Muscle unloading induces slow to fast transitions in myofibrillar but not mitochondrial properties. Relevance to skeletal muscle abnormalities in heart failure

Muscle deconditioning is a common observation in patients with congestive heart failure (CHF), chronic obstructive pulmonary disease, neuromuscular diseases or prolonged bed rest. To gain further insight into metabolic and mechanical properties of deconditioned slow-twitch (soleus) or fast-twitch (EDL) skeletal muscles, we induced experimental muscle deconditioning by hindlimb suspension (HS) in rats for 3 weeks. Cardiac muscle was also studied. Besides profound muscle atrophy, increased proportion of fast type II fibers as well as fast myosin isoenzymes, we found decreased calcium sensitivity of Triton X-100 skinned fiber bundles of soleus muscle directed towards the fast muscle phenotype. Glycolytic enzymes such as hexokinase and pyruvate kinase were increased, and the LDH isoenzyme pattern was clearly shifted from an oxidative to an anaerobic profile. Creatine kinase (CK) and myokinase activities were increased in HS soleus towards EDL values. Moreover, the M-CK mRNA level was greatly increased in soleus, with no change in EDL. However, oxygen consumption rate assessed in situ in saponin skinned fibers (12.5 +/- 0.8 in C and 15.1 +/- 0.9 micromol O2/min/g dw in HS soleus compared to 7.3 +/- 1.3 micromol O2/min/g dw in control EDL), as well as mitochondrial CK (mi-CK) and citrate synthase activities, were preserved in HS soleus. Following deconditioning no change in Km for ADP of mitochondrial respiration, either in the absence (511 +/- 92 in C and 511 +/- 111 microM in HS soleus compared to 9 +/- 4 microM in control EDL) or presence of creatine (88 +/- 10 in C and 95 +/- 16 microM in HS soleus compared to 32 +/- 9 microM in control EDL), was found. The results show that muscle deconditioning induces a biochemical and functional slow to fast phenotype transition in myofibrillar and cytosolic compartments of postural muscle, but not in the mitochondrial compartment, suggesting that these compartments are differently regulated under conditions of decreased activity.     

Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.     

Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).     

Impaired cardiac energetics in mice lacking muscle-specific isoenzymes of creatine kinase

Our purpose was to determine whether hearts from mice bioengineered to lack either the M isoform of creatine kinase (MCK-/- mice) or both the M and mitochondrial isoforms (M/MtCK-/- mice) have deficits in cardiac contractile function and energetics, which have previously been reported in skeletal muscle from these mice. The phenotype of hearts with deleted creatine kinase (CK) genes is of clinical interest, since heart failure is associated with decreased total CK activity and changes in the relative amounts of the CK isoforms in the heart. We measured isovolumic contractile performance in isolated perfused hearts from wild-type, MCK-/-, and M/MtCK-/- mice simultaneously with cardiac energetics (31P-nuclear magnetic resonance spectroscopy) at baseline, during increased cardiac work, and during recovery. Hearts from wild-type, MCK-/-, and M/MtCK-/- mice had comparable baseline function and responded to 10 minutes of increased heart rate and perfusate Ca2+ with similar increases in rate-pressure product (48+/-5%, 42+/-6%, and 51+/-6%, respectively). Despite a similar contractile response, M/MtCK-/- hearts increased [ADP] by 95%, whereas wild-type and MCK-/- hearts maintained [ADP] at baseline levels. The free energy released from ATP hydrolysis decreased by 3.6 kJ/mol in M/MtCK-/- hearts during increased cardiac work but only slightly in wild-type (1.7 kJ/mol) and MCK-/- (1.5 kJ/mol) hearts. In contrast to what has been reported in skeletal muscle, M/MtCK-/- hearts were able to hydrolyze and resynthesize phosphocreatine. Taken together, our results demonstrate that when CK activity is lowered below a certain level, increases in cardiac work become more "energetically costly" in terms of high-energy phosphate use, accumulation of ADP, and decreases in free energy released from ATP hydrolysis, but not in terms of myocardial oxygen consumption.     

Neurotransmitter transporters as molecular targets for addictive drugs

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Mathematical model of compartmentalized energy transfer: its use for analysis and interpretation of 31P-NMR studies of isolated heart of creatine kinase deficient mice

