Metabolic and functional consequences of complete inhibition of creatine kinase by iodoacetamide in the perfused heart]

Treatment of perfused rat hearts with 0.5 mM iodoacetamide (IAAm) for 15 min at different workloads resulting in a nearly complete inhibition of creatine kinase (CK, 99%) was followed by a rapid decline of the phosphocreatine (PCr) level (30%) and a 2-fold increase of the P(i) level which then stabilized. Conversely, the ATP content started to drop monotonously at the beginning of the IAAm washout and reached 30% 90 min after the IAAm removal under medium load. Under low workload the ATP decay occurred at later periods. Neither the ADP-stimulated mitochondrial respiration in skinned fibers, nor the Ca(2+)-stimulated ATPase activity of myofibrils was affected by IAAm treatment. The sensitivity of the resting tension of skinned fibers to Ca2+ tended to a slight increase. The cardiac work index (PRP-pressure-rate product) decreased by 25%, while the end diastolic pressure (EDP) rose by 15 mm Hg when IAAm acted under medium load. In contrast, under low work these parameters were practically stable. The hearts poisoned with IAAm performed a two times lower maximal work and had reduced (by 35%) oxygen consumption rates. The efficiency of energy utilization for mechanical work decreased by 40%. The changes in PRP and EDP correlated with the cytosolic [ATP]/[ADP] ratio in such a way that the decrease in the latter was associated with a decrease in PRP and the elevation of EDP. These data suggest that the creatine kinase system is necessary for the effective translation of a high [ATP]/[ADP] ratio from the intermembrane space of mitochondria to the cytoplasm, myofibrils and ionic pumps. This provides a high level of mechanical work and good relaxation of the left ventricle and protects cytosolic adenine nucleotides from the breakdown.     

Kinetics of creatine kinase in an experimental model of low phosphocreatine and ATP in the normoxic heart

To study the dependence of the forward flux of creatine kinase(CK) on its substrates and products we designed an acute normoxic modelof steady-state depletion of phosphocreatine (PCr) and adenylate in theisovolumic acetate-perfused rat heart. Various concentrations of PCrand ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presenceof insulin. The apparent rate constant(k_f) and theforward CK flux were measured under metabolic and contractile steadystate by progressive saturation-transfer³¹P nuclear magnetic resonance(NMR). At high adenylate content CK flux was constant for a twofoldreduction in PCr concentration ([PCr]); CK flux was 6.3 ± 0.6 mM/s (vs. 6.5 ± 0.2 mM/s in control) because of adoubling of k_f.Although, at the lowest ATP concentration and [PCr], CKflux was reduced by 50%, it nevertheless always remained higher thanATP synthesis estimated by parallel oxygen consumption measurement.NMR-measured flux was compared with the flux computed under thehypothesis of CK equilibrium. CK flux could not be fully predicted bythe concentrations of CK metabolites. This is discussed in terms ofmetabolite and CK isozyme compartmentation.

     

31P NMR detection of subcellular creatine kinase fluxes in the perfused rat heart: contractility modifies energy transfer pathways

The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by (31)P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, F(f) = PCr --> ATP, and reverse flux, F(r) = ATP --> PCr = 3.3 mm.s(-1)), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (F(f) = 1.7 x F(r)), and (iii) a mitochondrial CK displaced toward PCr synthesis (F(f) = 0.3 and F(r) = 2.6 mm.s(-1)). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed.     

Evidence for myocardial ATP compartmentation from NMR inversion transfer analysis of creatine kinase fluxes

The interpretation of creatine kinase (CK) flux measured by (31)P NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P(i) exchange on the forward (F(f): PCr --> ATP) and reverse (F(r)) CK fluxes derived from complete analysis of inversion transfer. Although F(f) should equal F(r) because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P(i) was masked by saturation of P(i) (sat-P(i)), F(f)/F(r) significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F(f)/F(r) for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P(i) might not result only from the masking of ATP-P(i) exchange.     

