
1010). In reperfused isolated rat hearts following varying
periods of ischemia, the decrease in Cr-stimulated mitochondrial
respiration was even identified as the first detectable
functional deterioration (822). The functional capacity
of Mi-CK may be reduced in two different ways in
reperfused myocardium. First, Mi-CK is inactivated rather
selectively, as evidenced by no or only minor decreases in
mitochondrial malate dehydrogenase or cytochrome oxidase
activities (69). Second, increased concentrations of
inorganic phosphate like those prevailing under ischemic
conditions are known to favor Mi-CK release from the
mitochondrial inner membrane and may thus disrupt the
functional coupling between Mi-CK and adenine nucleotide
translocase which is suggested to be important for
metabolic channeling of high-energy phosphates out of
the mitochondria (see Refs. 325, 675, 1059, 1124). Remarkably,
the loss of Mi-CK activity and of total CK flux was
found to correlate almost perfectly with the decrease in
LV developed pressure (r � 0.97) (69, 420) and in the
rate-pressure product (r � 0.99) (699), respectively.
Although in two studies, myofibril-bound MM-CK was
found to be preserved despite a loss in total CK activity
(509, 1062), its activity was reported to be depressed by
others (304). Upon prolonged ischemia, redistribution of
M-CK was observed in immunoelectron micrographs of
the dog heart, with a progressive loss of M-CK from the
myofibrillar A band (740). In addition to these findings, a
transient decrease in M-CK mRNA level was observed in
ischemic dog and rabbit hearts 0.3–6 h after ligation of the
left anterior descending (LAD) coronary artery (630), and
MB-CK activity was found to increase in both ischemic
and surrounding nonischemic portions of the dog heart
upon LAD occlusion (879).
Several lines of evidence suggest that CK inhibition
during reperfusion is brought about by reactive oxygen
species: 1) bovine, rabbit, and rat heart MM-CK as well as
rat heart Mi-CK were inactivated by incubation with either
(hypo)xanthine plus xanthine oxidase or H 2O 2, with a
concomitant loss of free sulfhydryl groups (48, 205, 351,
455, 622, 962, 997, 1154). Superoxide dismutase, catalase,
desferrioxamine, reduced glutathione, dithiothreitol, and
cysteine protected against inactivation. Rabbit MM-CK
seems to be inactivated mainly by H 2O 2, whereas both the
superoxide and the hydroxyl radical have been implicated
in the inactivation of bovine MM-CK. The xanthine oxidase
activity required for half-maximal inactivation of
bovine MM-CK was �30-fold lower than that found in rat
myocardium. Rabbit MM-CK, when incubated for 15 min
at 37°C, displayed half-maximal inactivation with �25 �M
H 2O 2. 2) Postischemic reperfusion in isolated rat hearts
causes a decrease in total CK activity, an effect that can
be prevented by addition of superoxide dismutase to the
perfusion medium (622). 3) In permeabilized muscle fibers
of the rat heart, myofibrillar MM-CK was identified as
the primary target of both xanthine oxidase/xanthine and
H 2O 2 (632). Inactivation of CK was prevented by catalase
or dithiothreitol. Under the same conditions, myosin ATPase
activity was not affected, and there was also no
indication for modification of myofibrillar regulatory proteins.
4) Myristic acid treatment increases catalase activity
in rat hearts. In myristic acid-treated rats, recovery of
heart function after ischemia and reperfusion was significantly
improved, and the decrease in CK activity was
either less pronounced than in controls or even absent
(461, 462). 5) Neither CK inactivation nor production of
reactive oxygen species is observed during ischemia, but
during subsequent reperfusion (18, 48, 461, 462).
Inactivation of CK in the reperfused myocardium
may be mediated in part by iron. Oxidative stress can
induce the release of iron from storage proteins, making it
thereby available for catalysis of free radical reactions. In
fact, ferrous iron enhanced the inactivation of rabbit MM-
CK by H 2O 2 or xanthine oxidase/hypoxanthine (504, 997).
Micromolar concentrations of iron and iron chelates that
were reduced and recycled by superoxide or doxorubicin
radicals were effective catalysts of CK inactivation (see
also Ref. 653). Korge and Campbell (503, 504) also obtained
evidence that iron may directly inhibit CK activity
and Ca 2� uptake into sarcoplasmic reticulum vesicles of
the rabbit heart. Inactivation depended on the redox state
and on modification of the reactive sulfhydryl group of CK
and was prevented by dithiothreitol, desferrioxamine, and
EDTA.
Protein S-thiolation, i.e., the formation of mixed disulfides
between protein sulfhydryl groups and thiols
such as glutathione, may also be a mechanism for regulation
of metabolism during oxidative stress. In cultured
cardiac cells exposed to diamide-induced oxidative
stress, a significant proportion of CK became S-thiolated
and was thereby (reversibly) inactivated (133). Whether
and to which extent reactive oxygen species like the
superoxide anion or H 2O 2 are implicated in the S-thiolation
reaction (750), and whether S-thiolation of CK plays
a protective rather than deleterious role (351), remains to
be further clarified.
Recently, NO and peroxynitrite were found to inactivate
CK reversibly and irreversibly, respectively, most
probably by binding to the reactive sulfhydryl group of the
enzyme (29, 315, 444, 497, 935, 1115). NO also inhibits
CK-mediated Ca 2� uptake into SR vesicles and decreases
the sensitivity of mitochondrial respiration to stimulation
by ADP. Whereas NO has been implicated to be a reversible
regulator of mitochondrial function, muscular oxygen
consumption and energy metabolism, its reaction product
with the superoxide radical, peroxynitrite, may display
inhibitory effects that are not readily reversible (679,