Inhibition Of Creatine Kinase Research Articles

Acrylamide does not cause apparent changes in genetic expression of creatine kinase in rat cerebellum.

Acrylamide inhibits creatine kinase (CK) activities in the brain of rats or mice. However, its effects on genetic expression of CK have not yet been studied. CK mRNA and protein level were examined by RT-PCR and Western blotting, respectively. Neither cytosolic CK (B subunit) and mitochondrial CK (ubiquitous form) mRNA nor B subunit protein was clearly changed in the cerebellum from rats intoxicated with acrylamide (50 mg/kg/day i.p. for 8 days) and showing clinical neurotoxic signs.

D-2-hydroxyglutaric acid inhibits creatine kinase activity from cardiac and skeletal muscle of young rats.

Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) is the biochemical hallmark of the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (DHGA). Patients affected by this disease usually present hypotonia, muscular weakness, hypertrophy and cardiomyopathy, besides severe neurological findings. However, the underlying mechanisms of muscle injury in this disorder are virtually unknown. In the present study we have evaluated the in vitro role of DGA, at concentrations ranging from 0.25 to 5.0 mM, on total, cytosolic and mitochondrial creatine kinase activities from skeletal and cardiac muscle of 30-day-old Wistar rats. We also tested the effects of various antioxidants on the effects elicited by DGA. We first verified that total creatine kinase (CK) activity from homogenates was significantly inhibited by DGA (22-24% inhibition) in skeletal and cardiac muscle, and that this activity was approximately threefold higher in skeletal muscle than in cardiac muscle. We also observed that CK activities from mitochondrial (Mi-CK) and cytosolic (Cy-CK) preparations from skeletal muscle and cardiac muscle were also inhibited (12-35% inhibition) by DGA at concentrations as low as 0.25 mm, with the effect being more pronounced in cardiac muscle preparations. Finally, we verified that the DGA-inhibitory effect was fully prevented by preincubation of the homogenates with reduced glutathione and cysteine, suggesting that this effect is possibly mediated by modification of essential thiol groups of the enzyme. Furthermore, alpha-tocopherol, melatonin and the inhibitor of nitric oxide synthase L-NAME were unable to prevent this effect, indicating that the most common reactive oxygen and nitrogen species were not involved in the inhibition of CK provoked by DGA. Considering the importance of creatine kinase activity for cellular energy homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA might be an important mechanism involved in the myopathy and cardiomyopathy of patients affected by DHGA.

Kinetic studies on the inhibition of creatine kinase activity by branched-chain α-amino acids in the brain cortex of rats

Maple syrup urine disease (MSUD) is a metabolic disorder biochemically characterized by the accumulation of branched-chain α-amino acids (BCAA) and their branched-chain α-keto acids (BCKA) in blood and tissues. Neurological dysfunction is usually present in the patients, but the mechanisms of brain damage in this disease are far from be understood. The main objective of this study was to investigate the mechanisms by which BCAA inhibit creatine kinase activity, a key enzyme of energy homeostasis, in the brain cortex of 21-day-old Wistar rats. For the kinetic studies, Lineweaver–Burk and a modification of the Chevillard et al. plots were used to characterize the mechanisms of enzyme inhibition. The results indicated that BCAA inhibit creatine kinase by competition with the substrates phosphocreatine and ADP at the active site. Considering the crucial role creatine kinase plays in energy homeostasis in brain, if these effects also occur in the brain of MSUD patients, it is possible that inhibition of this enzyme activity may contribute to the brain damage found in this disease. In this case, it is possible that creatine supplementation to the diet might benefit MSUD patients.