Cardiac pump function of the isolated rat heart at two modes of energy deprivation and effect of adrenergic stimulation

The contractile function of the isolated rat heart and high energy phosphate content were evaluated under conditions of depressed energy supply caused by disturbances either in mitochondrial ATP production or ATP-phosphocreatine transformation. Amytal (0.3 mM), an inhibitor of mitochondrial respiration, or iodoacetamide (IAA, 0.1 mM) reducing in this dose creatine kinase activity to 19% of the initial level, were used, respectively. Myocardial ATP content remained unaffected in both groups and PCr content decreased to 37% only in amytal-treated group. Very similar alterations in cardiac pump function during volume load were observed in both treated groups; maximal cardiac output was significantly less by 30%, cardiac pressure-volume work by 38–40%, left ventricular (LV) systolic pressure by 24–29%, and LV +dP/dt by 36–39%. In contrast, the extent of decreased LV distensibility was different, a curve relating LV filling volume and end-diastolic pressure was shifted up and to the left much more prominently after IAA treatment. Heart rate was decreased by 24% only in amytal-treated group. Results indicate that a decreased myocardial distensibility is a dominating feature in the acute cardiac pump failure caused by an inhibition of myocardial creatine kinase. Isoproterenol (0.1 M) substantially increased heart rate and pressure-rate product in IAA-treated hearts but failed to increase cardiac work probably due to its inability to improve myocardial distensibility.     

Cerebral creatine kinase deficiency influences metabolite levels and morphology in the mouse brain: a quantitative in vivo 1H and 31P magnetic resonance study

Creatine kinase (CK)-catalysed ATP-phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B-CK (B-CK-/-), mitochondrial ubiquitous CK (UbCKmit-/-), or both isoenzymes (CK -/-), using non-invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B-CK-/- mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high-energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20-30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N-acetyl-aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo-inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr-CK energy system and suggest a multifaceted role for creatine in the brain.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

Recovery of isolated rat atrial function related to ATP under different anoxic conditions

Spontaneously beating isolated rat atria were subjected to 1 h of anoxia at 37 degrees C in various cardioplegic solutions. Contraction continued for different times upon initiation of anoxia, depending on the nature of the cardioplegic solution. Two hundred micromolar P1,P5-di(adenosine-5')pentaphosphate (Ap5A) stopped atrial function in less than 30 s of anoxia in contrast to 50 s in the case of Hearse's cardioplegic solution (16 mM MgCl2, 16 mM KCl, 1 mM Procaine), and 20 min in the case of controls. The stopping time was also prolonged from 30 to approximately 50-55 seconds if a lower concentration of Ap5A (100 microM) was used. Function, adenine nucleotides (AN), and phosphocreatine (PCr) were then measured 20 min after reoxygenation. The recovery of both function and AN was most rapid and complete with 200 microM Ap5A (97% recovery in ATP and 100% in function) and least complete in control (50% recovery in ATP and 78% in function). A positive correlation between recovery of ATP, or total adenine nucleotides, and recovery of function was observed in all cases. The higher the level of ATP remaining at the end of 1 h of anoxia and the more recovered after 20 min of reoxygenation, the more complete the recovery of function. The PCr returned to normal or even higher than normal values in all cases, even though function returned only in proportion to ATP. Since PCr is mitochondrial in origin, it appears that loss of a portion of the AN localized at the energy-utilizing sites occurred before serious mitochondrial damage and was responsible for the incomplete postanoxic functional recovery.     

Systems bioenergetics of creatine kinase networks: physiological roles of creatine and phosphocreatine in regulation of cardiac cell function

Physiological role of creatine (Cr) became first evident in the experiments of Belitzer and Tsybakova in 1939, who showed that oxygen consumption in a well-washed skeletal muscle homogenate increases strongly in the presence of creatine and with this results in phosphocreatine (PCr) production with PCr/O₂ ratio of about 5–6. This was the beginning of quantitative analysis in bioenergetics. It was also observed in many physiological experiments that the contractile force changes in parallel with the alteration in the PCr content. On the other hand, it was shown that when heart function is governed by Frank–Starling law, work performance and oxygen consumption rate increase in parallel without any changes in PCr and ATP tissue contents (metabolic homeostasis). Studies of cellular mechanisms of all these important phenomena helped in shaping new approach to bioenergetics, Molecular System Bioenergetics, a part of Systems Biology. This approach takes into consideration intracellular interactions that lead to novel mechanisms of regulation of energy fluxes. In particular, interactions between mitochondria and cytoskeleton resulting in selective restriction of permeability of outer mitochondrial membrane anion channel (VDAC) for adenine nucleotides and thus their recycling in mitochondria coupled to effective synthesis of PCr by mitochondrial creatine kinase, MtCK. Therefore, Cr concentration and the PCr/Cr ratio became important kinetic parameters in the regulation of respiration and energy fluxes in muscle cells. Decrease in the intracellular contents of Cr and PCr results in a hypodynamic state of muscle and muscle pathology. Many experimental studies have revealed that PCr may play two important roles in the regulation of muscle energetics: first by maintaining local ATP pools via compartmentalized creatine kinase reactions, and secondly by stabilizing cellular membranes due to electrostatic interactions with phospholipids. The second mechanism decreases the production of lysophosphoglycerides in hypoxic heart, protects the cardiac cells sarcolemma against ischemic damage, decreases the frequency of arrhythmias and increases the post-ischemic recovery of contractile function. PCr is used as a pharmacological product Neoton in cardiac surgery as one of the components of cardioplegic solutions for protection of the heart against intraoperational injury and injected intravenously in acute myocardial ischemic conditions for improving the hemodynamic response and clinical conditions of patients with heart failure.     

