Multiple Funcitons of Creatine Kinase

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Prof. Dr. Theo Wallimann:

"Multiple Functions of Creatine Kinase for Cellular Energetics: a Scientific Rationale for Creatine Supplementation"

Abstract

Creatine kinase (CK) isoenzymes are found in cells with intermittently high energy requirements. They are specifically located at places of energy demand and energy production and are linked by a phosphocreatine/creatine (PCr/Cr) circuit. Cytosolic CK, in close conjunction with Ca2+-pumps, plays a crucial role for the energetics of Ca2+-homeostasis. Mitochondrial Mi-CK, a cuboidal-shaped octamer with a central channel, binds and cross-links mitochondrial membranes and forms a functionally coupled microcompartment with porin (VDAC) and adenine nucleotide translocase (ANT) for vectorial export of PCr into the cytosol. The CK system is regulated by AMP-activated protein kinase via the ATP/AMP-, as well as the PCr/Cr ratio. Mi-CK stabilizes and cross-links cristae- or inner/outer membranes to form parallel membrane stacks and, if overexpressed due to creatine-depletion or cellular energy stress, forms those crystalline intramitochondrial inclusions often seen as hallmarks in mitochondrial cytopathy patients. Mi-CK is a prime target for free radical damage by peroxynitrite. Mi-CK octamers, together with CK substrates have a marked stabilizing and protective effect against mitochondrial permeability transition pore (PTP) opening, thus providing a rationale for creatine supplementation of patients with neuromuscular and neurodegenerative diseases. In addition to the well documented improvement of high-intensity intermittent exercise performance after creatine supplementation, recent results seem to indicate that creatine supplementation may also favourably affect long-endurance exercise. Chronic high-dose creatine ingestion, however, was shown to down-regulate the expression and/or accumulation of creatine transporter polypeptides in skeletal muscle of the rat. Thus, a one month pause, after three month of creatine supplementation, as suggested earlier, seems a reasonable advise.

key words and abbreviations:
creatine kinase (CK), creatine (Cr), phosphocreatine (PCr), PCr-shuttle, energetics of Ca2+-homeostasis, CK null-mutant transgenic mice, mitochondrial creatine kinase (Mi-CK), intramitochondrial inclusions, mitochondrial myopathies, AMP-activated protein kinase (AMPK), mitochondrial permeability transition (MTP), peroxynitrite PN), porin (P), adenine nucleotide translocase (ANT), cell- and neuroprotective effects of creatine, creatine supplementation, neuromuscular diseases, short-term physical performance, high-intensity-long-endurance exercise, creatine transporter (CreaT)