Hyperphenylalaninemia reduces creatine kinase activity in the cerebral cortex of rats

Phenylketonuria (PKU) is a metabolic disorder accumulating phenylalanine (Phe) and its metabolites in plasma and tissues of the patients. Considering that phenylalanine is the main neurotoxic metabolite, and brain energy homeostasis seems to be affected in phenylketonuria, our main objective was to investigate the effect of acute and chronic hyperphenylalaninemia (HPA) on creatine kinase (CK) activity in brain cortex of Wistar rats. Hyperphenylalaninemia was induced by subcutaneous administration of 5.2μmol phenylalanine + 2.4μmol α-methylphenylalanine (phenylalanine hydroxylase (PAH) inhibitor)/g of body weight. We also investigated the in vitro effect of phenylalanine and/or α-methylphenylalanine on creatine kinase activity in the brain cortex of non-treated rats. The results showed that phenylalanine significantly inhibited creatine kinase activity in vitro and reduced the enzyme activity in vivo. Considering the importance of creatine kinase for the maintenance of energy homeostasis in brain, if this enzyme inhibition also occurs in phenylketonuric patients, it is possible that creatine kinase inhibition may be one of the mechanisms by which phenylalanine is neurotoxic in phenylketonuria.

Phosphocreatine content of freeze-clamped muscle: influence of creatine kinase inhibition.

The study of cellular energetics is critically dependent on accurate measurement of high-energy phosphates. Muscle values of phosphocreatine (PCr) vary greatly between in vivo measurements (i.e., by nuclear magnetic resonance) and chemical measurements determined from muscles isolated and quick-frozen. The source of this difference has not been experimentally identified. A likely cause is activation of ATPases and phosphotransfer from PCr to ADP. Therefore, rat hindlimb skeletal muscle was perfused either with or without 2 mM iodoacetamide, a creatine kinase inhibitor, and muscle was freeze-clamped either at rest or after contraction. Creatine kinase inhibition resulted in approximately 6 micromol/g higher PCr and lower creatine in the freeze-clamped soleus, red gastrocnemius, and white gastrocnemius. This PCr content difference was reduced when the initial PCr content was decreased with prior contractions. Therefore, the amount of PCr artifact appears to scale with initial PCr content within a fiber-type section. This artifact directly affects the measurement and, thus, the calculations of muscle energetic parameters from studies using isolated and frozen muscle.

Essential role of ATP synthesized by creatine kinase in contraction of alpha-toxin permeabilized preparations of tonic type smooth muscle.

The role of ATP newly synthesized from ADP and phosphocreatine (PC) by creatine kinase (CK) in the contraction of tonic type smooth muscle, rat femoral artery was studied, since its necessity for phasic type smooth muscle was previously shown. In alpha-toxin-permeabilized preparations obtained from rat femoral artery, Ca(2+) induced a tonic type contraction in the presence of ATP and PC. Omission of PC inhibited significantly the contraction. Treatment of the preparations with 2,4-dinitrofluorobenzene, an inhibitor of CK, also inhibited the contraction. In the presence of ADP and PC, Ca(2+) also induced the contraction to a level comparable to that in the presence of ATP and PC. The extent of phosphorylated myosin light chain was fairly consistent with that of Ca(2+)-induced contraction under all experimental conditions planned above. These results suggest that ATP newly synthesized by CK essentially participates in the whole of the contraction in tonic type smooth muscle, although it participates only in a rapid phasic contraction in phasic type muscle as previously shown.

The site where newly synthesized ATP is necessary for tension development in alpha-toxin permeabilized preparations of rat proximal colon.

Since it was suggested in our previous study that ATP newly synthesized from ADP and phosphocreatine (PCr) by creatine kinase had an important role in Ca2+-induced phasic contraction in alpha-toxin permeabilized smooth muscle of rat proximal colon, we studied the role of newly synthesized ATP on myosin ATPase activity, by assessing a rate of force development as an index of myosin ATPase activity. The alpha-toxin-permeabilized preparations were thiophosphorylated by treatment with ATPgammaS. After the thiophosphorylation, the contraction induced by ATP plus PCr in the absence of Ca2+ reached the maximum at 30 s. When PCr was omitted from the bathing solution, the initial rate of the contraction was significantly slower, while the level of myosin light chain thiophosphorylation remained unchanged. An inhibitor of creatine kinase slowed the initial contractile rate to a rate similar to that induced by ATP alone. ADPbetaS had no effect on ATP plus PCr-induced contraction, suggesting that accumulation of ADP does not affect the initial rate of the contraction. PCr alone did not contract the thiophosphorylated-preparations. However, in the presence of ADP, PCr induced contraction at the initial rate which was slower than that induced by ATP plus PCr. These results indicate that newly synthesized ATP together with preexisting ATP is utilized as a substrate for myosin ATPase.