Analyzing the functional properties of the creatine kinase system with multiscale 'sloppy' modeling

In this study the function of the two isoforms of creatine kinase (CK; EC 2.7.3.2) in myocardium is investigated. The 'phosphocreatine shuttle' hypothesis states that mitochondrial and cytosolic CK plays a pivotal role in the transport of high-energy phosphate (HEP) groups from mitochondria to myofibrils in contracting muscle. Temporal buffering of changes in ATP and ADP is another potential role of CK. With a mathematical model, we analyzed energy transport and damping of high peaks of ATP hydrolysis during the cardiac cycle. The analysis was based on multiscale data measured at the level of isolated enzymes, isolated mitochondria and on dynamic response times of oxidative phosphorylation measured at the whole heart level. Using 'sloppy modeling' ensemble simulations, we derived confidence intervals for predictions of the contributions by phosphocreatine (PCr) and ATP to the transfer of HEP from mitochondria to sites of ATP hydrolysis. Our calculations indicate that only 15±8% (mean±SD) of transcytosolic energy transport is carried by PCr, contradicting the PCr shuttle hypothesis. We also predicted temporal buffering capabilities of the CK isoforms protecting against high peaks of ATP hydrolysis (3750 μM*s(-1)) in myofibrils. CK inhibition by 98% in silico leads to an increase in amplitude of mitochondrial ATP synthesis pulsation from 215±23 to 566±31 μM*s(-1), while amplitudes of oscillations in cytosolic ADP concentration double from 77±11 to 146±1 μM. Our findings indicate that CK acts as a large bandwidth high-capacity temporal energy buffer maintaining cellular ATP homeostasis and reducing oscillations in mitochondrial metabolism. However, the contribution of CK to the transport of high-energy phosphate groups appears limited. Mitochondrial CK activity lowers cytosolic inorganic phosphate levels while cytosolic CK has the opposite effect.     

Dual cardiac contractile effects of the alpha2-AMPK deletion in low-flow ischemia and reperfusion

Because the question "is AMP-activated protein kinase (AMPK) alpha(2)-isoform a friend or a foe in the protection of the myocardium against ischemia-reperfusion injury?" is still in debate, we studied the functional consequence of its deletion on the contractility, the energetics, and the respiration of the isolated perfused heart and characterized the response to low-flow ischemia and reperfusion with glucose and pyruvate as substrates. alpha(2)-AMPK deletion did not affect basal contractility, respiration, and high-energy phosphate contents but induced a twofold reduction in glycogen content and a threefold reduction in glucose uptake. Low-flow ischemia increased AMPK phosphorylation and stimulated glucose uptake and phosphorylation in both alpha(2)-knockout (alpha(2)-KO) and wild-type (WT) groups. The high sensitivity of alpha(2)-KO to the development of ischemic contracture was attributed to the constitutive impairment in glucose transport and glycogen content and not to a perturbation of the energy transfer by creatine kinase (CK). The functional coupling of MM-CK to myofibrillar ATPase and the CK fluxes were indeed similar in alpha(2)-KO and WT. Low-flow ischemia impaired CK flux by 50% in both strains, showing that alpha(2)-AMPK does not control CK activity. Despite the higher sensitivity to contracture, the postischemic contractility recovered to similar levels in both alpha(2)-KO and WT in the absence of fatty acids. In their presence, alpha(2)-AMPK deletion also accelerated the contracture but delayed postischemic contractile recovery. In conclusion, alpha(2)-AMPK is required for a normal glucose uptake and glycogen content, which protects the heart from the development of the ischemic contracture, but not for contractile recovery in the absence of fatty acids.     