The creatine kinase / phospho-creatine circuit

The enzyme creatine kinase (CK), catalyzing the reversible transfer of the N-phosphoryl group >from phosphocreatine (PCr) to ADP to regenerate ATP, plays a key role in the energy homeostasis of cells with intermittently high, fluctuating energy requirements, e.g. skeletal and cardiac muscle, neurons, photoreceptors, spermatozoa and electrocytes. Cytosolic CK isoenzyme(s) (MM-, MB- and BB-CK) are always co-expressed in a tissue-specific fashion together with a mitochondrial isoform. Using biochemical fractionation and in situ localization, one was able to show that the CK isoenzymes, earlier considered to be strictly soluble, are in fact compartmentalized subcellularly and coupled functionally and/or structurally either to sites of energy production (glycolysis and mitochondria) or energy consumption (cellular ATPases, such as the acto-myosin ATPase and SR-Ca2+-ATPase). Thus they form an intricate, highly regulated energy distribution network, the so-called PCr-circuit or PCr-shuttle (Figure 1, for review see [1] and the special volumes of Mol. Cell Biochem. 133/134, 1994, and 184, 1998).
This non-equilibrium energy transport model has been challenged, based upon global 31P-NMR experiments, measuring CK-mediated flux in muscles at different work-loads [2,3]. The conclusions reached by these authors were i) that the CK system is in equilibrium with the substrates, behaving like a solution of well-mixed enzymes, ii) that effects of compartmentation were negligible with respect to total cellular bioenergetics and iii) that thermodynamic characteristics of the cytosol could be predicted as if the CK metabolites were freely mixing in solution. However, based on the organizational principles of sarcomeric muscle, as well as on our findings concerning the highly structured subcellular CK-compartments, this interpretation seemed rather unlikely and thus has been questionned [4]. In support of this, 31P-NMR CK-flux measurements with transgenic mice showing graded reductions of MM-CK expression in their muscles, revealed a strikingly unexpected, "anomalous" CK-flux behaviour [5]. These results indicate that some flux through CK, presumably bound CK, and possibly also some PCr and/or ATP, are NMR-invisible or otherwise not amenable to this analysis [4,6]. In the meantime, more evidence from NMR-measurements [7,8,9,10], as well as >from recent in vivo 14[C]Cr-tracer studies [11], is accumulating in favour of compartmentation of the CK system and for the existence of different pools of CK substrates. As a matter of fact, it has now become clear that in muscle, Cr and PCr molecules do not tumble freely, but display partial orientational ordering, which is in contrast to what is expected for small molecules dissolved in water [7]. Furthermore, 31P-NMR saturation transfer experiments with sea-urchin spermatozoa show that the CK-flux increases by a factor of 10-20 upon sperm activation [12]. These specialized sperm cells derive their energy for motility entirely from fatty oxidation within the single large mitochondrion located just behind the sperm head, from where PCr is diffusing along the 50 µm long sperm tail to fuel the dynein/tubulin ATPase. It is obvious that in these polar, elongated cells, the diffusional limitation of ADP is the key limiting factor with respect to high-energy phosphate provision [13]. Also in support of the PCr-shuttle model, the calculated diffusional flux of ADP in these sperm cells is by 2 and 3 orders of magnitude smaller than those of ATP and PCr, respectively [13].
In conclusion, it becomes obvious that calculations of free cellular [ADP] by using global [ATP] and [PCr], determined by in vivo 31P-NMR, together with the CK equilibrium constant, may be valid only in certain limited cases, e.g. in fast twitch glycolytic white muscle fibres, where the buffer function of CK by far prevails the transport function and where the flux through the CK reaction at rest and during high work load are higher by a factor of 100 and 20, respectively, than the total cellular ATPase turnover at these respective states. In cases where the transport function of the CK prevails, e.g. oxidative tissues or in polar cells (sea urchin sperms) with high concentrations of Mi-CK, local [ADP] and [ATP] levels, e.g. in the mitochondrial intermembrane space or near CK-ATPase complexes, may differ by orders of magnitude compared to the bulk concentrations calculated from the CK equilibrium constant. Considering the complications of subcellular compartmentation of CK isoenzymes in a cell, where after activation, some CK will work in the forward and some in the reverse direction, the interpretation of global CK flux measurements may also represent a rather difficult endeavour.

The importance of creatine kinase for calcium homeostasis and muscle contraction:

Transgenic CK(-/-) double knock-out mice show significantly increased relaxation times of their limb muscles, altered Ca2+-transients in myotubes after stimulation, as well as remarkable remodelling of the contractile apparatus with increased numbers of mitochondria and grossly over-produced tubular SR membranes [14]. The obvious difficulties of these mice with muscle Ca2+-handling, as the main phenotype, is in line with biochemical and functional data showing that some MM-CK is specifically associated with SR membranes [15], where it is crucial for fueling the energetically highly demanding Ca2+-ATPase [15,16,17]. The strong dependence of Ca2+ regulation by the SR on the supply of ATP via endogenous SR-bound has also been confirmed very recently with mechanically skinned muscle fibers [93]. Thus, depletion of PCr may contribute to impaired SR Ca2+-regulation known to occur in inteact skeletal muscle under conditions of fatigue. Therefore, one of the most crucial function of the CK-system in muscle seems to be related to the energetics of Ca2+-homeostasis [6].
In addition, some CK is also associated with the myofibril [1]. The domain responsible for the isoenzyme-specific binding of MM-CK to the myofibrillar M-band has been localized by an in situ biochemical approach, using heterologously expressed, fluorescently labelled site-directed mutants, as well as M/B-CK chimaeras for diffusion into chemically skinned skeletal muscle fibers [18]. This M-band interaction domain could be narrowed down to two "charge-clamps", symmetrically organized on a exposed face of each M-CK monomer [80]. Using the same approach to study the weak MM-CK binding to the myofibrillar I-band, observed by in situ immunofluorescence localization, we found that MM-CK binding to this sarcomeric region is mediated by some glycolytic enzymes [19].