Proline reduces creatine kinase activity in the brain cortex of rats.

Type II hyperprolinemia is an inherited disorder caused by a deficiency of delta 1-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurological symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of the present study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity of brain cortex of Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered twice a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the brain cortex of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in cerebral cortex of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase forthe maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.

Effect of proline on creatine kinase activity in rat brain.

Type II Hyperprolinemia is an inherited disorder caused by a deficiency of delta1-pyrroline-5-carboxilic acid dehydrogenase, whose biochemical hallmark is proline accumulation in plasma and tissues. Although neurologic symptoms occur in most patients, the neurotoxicity of proline is still controversial. The main objective of this study was to investigate the effect of acute and chronic administration of proline on creatine kinase activity in the homogenates of cerebellum and midbrain from Wistar rats. Acute treatment was performed by subcutaneous administration of one injection of proline to 22-day-old rats. For chronic treatment, proline was administered four times a day from the 6th to the 21st postpartum day. The results showed that creatine kinase activity was significantly inhibited in the cerebellum and midbrain of rats subjected to acute proline administration. In contrast, this activity was increased in animals subjected to chronic administration. We also measured the in vitro effect of proline on creatine kinase activity in the same cerebral structures of 22-day-old nontreated rats. Proline significantly inhibited creatine kinase activity. Considering the importance of creatine kinase for the maintenance of energy homeostasis in the brain, it is conceivable that an alteration of this enzyme activity in the brain may be one of the mechanisms by which proline might be neurotoxic.

Importance of creatine kinase activity for functional recovery of myocardium after ischemia-reperfusion challenge.

To define the relation between the phosphoryl transfer via creatine kinase and the ability to recover from an ischemia-reperfusion challenge, the authors chemically inhibited creatine kinase activity with iodoacetamide (IAm) and then measured myocardial recovery after 2, 10, or 30 min of global ischemia followed by 30 min of reperfusion in the isolated, arterially perfused interventricular septa of the rabbit heart. During normoxia, IAm (0.5 M perfused for 15 min) did not by itself modify developed tension, maximal rate of tension development, or resting tension. In ischemia, IAm pretreatment increased the rate of developed tension loss and highly diminished developed tension recovery after reperfusion for all the ischemia periods tested. Moreover, IAm significantly enhanced the maximal increase in the resting tension induced by 10 or 30 min of ischemia plus reperfusion. Lactate dehydrogenase activity in reperfusion was also significantly increased over untreated septa. On the basis of the present results, the authors suggest that the aggravating effects exhibited by IAm on the ischemic myocardium are compatible with its creatine kinase inhibition properties and that creatine kinase activity is essential for full recovery from an ischemia-reperfusion challenge.

Creatine kinase activity from rat brain is inhibited by branched-chain amino acids in vitro.

Maple syrup urine disease (MSUD) is an inherited metabolic disorder biochemically characterized by the accumulation of branched-chain amino acids (BCAAs) and their branched-chain keto acids (BCKAs) in blood and other tissues. Neurological dysfunction is usually present in the affected patients, but the mechanisms of brain damage in this disease are not fully understood. Considering that brain energy metabolism seems to be altered in MSUD, the main objective of this study was to investigate the in vitro effect of BCAAs and BCKAs on creatine kinase activity, a key enzyme of energy homeostasis, in brain cortex of young rats. BCAAs, but not their BCKAs, significantly inhibited creatine kinase activity at concentrations similar to those found in the plasma of MSUD patients (0.5-5 mM). Considering the crucial role creatine kinase plays in energy homeostasis in brain, if this effect also occurs in the brain of MSUD patients, it is possible that inhibition of this enzyme activity may contribute to the brain damage found in this disease.