Creatine kinase binding and possible role in chemically skinned guinea-pig taenia coli.

The activity and role of creatine kinase (CK) associated with contractile proteins of smooth muscle have been investigated using skinned guinea-pig taenia coli fibers. Total CK activity was 163 +/- 22 IU/g (ww) and agarose electrophoresis showed BB, MB, and MM isoforms (BB-CK being the predominant isoenzyme). After skinning for 1 h with Triton X-100, BB-CK was specifically associated with the myofibrils, representing 22% of the preskinned CK activity. When relaxed fibers were exposed to pCa 9 in the presence of 250 microM ADP, 0 ATP and 12 mM PCr, tension was not significantly different from resting tension, but changing to pCa 4.5 caused the fibers to generate 59.1 +/- 5.2 percent of maximal tension. When a high-tension rigor state was achieved (250 microM ADP, 0 ATP, 0 PCr, and pCa 9), the addition of 12 mM PCr effected significant relaxation. These observations implicate an endogenous form of BB-CK, associated with the myofilaments and capable of producing enough ATP for submaximal tension generation and significant relaxation from rigor conditions. It was also shown that ADP is bound to the myofibrils and available for rephosphorylation by BB-CK. These results suggest co-localization of ATPase, MLCK and CK on the contractile proteins of the taenia coli. This enzymic association may play a role in the compartmentation of adenine nucleotides in smooth muscle.     

Muscle acidosis during static exercise is associated with calf vasoconstriction

In this study we measured (n = 6) the phosphocreatine-to-inorganic phosphate ratio (PCr/Pi), Pi, and pH with 31P-nuclear magnetic resonance (31P-NMR) in the human forearm during static work at 30% of maximal voluntary contraction (MVC) for 2 min followed immediately by 3 min of circulatory arrest (forearm arterial occlusion). Static exercise, with its central volitional and skeletal muscle metabolic and mechanical afferent components, caused a rise in heart rate (HR, 32%), blood pressure (BP, 29%), and calf vascular resistance (calf R, 30%). During forearm occlusion after static exercise, HR returned to base line, the increase in BP was attenuated by 30%, and calf R remained elevated and unchanged. The percent change in calf R was correlated with forearm cellular pH (R = 0.56, P less than 0.001) but only weakly associated with PCr/Pi (R = 0.33, P less than 0.042). 30% MVC for 1 min followed by arterial occlusion (3 min) reduced PCr/Pi by 65% and pH by 0.16 U (P less than 0.05). Calf R was unchanged. Circulatory arrest alone (20 min) caused no change in either pH or calf R but large changes in PCr/Pi (50% reduction). We conclude that 1) there is an association between forearm cellular acidosis and calf vasconstriction during static forearm exercise and 2) large changes in PCr/Pi without concomitant changes in pH are not associated with changes in calf R.     

Cardiac contractile function, oxygen consumption rate and cytosolic phosphates during inhibition of electron flux by amytal--a 31P-NMR study

In order to investigate the potential role of cytosolic phosphates ([ATP], [ADP] and [Pi]) in the integration of mitochondrial respiration and mechanical function in the perfused heart, inhibition of the substrate end of the respiratory chain by amytal has been employed. A stepwise increase in amytal concentration (from 0.2 to 1.2 mM) resulted in the progressive abolition of the cardiac oxygen consumption, rate (VO2) in hearts oxidizing pyruvate (5 mM). The inhibition curve for VO2 was S-shaped, with K0.5 = 1.1 mM, and independent of the initial VO2 values varied by coronary flow and isoproterenol (Iso) addition. ADP-stimulated respiration of isolated mitochondria (malate + pyruvate) was twice as sensitive to amytal inhibition, whereas state 2 respiration (before ADP addition) had the same sensitivity as cardiac VO2. Decrease in VO2 was followed by a decline in phosphocreatine (PCr) content and augmentation of Pi at nearly constant ATP level and intracellular pH as assessed by the 31P-NMR method. These changes were associated with an elevation of cytosolic free [ADP] and a reduction of the [ATP]/[ADP] ratio and ATP affinity calculated from creatine kinase equilibrium. Concomitantly, pressure-rate product (PRP), maximal rates of contraction and relaxation fell down and the end diastolic pressure (EDP) rose at all initial loads. Amytal-inhibited hearts retained the capability to respond to Iso stimulation (0.1 microM, about 50% enhancement of PRP) even at 1 mM amytal, but their response to elevation of coronary flow was greatly diminished. Alterations in the PRP value induced by the inhibitor at a fixed coronary flow correlated negatively with cytosolic [ADP] and [Pi], and positively with [ATP]/[ADP] and A(ATP). In contrast, EDP correlated with all these parameters in the opposite manner. However, when PRP was varied by coronary flow in the absence of the inhibitor or at its fixed concentrations, such correlations were absent. These data imply that cytosolic phosphates can serve as a feedback between energy production and utilization when the control point(s) is (are) at the mitochondria. In contrast, other regulatory mechanisms should be involved when control is distributed among different steps located both in energy producing and utilizing systems.     