AMP-activated protein kinase a ratiometric PCr/Cr energy sensor at last:

According to recent findings, AMP-activated protein kinase (AMPK) is able to bind rather tightly to muscle-type MM-CK and phosphorylate the latter to inhibit its activity to a certain extent. Most surprisingly, it was found by the same authors that AMPK itself is regulated not only by the ATP/AMP ratio, but also by the PCr/Cr ratio [20]. This invalidates the long-held dogma that PCr and Cr are metabolically completely inert compounds. Thus, AMPK, as an energy sensor system, could represent the missing link for regulation of adaptive metabolic changes, e.g. after depletion of creatine levels in skeletal and cardiac muscle. Interestingly enough, both the ablation of the muscle-type CK isoenzymes in transgenic animals [14] or the depletion of creatine, the substrate of the CK reaction, after supplementation with b-GPA [50], seem to elicite very similar adaptational effects in skeletal muscle. The activation of AMPK by decreasing PCr/Cr ratios and increasing [AMP], as observed during muscle activation at high work-load would lead to progressively stronger inactivation of cytosolic muscle-type MM-CK [20]. This could very well explain the long-standing enigma why, in muscle, the CK-mediated reaction flux, which can be more than 10-20-fold higher, depending on the muscle type, than the highest ATPase turnover, does not increase with higher workload, but rather tends to decrease instead [78,79].

Mitochondrial creatine kinase for metabolic channeling of high-energy phosphate compounds:

Mitochondrial creatine kinase (Mi-CK) is located in the mitochondrial intermembrane space along the inner membrane, but also at contact sites where inner and outer membranes are in close proximity [1,48]. Mi-CK can directly transphosphorylate intramitochondrially produced ATP into PCr, which subsequently is exported to the cytosol. A well documented role of Mi-CK is the functional coupling of mitochondrial CK to oxidative phosphorylation [21,22]), which facilitates the antiport of ATP versus ADP through the inner membrane via the adenine nucleotide translocator (ANT). In addition, a physical interaction of Mi-CK with outer mitochondrial membrane porin (VDAC) has also been demonstrated [23]. The solved atomic X-ray structure of octameric Mi-CK [24] is consistent with the proposed energy channeling function of this enzyme. Detailed structure/function analyses concerning the molecular physiology, catalytic site and mechanism, octamer/dimer equilibrium, as well as the interaction of Mi-CK with mitochondrial membranes have been published [21,25]. The identical top and bottom faces of the octamer contain putative membrane binding motifs likely to be involved in binding of Mi-CK to mitochondrial membranes. The central 26 " wide channel of the Mi-CK octamer may be of functional significance for the exchange of energy metabolites between mitochondria and cytosol. If Mi-CK would follow a "back door" mechanism by which PCr is be expelled into the central channel of the Mi-CK octamer, as depicted in hypothetical models (see Figs. 6A and 7 in ref. [21]), vectorial transport of PCr from the mitochondrial matrix into the cytosol could be greatly facilitated.