A mathematical model of the compartmentalized energy transfer in cardiac cells is described and used for interpretation of novel experimental data obtained by using phosphorus NMR for determination of the energy fluxes in the isolated hearts of transgenic mice with knocked out creatine kinase isoenzymes. These experiments were designed to study the meaning and importance of compartmentation of creatine kinase isoenzymes in the cells in vivo. The model was constructed to describe quantitatively the processes of energy production, transfer, utilization, and feedback between these processes. It describes the production of ATP in mitochondrial matrix space by ATP synthase, use of this ATP for phosphocreatine production in the mitochondrial creatine kinase reaction coupled to the adenine nucleotide translocation, diffusional exchange of metabolites in the cytoplasmic space, and use of phosphocreatine for resynthesis of ATP in the myoplasmic creatine kinase reaction. It accounts also for the recently discovered phenomenon of restricted diffusion of adenine nucleotides through mitochondrial outer membrane porin pores (VDAC). Practically all parameters of the model were determined experimentally. The analysis of energy fluxes between different cellular compartments shows that in all cellular compartments of working heart cells the creatine kinase reaction is far from equilibrium in the systolic phase of the contraction cycle and approaches equilibrium only in cytoplasm and only in the end-diastolic phase of the contraction cycle. Experimental determination of the relationship between energy fluxes by a 31P-NMR saturation transfer method and workload in isolated and perfused heart of transgenic mice deficient in MM isoenzyme of the creatine kinase, MM-/-showed that in the hearts from wild mice, containing all creatine kinase isoenzymes, the energy fluxes determined increased 3-4 times with elevation of the workload. By contrast, in the hearts in which only the mitochondrial creatine kinase was active, the energy fluxes became practically independent of the workload in spite of the preservation of 26% of normal creatine kinase activity. These results cannot be explained on the basis of the conventional near-equilibrium theory of creatine kinase in the cells, which excludes any difference between creatine kinase isoenzymes. However, these apparently paradoxical experimental results are quantitatively described by a mathematical model of the compartmentalized energy transfer based on the steady state kinetics of coupled creatine kinase reactions, compartmentation of creatine kinase isoenzymes in the cells, and the kinetics of ATP production and utilization reactions. The use of this model shows that: (1) in the wild type heart cells a major part of energy is transported out of mitochondria via phosphocreatine, which is used for complete regeneration of ATP locally in the myofibrils--this is the quantitative estimate for PCr pathway; (2) however, in the absence of MM-creatine kinase in the myofibrils in transgenic mice the contraction results in a very rapid rise of ADP in cytoplasmic space, that reverses the mitochondrial creatine kinase reaction in the direction of ATP production. In this way, because of increasing concentrations of cytoplasmic ADP, mitochondrial creatine kinase is switched off functionally due to the absence of its counterpart in PCr pathway, MM-creatine kinase. This may explain why the creatine kinase flux becomes practically independent from the workload in the hearts of transgenic mouse without MM-CK. Thus, the analysis of the results of studies of hearts of creatine kinase-deficient transgenic mice, based on the use of a mathematical model of compartmentalized energy transfer, show that in the PCr pathway of intracellular energy transport two isoenzymes of creatine kinase always function in a coordinated manner out of equilibrium, in the steady state, and disturbances in functioning of one of them inevitably result     

Improvement of muscular oxidative capacity by training is associated with slight acidosis and ATP depletion in exercising muscles

Metabolic and mechanical properties of female rat skeletal muscles, submitted to endurance training on a treadmill, were studied by a 60-min in vivo multistep fatigue test. 31P-NMR was used to follow energy metabolism and pH. Mechanical performance was greatly improved in trained muscles. The oxidative capacity of the skeletal muscles was evaluated from the relationship between ADP calculated from the creatine kinase equilibrium and work and from the measure of the rate of phosphocreatine (PCr) resynthesis following exercise. In trained muscles, ADP production was lower per unit of mechanical performance, showing an improvement of oxidative metabolism. However, the PCr resynthesis rate was not modified. Slight acidosis and ATP depletion were observed from the beginning of the fatigue test. These modifications suggest changes of the creatine kinase equilibrium favoring mitochondrial ATP production. Our results indicate that muscle status improvement could be accompanied by ATP depletion and minimal acidosis during contraction; this would be of particular importance for objective evaluation of muscle regeneration processes and of gene therapy in muscle diseases.     

Fluorometric studies of recovery metabolism of rat fast- and slow-twitch muscles

Recovery metabolism of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of the rat has been investigated using fluorometric monitoring of reduction of nicotinamide adenine dinucleotide (NAD). In both EDL and SOL, groups of twitch contractions produced a decrease in fluorescence (oxidation of NADH) which returned to the resting base line after contraction ceased. These responses proceeded more quickly in EDL than SOL and were abolished by anoxia. A 1-s tetanus of SOL produced an initial reduction which could be abolished with iodoacetate followed by a prolonged oxidation which could be blocked by anoxia. The fluorescence of EDL was decreased immediately following a 1-s tetanus but then rapidly increased well beyond the resting level of reduction and persisted throughout the recovery period. This reduction was largely depressed by iodoacetate. The results indicate marked differences in the recovery metabolism of these muscles, consistent with predominantly mitochondrial oxidative activity in the slow-twitch muscles and predominantly glycolytic activity in the fast-twitch muscles.     