Phosphocreatine as a determinant of K(ATP) channel activity in pancreatic beta-cells.

The aim of the present study was to test the hypothesis that a creatine kinase/phosphocreatine system is involved in the regulation of K(ATP) channels in pancreatic beta-cells. The phosphocreatine concentration in isolated mouse islets clearly increased in parallel with the ATP/ADP ratio in response to a rise of the glucose concentration from 0.5 mM to 15 mM. The currents through K(ATP) channels expressed in oocytes of Xenopus laevis were inhibited by injection of phosphocreatine or ATP but not by phosphate or creatine alone. In inside-out patches of beta-cell membranes obtained from native beta-cells, phosphocreatine reduced the open probability of single K(ATP) channels in the presence of ADP but not in the absence of the nucleotide. These experiments suggest the existence of a K(ATP) channel-associated creatine kinase that phosphorylates ADP. The creatine kinase inhibitor iodoacetamide suppressed the glucose-induced oscillations of the cytoplasmic Ca(2+) concentration, [Ca(2+)](c). It is concluded that phosphocreatine serves as a shuttle for energy-rich phosphate from the mitochondria to the plasma membrane. The data provide a novel model for signal transduction to K(ATP) channels in pancreatic beta-cells.

Peroxynitrite-induced inhibition and nitration of cardiac myofibrillar creatine kinase

Although cardiac peroxynitrite formation and attendant protein nitration is an established event in both acute and chronic settings of cardiac failure, the putative intracellular targets involved remain incompletely defined. We have recently shown that the myofibrillar isoform of creatine kinase (a critical energetic controller of cardiomyocyte contractility) may be a particularly sensitive target of peroxynitrite-induced nitration and inactivation in vivo. However, the kinetic and mechanistic aspects of this interaction remain undefined. Here we tested the hypothesis that myofibrillar creatine kinase is sensitive to inhibition by peroxynitrite, and investigated the mechanistic role for tyrosine nitration in this process. Peroxynitrite potently and irreversibly inhibited myofibrillar creatine kinase capacity (Vmax), at concentrations as low as 100 nM, while substrate affinity (Km) was unaffected. Concentration-dependent nitration of myofibrillar creatine kinase was observed. The extent of nitration was linearly related to peroxynitrite concentration and highly correlated to the extent of myofibrillar creatine kinase inhibition. This inhibition was not reversible by treatment with free cysteine (250 μM), but pre-incubation with substrate (phosphocreatine and/or ATP) provided significant protection of MM-CK from both nitration and inhibition. These results suggest that myofibrillar creatine kinase is a highly sensitive target of peroxynitrite-mediated inhibition, and that nitration may mediate this inhibition.

Is Creatine Kinase a Target for AMP-activated Protein Kinase in the Heart?