Exquisite sensitivity of Mi-CK to peroxynitrite, effects on cellular calcium homeostasis and linkage to pathological states:

Peroxynitrite (ONOO-, PN), the product of the reaction between nitrogen monoxide (NO) and the superoxide anion O2- has been shown to be highly reactive towards Mi-CK [26]. Recently, a mitochondrial NO synthase isoform has been discovered [27]. Thus, mitochondria as a notorious source of O2-, especially after ischemia/reperfusion episodes, additionally produce PN internally. We have found that Mi-CK in intact mitochondria is a prime target of inactivation and modification by PN, at concentrations of PN that are much lower than those needed for inactivation of mitochondrial respiratory chain enzymes [26]. The pronounced sensitivity of Mi-CK towards reactive oxygen species (ROS), especially peroxynitrite, may explain the effects seen after perturbation of cellular pro-oxidant/antioxidant balance, e.g. after ischemia/reperfusion. These effects include energy failure, paralleled by elevated ADP levels and chronic calcium overload due to inactivation of the CK system. Perfusion of hearts with NO donors lead to an inhibition of cardiac CK by 65% and a concomitant decrease in heart contractile reserve [28]. Stimulation of inducible NO-synthase (NOS), which is indeed increased in vivo in skeletal muscle biopsies from patients with chronic heart failure [29], also leads to a NO-dependent depression of cardiac function [30]. Thus, a correlation between a compromised CK system and energy failure of the heart becomes obvious.
Most recently, we found that PN is also affecting the oligomeric state of Mi-CK. PN-treatment of Mi-CK octamers leads to some dimerisation, whereas treatment of dimeric Mi-CK with the same reagent prevents reoctamerization of Mi-CK dimers in a PN-concentration dependent manner [31]. These findings may explain why in different models of cardiac infarction, one consistently detects a significantly enhanced proportion of Mi-CK dimers as compared to in non-infarcted heart tissue [81].
The results that cytosolic CK?s, and therefore also SR-bound MM-CK, which is functionally coupled to the SR-Ca2+-pump [15-17,93], are also very sensitive to reactive oxygen species (ROS) as well [32,33], indicate that impairment of the CK system by ROS would severely disturb cellular Ca2+-handling and homeostasis. As a consquence of cellular Ca2+-overload, resulting among other factors in a break-down of mitochondrial membrane potential, mitochondria may release additional Ca2+ into the cytosol [34], thus aggravating the situation even more [35]. The interaction of elevated Ca2+-levels and raise in [ROS] would then lead into a vicious cycle with progressive inactivation of both Mi-CK and SR-bound MM-CK. Therefore, the destabilization of cellular energetics by chronic exposure to ROS, thought to occur in many neuromuscular diseases [36], may finally lead to apoptosis or cell death, especially in those cells with high mitochondrial activity. Skeletal muscle and cardiac or neuronal cells are ideal candidates as chronically elevated Ca2+-levels or Ca2+-overload has been identified as a major player of cell destruction [36]. A clear link between chronically elevated Ca2+-concentration and a calcineurin-dependent signalling pathway, eventually leading to cardiac hypertrophy and chronic heart failure has been demonstrated very recently [35]. In accordance with the CK/ Ca2+-connection, in brain, the concentration of CK was found to be very high in those cells that display high-frequency Ca2+-spiking, e.g. cerebellar Purkinje neurons, as well as granule and pyramidal cells of the hippocampus [37]. A most recent finding, showing that in neurodegenerative diseases, like Alzheimer?s disease, CK enzyme activity is severely reduced and cytosol-membrane partitioning is aberrant [38], also corroborates the imporant role of the CK/PCr-system in the energetics of brain pathology.

Involvement of Mi-CK and CK substrates in mitochondrial permeability transition and early apoptosis:

A protein complex containing ANT and mitochondrial porin has recently been described to display the characteristics of the mitochondrial permeability transition pore (MTP) or mega-channel [39]. The physical interaction and functional coupling of Mi-CK with porin and ANT indicates an involvement of Mi-CK in the regulation of MTP, since octameric Mi-CK [1] in this protein complex [23,39,40], plus creatine or creatine analogues, can delay MTP [41]. This has been demonstrated by using transgenic mice that express Mi-CK in liver. Since liver of wild-type animals do not contain this enzyme, but otherwise are identical, mitochondria >from wt livers serve as an ideal control. Our experiments provided exciting new evidence that Mi-CK is not only involved in mitochondrial energy transfer and shuttling of high-energy phosphate, but may also participate directly in mitochondrial permeability transition (MPT) [41]. The Ca2+-dependent increase of inner membrane permability to ions and solutes is dependent on the transmembrane potential difference, matrix pH, SH-group reactants and is modulated by a variety of effectors. Cyclosoporin-A turned out to be a very potent inhibitor of MPT [42]. Interestingly, creatine or cyclo-creatine delayed cyclosporin-A-sensitive swelling and inhibited concomitant increase of state-4 respiration of mitochondria from Mi-CK-containing transgenic livers [41]. No comparable effect was seen with control liver mitochondria that do not contain any CK. This novel Mi-CK-related phenomenon deserves further attention since it may shed some new light on the recently observed neuroprotective effects of creatine and its analogues in animals models [43,44,85].
In addition, protein complexes, containing octameric Mi-CK, porin and ANT, could be isolated >from detergent solubilized rat brain extracts [39,40]. After reconstitution into malate-loaded lipid vesicles, the presence of octameric Mi-CK prevented Ca2+-induced malate release, which, however, was observed after dimerization of Mi-CK [41]. The fact that highly purified ANT, functionally reconstituted as ATP/ADP exchange carrier, displayed a Ca2+-dependent release of internal substances, while atractyloside or HgCl2, both induced unspecific pore opening of ANT, indicate that ANT is capable of adopting a pore-like structure under conditions known to induce MPT [45]. Mi-CK has been shown to be functionally coupled to ANT (for review see [1, 22, 46, 47] and to form complexes with porin and ANT [40]. Therefore, it is obvious that Mi-CK octamers could directly affect this ANT-mediated permeability transiton. Thus, the arrangement of Mi-CK as an energy channeling unit sandwiched in between porin and ANT and linking OM and IM together, seems not only important for high-energy phosphate conversion and transport (see Figure 1), but the molecule may also act as a protective regulatory component of the permeability transition complex. Depending on the cellular energy state and intracellular [Ca2+], octameric Mi-CK may prevent MTP [48], an early event in the execution of apoptosis [49] in cells with high energy demands, thus sparing the cells from- or delaying cell death. On the other hand, dimerization of the Mi-CK octamer may allow the ANT to switch to its MTP-like state [48], eventually leading to apoptosis.

Enhancement of physical performance by creatine supplementation:

The CK/PCr system is now recognized as an important metabolic regulator during health and disease. Creatine, synthesized in part by the body, but also ingested by food, especially meat and fish (for review see [50]), is taken up into cells by a creatine transporter (CreaT) (for review see [51]). Creatine supplementation in humans leads to an increase in intracellular [Cr] and [PCr], concomitantly improving anaerobic performance of muscle [52,53], shortens muscle relaxation time [83], increases fat free- or lean body mass [94] as well as the cross-sectional area (fiber diameter) of all muscle fiber types [93]. In addition, creatine seems to improve recovery after exhaustive excercise [54] (for review see [55,56]). One could show that creatine supplementation may also have beneficial effects for high-intensity, aerobic long-endurance exercise [57]. In a double-blinded placebo-controlled study, 20 highly trained top athletes were subjected at 1?650 meters above sea level (in Davos, Switzerland) to a series of spiro-ergometric short- and long-term performance tests before and after 10 days of supplementation with 3x3.3 g of Cr per day. In accordance with earlier studies, short performance and maximal work output were both improved by approx. 30 Watt. In a 1 hour spiro-ergometric test at 85% power output of the individually determined anaerobic threshold, the Cr group was able to perform, after Cr supplementation, at the same level of exercise with a significantly lower heart rate (-8.4 beats/min) than before Cr intake. In this group, lactate levels were lower by 0.48 mM/l and Borg scale numbers by 1.35 points. These effects were not observed in the controls. Ventilation, VO2 and respiratory quotient (RQ) were basically unchanged [57]. The effects of Cr on endurance performance seem to be due to increased efficiency of energy utilization by heart and skeletal muscle which may be related to the involvement of CK in the energetics of Ca2+-homeostasis. As a consequence of creatine supplementation, the elevated cellular PCr level is likely to increase the supply of the SR-Ca2+-ATPase with high-energy phosphates via the coupled CK reaction and thus would also increase the efficiency of Ca2+-pumping and delay impaired Ca2+-regulation known to occur under conditions of fatigue [93]. During long-endurance exercise, this process consumes a significant proportion of the available bioenergy. In addition, Cr-stimulated respiration and enhanced resynthesis of PCr after creatine ingestion [54] and/or the recently discovered control of AMP-activated protein kinase by the PCr/Cr ratio [20] and its effects on CK and lipid metabolism in general [20] could be important factors leading to the observed improvement of aerobic exercise described above.
An important new aspect of creatine supplementation was descovered only recently, that is, creatine supplementation in combination with carbohydrate loading after submaximal glycogen-depleting exercise not only markedly improves Cr uptake, but also increases glycogen accumulation in human muslcle [96]. Thus, the highly elevated levels of glycogen reached after combined carbohydrate and creatine loading after glycogen-depleting exercise may, of course, also add to the positive effect of creatine supplementation on long-endurance exercise [57].