ATP-phosphocreatine metabolism catalyzed by creatine kinase. Comparison of saturation transfer (NMR) and isotope labeling technics

Unidirectional fluxes from ATP to phosphocreatine (PCr) catalyzed by MM-isoenzyme of creatine kinase (CK) were measured by using 31P-NMR saturation transfer technique and by means of radioactively labeled [gamma-32P]ATP. At 30-37 degrees C and pH 7.4 in a wide range of [PCr]/[creatine] ([PCr]/[Cr]) ratios (0.2 to 3.0) both of these methods gave similar results, thus showing that magnetization (saturation) transfer allows to determine fluxes close to real ones under "physiological" conditions. However, at [PCr]/[Cr] ratio higher than 5 ([ADP] less than 30 microM) or at decreased temperatures (7-15 degrees C, [PCr]/[Cr] approximately 1) fluxes determined by saturation transfer substantially exceeded those measured with the radioactive label. These data imply that under "physiological" conditions phosphoryl group transfer is actually rate-determining step of the CK reaction. On the contrary, at high [PCr]/[Cr] values or at low temperature the control step could be shifted from the phosphoryl group transfer or distributed among other steps of the reaction.     

The creatine kinase system in smooth muscle

Despite the energetic flux being much lower in smooth muscle compared to striated muscles (such as the heart and skeletal muscle) creatine kinase (CK) has been found present and active in all smooth muscles studied to date. A complete CK circuit has been identified, with CK found in the mitochondria, contractile elements, membrane pumps and the cytoplasm. CK isoenzymes are coupled to many cellular energetic processes and appears to be involved in energy production and consumption by acting as an energy transducer. The CK system responds to pathological insults and development (e.g. hypertrophy and gestation respectively) by changes in sub-cellular distribution localization, isoenzymes, and specific activity. The conclusion from these observations is that creatine kinase is intimately involved in the energetic system of smooth muscle.Abbreviations CK creatine kinase - Mi-CK mitochondrial creatine kinase - Cr creatine - PCr phosphocreatiner - NMR nuclear magnetic resonance - SHR spontaneously hypertensive rat - -GPA -guanidinopropionic acid     

Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart

Creatine kinase (CK) exists as a family of isoenzymes in excitable tissue. We studied isolated perfused hearts from mice lacking genes for either the main muscle isoform of CK (M-CK) or both M-CK and the main mitochondrial isoform (Mt-CK) to determine 1) the biological significance of CK isoenzyme shifts, 2) the necessity of maintaining a high CK reaction rate, and 3) the role of CK isoenzymes in establishing the thermodynamics of ATP hydrolysis. (31)P NMR was used to measure [ATP], [PCr], [P(i)], [ADP], pH, as well as the unidirectional reaction rate of PCr--> [gamma-P]ATP. Developmental changes in the main fetal isoform of CK (BB-CK) were unaffected by loss of other CK isoenzymes. In hearts lacking both M- and Mt-CK, the rate of ATP synthesis from PCr was only 9% of the rate of ATP synthesis from oxidative phosphorylation demonstrating a lack of any high energy phosphate shuttle. We also found that the intrinsic activities of the BB-CK and the MM-CK isoenzymes were equivalent. Finally, combined loss of M- and Mt-CK (but not loss of only M-CK) prevented the amount of free energy released from ATP hydrolysis from increasing when pyruvate was provided as a substrate for oxidative phosphorylation.     