J. S. I NGWALL. Is Creatine Kinase a Target for AMP-activated Protein Kinase in the Heart?Journal of Molecular and Cellular Cardiology (2002) 34, 1111–1120. By phosphorylating target proteins, AMP-activated protein kinase (AMPK) inhibits ATP-utilizing proteins and activates ATP-synthesizing proteins, thereby increasing ATP synthesis under conditions such as hypoxia and ischemia. It has been proposed that AMPK also phosphorylates and inhibits creatine kinase (CK), the enzyme which catalyzes the reversible transfer of a phosphoryl group between creatine and ADP. Here, we examine the hypothesis that AMPK inactivates CK activity under three conditions where [AMP] and AMP-dependent AMPK velocity increase: increased workload both in the isolated rat heart and in the living rat, hypoxia in the living rat heart and low-flow ischemia in the isolated red blood cell perfused rat heart. For the experiments varying workload in the isolated rat heart (both ejecting and isovolumic models), we also changed oxidizable substrate available to the isolated heart in order to vary the [AMP]/[ATP]. CK reaction velocity in the intact rat heart was directly measured using 31P magnetization transfer. The metabolically active AMP and ATP pools were determined from 31P NMR measurements and we calculate AMP-dependent AMPK velocity from the Michaelis–Menten relationship. We found that under normoxic conditions where [AMP] and AMPK velocity increase, the linear relationship between CK and AMPK velocities is positive, not inverse. Under conditions of low pO2 (hypoxia and low-flow ischemia), CK velocity fell 2–4-fold while the increase in AMP-activated AMPK activity was modest. This analysis illustrates the complex nature of AMPK regulation in the heart.

Intracellular ATP measured with luciferin/luciferase in isolated single mouse skeletal muscle fibres.

The firefly luciferin/luciferase reaction was utilized to monitor intracellular ATP concentration ([ATP](i)). Single fibres of mouse skeletal muscle were dissected and injected with luciferase. Luciferin was added to the perfusate and light emission from the fibres was monitored as an indication of [ATP](i). Inhibition of oxidative phosphorylation with cyanide and anaerobic glycolysis with iodoacetate caused light emission to fall to zero within 10 min and the fibres developed a rigor contraction. Inhibition of creatine kinase with 2,4-dinitro-1-fluorobenzene produced a small transient fall in light emission in association with each tetanus. Muscle fibres were fatigued by repeated tetani and 5/12 fibres showed a fall in light emission in the late phase of fatigue. If fibres were allowed to recover from fatigue in the absence of glucose and then restimulated in the absence of glucose they fatigued much more rapidly. However, such fibres showed no obvious change in light emission. We conclude that the luciferin/luciferase system can be used to monitor [ATP](i) in functioning single skeletal muscle cells. A depletion of global [ATP](i) is not observed in all fatiguing fibres and cannot be the sole cause of the final phase of fatigue.

Effects of acrylamide on creatine kinase from rabbit muscle

The mechanism of inhibition of creatine kinase (CK) by acrylamide (Acr) has been examined (in vitro). Within the concentration range of 0 to 1 M, Acr markedly inhibited CK and depleted the protein thiols. Both inactivation and thiol depletion were time- and Acr concentration-dependent. Addition of dithiothreitol (DTT) did not reactivate CK inactivated by Acr. However, CK with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) pre-blocked thiols can be reactivated by DTT after incubation with Acr. The transition-state analogue also had a significant protective effect on CK against Acr inhibition. We conclude that thiol alkylation is a critical event in inactivation of CK by Acr. Furthermore, Acr binding to CK changed its surface charge, which may be the same effect for the toxicity of Acr towards other proteins.

The nitric oxide-induced reduction in cardiac energy supply is not due to inhibition of creatine kinase.

While nitric oxide (NO) is a potent vasodilator already in the nM range, a cGMP-independent negative inotropic effect is observed at higher concentrations. Since inhibition of creatine kinase (CK) by NO-induced nitrosylation has been proposed as a possible mechanism of action, we measured the flux through CK in the intact heart. In saline perfused, paced guinea pig hearts 31P NMR spectroscopy was employed to directly assess the cardiac energy status, i.e. free energy of ATP hydrolysis (DeltaG(ATP)) and flux through CK using magnetization transfer in absence and presence of NO. NO (50 microM) doubled coronary flow and induced a rapid drop in left ventricular developed pressure (39+/-10 vs. 81+/-10 mmHg) and MVO(2) (1.3+/-0.8 vs. 3.7+/-0.5 micromol/min/g) (n=7). This effect was associated with an immediate decrease in phosphocreatine (PCr) (-69%) and DeltaG(ATP). During the subsequent 35 min of NO infusion cardiac function and MVO(2) remained depressed, while PCr partially recovered. NO had no effect on the unidirectional forward flux through CK (98 +/- 21 vs. 99 +/- 20 micromol/min/g, n=7) which was 5- to 10-fold greater than the rate of ATP turnover. Upon cessation of NO infusion both cardiac function and PCr rapidly returned to baseline values. The NO-induced fall in the myocardial energy status was associated with an increase in mitochondrial NADH (n=7) as assessed by surface fluorescence. The observed change in fluorescence was similar to that observed with short term ischemia. The NO-mediated depression of myocardial function, MVO(2) and energy status is not mediated by changes in CK flux. Most likely a partial blockade of mitochondrial oxidative phosphorylation at the level of cytochrome c oxidase is responsible for this effect.