Down-regulation of the creatine transporter after chronic creatine ingestion:

The creatine transporter (CreaT), responsible for the uptake of creatine into a variety of tissues and cells, was detected in rat skeletal and cardiac muscle, cerebellum, forebrain and kidney. Two polypeptides with an apparent Mr of 70 kDa and 55 kDa were always recognized by both of our specific polyclonal antibodies directed against synthetic peptides of either the NH2- or the COOH-terminus of CreaT, indicating a high degree of homology between the two proteins [51]. In contrast to published data obtained by Northern blot analysis, suggesting a complete absence of CreaT mRNA message in liver, we could clearly detect both CreaT polypeptides also in rat liver and hepatocyte lysates. In support of this, cultured hepatocytes show an endogenous CreaT activity which is antagonized by the creatine analogue, b-guanidino propionic acid (b-GPA), a well known inhibitor of CreaT. Glyco-staining of CreaT, enriched by immuno-affinity chromatography, mainly containing both the 70 and 55 kDa bands, showed strong glycosylation of preferentially the upper 70 kDa polypeptide indicating that the latter is a posttranslationally modified form of the 55 kDa core protein. HeLa cells transfected with rat CreaT cDNA showed an increase in [14C]-creatine uptake, when compared to control cells, that was antagonized by b-GPA. In parallel, an increase in the expression of both the 70 and the 55 kDa polypeptides over endogenous CreaT of controls was noticed on Western blots. Furthermore, we have found that chronic creatine supplementation of rats, at very high dosage, down-regulates in vivo the expression and/or accumulation of the CreaT in skeletal muscle, but not in brain and heart [58]. Although the amounts of creatine taken by athletes, 20 grams / day during a 10 days loading phase and 5 grams as a maintenance dose during the following three months (amounting to approximately 0.1 gram of Cr /kg body weight/ day), is significantly lower than the amounts given in the above experiments to the rats (approximately 0.5 grams /kg body weight /day), the finding made with laboratory animals nevertheless may have consequences with respect to creatine supplementation schedules for humans. In the future, however, detailed studies on humans are needed to optimize the creatine supplementation schedules in use with respect to the observed down-regulation of CreaT expression and/or accumulation in animal experiments. According to most recent results, using "normal" Cr supplementation schedules with humans, CreaT seems also to be down-regulated, especially in combination with exercise (Greenhaff et al. unpublished), but, over the time course of this human trial, creatine transporter function did not seem to become a limiting factor for maintaining normal intracellular creatine levels. Nevertheless, as suggested earlier [86], a one month pause, after three months of continuous creatine supplementation, would still seem to be a reasonable thing to do.
With respect to cardiac pathology, a down-regulation of creatine transporter protein expression has recently been shown in experimental animal models of heart disease, as well as in failing human myocardium [91], indicating that the generally lowered PCr and Cr levels measured in failing hearts are related to down-regulated creatine transporter capacity. Thus, creatine supplementation, by improving cellular energetics, may also turn out to be beneficial for certain heart diseases.