Discrimination of cardiac subcellular creatine kinase fluxes by NMR spectroscopy: a new method of analysis.

A challenge in the understanding of creatine kinase (CK) fluxes reflected by NMR magnetization transfer in the perfused rat heart is the choice of a kinetic model of analysis. The complexity of the energetic pathways, due to the presence of adenosine triphosphate (ATP)-inorganic phosphate (Pi) exchange, of metabolite compartmentation and of subcellular localization of CK isozymes cannot be resolve from the sole information obtained from a single NMR protocol. To analyze multicompartment exchanges, we propose a new strategy based on the simultaneous analysis of four inversion transfer protocols. The time course of ATP and Phosphocreatine (PCr) magnetizations computed from the McConnell equations were adjusted to their experimental value for exchange networks of increasing complexity (up to six metabolite pools). Exchange schemes were selected by the quality of their fit and their consistency with data from other sources: the size of mitochondrial pools and the ATP synthesis flux. The consideration of ATP-Pi exchange and of ATP compartmentation were insufficient to describe the data. The most appropriate exchange scheme in our normoxic heart involved the discrimination of three specific CK activities (cytosolic, mitochondrial, and close to ATPases). At the present level of heart contractility, the energy is transferred from mitochondria to myofibrils mainly by PCr.     

The creatine kinase reaction: a simple reaction with functional complexity

The classical role of PCr is seen as a reservoir of high-energy phosphates defending cellular ATP levels under anaerobic conditions, high rates of energy transfer or rapid fluctuations in energy requirement. Although the high concentration of PCr in glycolytic fast-twitch fibers supports the role of PCr as a buffer of ATP, the primary importance of the creatine kinase (CK) reaction may in fact be to counteract large increases in ADP, which could otherwise inhibit cellular ATPase-mediated systems. A primary role for CK in the maintenance of ADP homeostasis may explain why, in many conditions, there is an inverse relationship between PCr and muscle contractility but not between ATP and muscle contractility. The high rate of ATP hydrolysis during muscle contraction combined with restricted diffusion of ADP suggests that ADP concentration increases transiently during the contraction phase (ADP spikes) and that these are synchronized with the contraction. The presence of CK, structurally bound in close vicinity to the sites of ATP utilization, will reduce the amplitude and duration of the ADP spikes through PCr-mediated phosphotransfer. When PCr is reduced, the efficiency of CK as an ATP buffer will be reduced and the changes in ADP will become more prominent. The presence of ADP spikes is supported by the finding that other processes known to be activated by ADP (i.e. AMP deamination and glycolysis) are stimulated during exercise but not during anoxia, despite the same low global energy state. Breakdown of PCr is driven by increases in ADP above that depicted by the CK equilibrium and the current method to calculate ADPfree from the CK reaction in a contracting muscle is therefore questionable.     

Thyroid hormones and the creatine kinase system in cardiac cells

The paper reviews the current evidence on the role of thyroid hormones in regulating the creatine kinase energy transfer system at multiple structures in cardiac cells. 1) Thyroid hormones modulate the overall synthesis of phosphocreatine (PCr) by increasing the rate of mitochondrial oxidative phosphorylation. 2) Thyroid hormones regulate the total activity of creatine kinase and its isoenzyme distribution. In comparison with normal thyroid state (euthyroidism), hypothyroidism is characterized by decreased total creatine kinase activity owing to diminished fraction of creatine kinase. On the other hand, hyperthyroidism, while causing no change in total creatine kinase activity, leads to increased fractions of neonatal isoforms of creatine kinase, and, in case of prolonged hyperthyroidism, to decreased fraction of mitochondrial creatine kinase. The latter change is associated with partial uncoupling between mitochondrial creatine kinase and adenine nucleotide translocase reflected by decreased PCr/O ratio. 3) Hyperthyroidism leads to increased passive sarcolemmal permeability due to which the leakage of creatine along its concentration gradient occurs. As a result of (i) increased sarcolemmal permeability for creatine, (ii) uncoupling of mitochondrial PCr synthesis, and (iii) increased energy utilization rate the steady state intracellular PCr content decreases under hyperthyroidism which, in turn, increases the myocardial susceptibility to hypoxic damage. Thyroid state also modulates the protective effects of exogenous PCr on energetically depleted myocardium.