Inhibition of creatine kinase reduces the rate of fatigue-induced decrease in tetanic [Ca(2+)](i) in mouse skeletal muscle.

1. Ca(2+)-phosphate (P(i)) precipitation in the sarcoplasmic reticulum (SR) may cause reduced SR Ca(2+) release in skeletal muscle fatigue. To study this, we inhibited the creatine kinase (CK) reaction with 2,4-dinitro-1-fluorobenzene (DNFB). The hypothesis was that with inhibition of CK, phosphocreatine would not break down to creatine and P(i). Therefore P(i) transport into the SR would be limited and Ca(2+)-P(i) precipitation would not occur. 2. Intact single fibres from a mouse foot muscle were fatigued by repeated short tetani under control conditions or after exposure to DNFB (10 microM). The free myoplasmic concentrations of Ca(2+) ([Ca(2+)](i)) and Mg(2+) ([Mg(2+)](i)) were measured with indo-1 and mag-indo-1, respectively. Changes in [Mg(2+)](i) were assumed to reflect alterations in myoplasmic ATP concentration. 3. During the first 10 fatiguing tetani, tetanic [Ca(2+)](i) increased both in control and after DNFB exposure. Thereafter tetanic [Ca(2+)](i) fell and the rate of fall was about fourfold lower after DNFB exposure compared with control. 4. Under control conditions, there was a good relationship between declining tetanic [Ca(2+)](i) and increasing [Mg(2+)](i) during the final part of fatiguing stimulation. This correlation was lost after DNFB exposure. 5. In conclusion, the present data fit with a model where Ca(2+)-P(i) precipitation inhibits SR Ca(2+) release in fatigue produced by repeated short tetani. Furthermore, the results suggest that the rate of P(i) transport into the SR critically depends on the myoplasmic Mg(2+)/ATP concentration.

ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes.

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.

IGF-I differentially regulates Bcl-xL and Bax and confers myocardial protection in the rat heart.

Bcl-2 family proteins play a crucial role in the cytoprotective action of insulin-like growth factor-I (IGF-I) by regulating cell death signaling at the mitochondrial level. The present study examined the effect of IGF-I on the expression of Bcl-2 family proteins in the rat heart mitochondria in relation to myocardial protection against ischemia-reperfusion injury. Systemic IGF-I (1 mg) treatment in the rat increased Bcl-xL and attenuated Bax 12-24 h later in the heart mitochondria fraction. Permeability transition and cytochrome c release occurred in a Ca(2+) concentration-dependent manner in the vehicle-treated mitochondria. This was significantly inhibited by the IGF-I-pretreatment. Moreover, ATP synthesis was significantly greater in the IGF-I-pretreated mitochondria. IGF-I pretreatment 24 h before 25 min of global ischemia in the isolated rat heart model significantly improved recovery of isovolumic left ventricular function and inhibited creatine kinase release during reperfusion. This was associated with a significantly less number of terminal transferase labeling-positive myocytes and nonmyocytes 2 h after reperfusion. These results suggest that IGF-1 differentially regulates Bcl-xL and Bax in heart mitochondria, which may be causally related to myocardial protection against ischemia-reperfusion injury.