Creatine supplementation as an adjuvant therapy for neuromuscular diseases:

Creatine seems helpful not only for athletes to improve physical performance on different levels (see above), but is also emerging as a therapeutic aid for neuromuscular and neurodegenerative diseases [85]. In some of these diseases, especially in mitochondrial myopathies, a compensatory over-expression of Mi-CK, due to cellular energy deficit, can lead to the formation of pathological intramitochondrial crystalline Mi-CK inclusions [59], that, at least in the b-GPA-animal model, disappear completely uponadministration of creatine [60].
A protective effect of creatine on neuronal function, especially during hypoxia or anoxia has been described already some years ago first on brain slices [61,62]. Only recently, encouraged by the success of creatine supplementation for improvement of muscle performance in humans, have creatine and analogues attracted new interest for brain metabolism [63,64,65]. In animal models, creatine, as well as the creatine analog, b-GPA, was shown to remarkably protect the brain of mice >from hypoxic damage and seizures in vivo [64,84] and significant neuroprotective effects of creatine and cyclocreatine have been described in an animal model of Huntington?s disease [44], as well as for Parkinsonism [66]. Creatine and cyclocreatine afforded significant protection against malonate, as well as 3-nitropropionic acid (3-NP) lesions and ROS generation in the brain. Most recently, very remarkable neuro-protective effects have been reported in an animal model of ALS, where 1% and 2% creatine in the food significantly increased life span of FALS mice in a dose-dependent manner and also delayed motor neuron degeneration as measured by rotorod performance [85]. The observed neuroprotective effects would be fully in line with the high expression levels and the specific localizations of CK isoenzymes in brain, both regionally [37] and on a cellular level [67], as well as functionally during brain development and maturation [70] or in the adult brain [68,69].
The above neuroprotective effects are paralleled also with astonishing findings in transgenic mice expressing BB-CK in liver, which normally is devoid of CK activity. Livers of such mice become highly resistant to hypoxia [71] and liver toxins [72]. In addition, CK and creatine, improving the intracellular phosphorylation potential of these transgenic livers, confer protection of ATP levels and stabilization of pH during a fructose load [73]. Most recently, creatine supplementation of dystrophic muscle cells from mdx mice was shown to result in a marked cell protection, after a challenge by either hypo-osmotic swelling or high extracellular [Ca2+], against chronically elevated calcium levels seen in untreated control cells [74]. Promising preliminary results and favourable subjective feed-back responses with patients suffering from different neuromuscular diseases [75,86] have stimulated controlled double-blinded clinical studies. Thus, the validity of creatine supplementation as a possible adjuvant therapy for neuromuscular and neurodegenerative diseases is currently being tested. The first controlled clinical studies with patients have been published [87,88] and some are about to appear [89,90], all showing a rather positive outcome.
A bright future can be foreseen for creatine as a nutritional supplement for healthy people, elderly and reconvalescent, and for vegetarians on one hand [86], as well as an adjuvant therapeutic aid for a plethora of new medical applications [94]. Finally, for some cases, creatine and its analogues will be used in the future for full-fletched pharmaceutical intervention, e.g. for treating inborn errors of creatine metabolism [76] or for anti-cancer therapy [77].

Acknowledgments:

This work was supported by the Swiss National Science Foundation, the Swiss Society for Muscle Diseases, the ETH-Z and privat sponsoring >from Careal Holding, Benni &Co parents association Germany, Swiss Cancer Foundation, Innerschweizerische Krebsliga.

The PCr-circuit: a temporal and spatial energy buffering network and regulatory system for energy metabolism in cells with intermittently high energy requirements.

Upper, cytosolic side: the bulk of soluble, cytosolic CK (CKc) equilibrates global ATP/ADP and PCr/Cr ratios by its equilibrium reaction (depicted in the right middle of the figure). In skeletal muscle at rest, these metabolite levels are approximately 3-5 mM/10-20 µM and 20-40 mM/10-15 mM, respectively (see [1,22,47]). One of the main functions of CKc is to keep the concentration of free global ADP very low and thus to maintaing global [ATP] remarkably stable also during cell activation. This part of the PCr-circuit model represents the classical textbook function of CK as a temporal energy buffer, being backed up by adenylate kinase as a second safeguard against declining ATP and rising ADP levels. Some of the cytosolic CKc is functionally coupled to glycolysis and, during periods of anaerobic work-output and recovery, preferentially accepts glycolytic ATP to replenish the very large PCr pool (ATP from glycolysis, depicted in the left middle of the figure). Additionally, however, some fractions of cytosolic CK, are very specifically associated (CKa) with ATP requiring processes at sites of energy consumption. For example, CKa is associated with the contractile apparatus and the sarcoplasmic reticulum, where it forms functionally coupled microcompartments with the acto-myosin ATPase and the SR-Ca2+-ATPase, respectively, or with other ATP requiring processes, like the Na+/K+-ATPase etc. (see top of figure). There, ATP is directly regenerated in situ by CKa via PCr, thus keeping local ATP/ADP ratios very high in the immediate vicinity of these ATPases.

CK is phosphorylated and down-regulated in its activity by AMP-dependent protein kinase (AMPK, top right), which itself is the first enzyme that has been found to be regulated by the PCr/Cr ratio, that is, AMPK is activated by high creatine versus PCr levels [20].
Lower mitochondrial side: mitochondrial Mi-CK is bound to the outer side of the inner mitochondrial membrane (IM) and localized along the cristae membranes, as well as at mitochondrial contact sites, where IM and OM are in close vicinity [48]. At these sites, Mi-CK octamers are forming microcompartments with porin (P) and adenine nucleotide translocase (ANT) for energy transfer >from ATP to Cr, followed by vectorial transport of PCr into the cytosol. ATP generated by oxidative phosphorylation is preferentially accepted by Mi-CK octamers, transphosphorylated onto Cr, which is entering through mitochondrial porin (P, or VDAC), to give PCr which then is exported into the cytosol. Thus, under high work-load, PCr would be shuttled from mitochondria to sites of energy consumption (ATPases, top of figure), where it is then used

by CKa to regenerate ATP locally in situ to fuel these ATP-requiring processes and to keep local ATP/ADP ratios very high. Cr would diffuse back to the mitochondria to be recharged again. This part of the model represents the spatial buffering function of the PCr-circuit. In this model, the specifically localized CK isoenzymes at sites of energy consumption and energy production are connected via PCr and Cr as mediators, generating metabolic waves and dampening oscillations of metabolites [22,46].

The dynamic recruitment of either free or membrane-bound Mi-CK octamers (double-arrows 5 or 1, respectively), possibly depending on the metabolic state of the mitochondria, the dynamic octamer/dimer equilibrium of Mi-CK (double arrows 2 and 4), as well as octamerization of Mi-CK dimers bound on the IM (double-arrow 2), all observed in vitro, are schematically visualized as potential modulatory events for long-term metabolic regulation. The interaction of Mi-CK with porin and complex formation of the enzyme with ANT, most likely facilitated by cardiolipin associated with ANT, are also illustrated. Under the conditions expected to prevail in the mitochondrial intermembrane space, however, the equilibria of these reactions, as observed in vitro, would clearly favour the membrane-bound octamer [21,25]. However,since the formation of contact sites and the establishment of the protein complexes are thought to be rather dynamic, a on/off recruitment of Mi-CK octamer into contact sites could easily be envisaged. Finally, these events that are heavily influenced by the exquisite sensitivity of Mi-CK towards peroxynitrite and other ROS [26], may be relevant also for the control of the permeability transition pore [39-41, 45].

Figure 1:

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