The low-affinity dihydropyridine receptor and Na+/Ca2+ exchanger are associated in adrenal medullary mitochondria

The effect of Ca2+ channel-acting drugs on bovine adrenal mitochondria Ca2+ movements was investigated. Mitochondrial Ca2+ uptake is performed by an energy-driven Ca2+ uniporter with a Km of 20.9 +/- 3.2 microM and Vmax of 148.1 +/- 7.2 nmol 45Ca2+ min-1 mg-1. Ca2+ release is performed through an Na+/Ca2+ antiporter with a Km for Na+ of 4.2 +/- 0.5 mM, a Vmax of 7.5 +/- 0.4 nmol 45Ca2+ min-1 mg-1, and a Hill coefficient of 1.4 +/- 0.2 Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger was inhibited by several dihydropyridines (nitrendipine, felodipine, nimodipine, (+)isradipine) and by the benzothiazepine diltiazem with similar potencies. In contrast, neither CGP 28392, Bay-K-8644, amlodipine, nor verapamil had any effect on Ca2+ efflux. Nitrendipine at 20 microM modified neither the Km nor the Hill coefficient for Na+, whereas the Vmax was reduced to 2.9 nmol 45Ca2+ min-1 mg-1, thus demonstrating noncompetitive modulation of the Na+/Ca2+ exchanger. None of the Ca2+ channel-acting drugs assayed at 100 microM affected Ca2+ influx through the uniporter. Ca2+ channel blockers inhibited the Na+/Ca2+ antiporter and displaced the specific binding of [3H]nitrendipine to intact mitochondria with Ki values similar to the IC50s obtained for the inhibition of the Ca2+ efflux. Ca2+ channel-acting drugs that did not inhibit the Na+/Ca2+ exchanger (amlodipine, CGP 28392, Bay-K-9644, and verapamil, at concentrations of 100 microM or higher) had no effect on [3H]nitrendipine binding. These results suggest that the adrenomedullary mitochondrial dihydropyridine receptor is associated with the Na+/Ca2+ exchanger.     

Role of reactive oxygen species and poly-ADP-ribose polymerase in the development of AZT-induced cardiomyopathy in rat

The short term cardiac side-effects of AZT (3'-azido-3'-deoxythymidine, zidovudine) was studied in rats to understand the biochemical events contributing to the development of AZT-induced cardiomyopathy. Developing rats were treated with AZT (50 mg/kg/day) for 2 wk and the structural and functional changes were monitored in the cardiac muscle. AZT treatment provoked a surprisingly fast appearance of cardiac malfunctions in developing animals characterized by prolonged RR, PR and QT intervals and J point depression. Electron microscopy showed abnormal mitochondrial structure but the cardiomyocyte had normal myofibers. The AZT treatment of rats significantly increased ROS and peroxynitrite formation in heart tissues as determined by the oxidation of nonfluorescent dihydrorhodamine123 and dichlorodihydro-fluorescein diacetate (H2DCFDA) to fluorescent dyes, and induced single-strand DNA breaks. Lipid peroxidation and oxidation of cellular proteins determined from protein carbonyl content were increased as a consequence of AZT treatment. Activation of the nuclear poly-ADP-ribose polymerase and the accelerated NAD+ catabolism were also observed in AZT-treated animals. Western blot analysis showed that mono-ADP-ribosylation of glucose regulated protein (GRP78/BIP) was enhanced by AZT treatment, that process inactivates GRP78. In this way moderate decrease in the activity of respiratory complexes was detected in the heart of AZT-treated animals indicating a damaged mitochondrial energy production. There was a significant decrease in creatine phosphate concentration resulting in a decrease in creatine phosphate/creatine ratio from 2.08 to 0.58. ATP level remained close to normal but the total extractable ADP increased with 45%. The calculated free ATP/ADP ratio decreased from 340 to 94 in the heart of AZT-treated rats as a consequence of increased free ADP concentration. It was assumed that the increased free ADP in AZT-treated cardiomyocyte may help cells to compensate the defective ATP production in damaged mitochondria by activating the ATP synthesis in undamaged mitochondria. Southern blot analysis did not show decreased quantity of mtDNA deriving from AZT-treated rat hearts indicating that under our experimental conditions AZT-induced heart abnormalities are not the direct consequence of the mtDNA depletion. These data show that ROS-mediated oxidative damages, activated ADP-ribosylation reactions and accelerated NAD+ catabolism play basic roles in the development of AZT-induced cardiomyopathy in our animal model and indicated that these ROS-mediated processes can be important factors in the development of myopathy and cardiomyopathy in zidovudine-treated AIDS patients.     

A comparison of the electrophysiological effects of two organophosphates, mipafox and ecothiopate, on mouse limb muscles

Adult male albino mice were given single subcutaneous injections of either mipafox (110 mumol/kg) or ecothiopate (0.5 mumol/kg), two organophosphorus compounds (OPs). Acetylcholinesterase activity was measured in the soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles. At 7 and 28 days after dosing, in vitro electrophysiological measurements were carried out in the soleus and EDL. Action potentials and end-plate potentials were evoked at 30 Hz and recorded intracellularly from single muscle fibers. The amplitudes, time course, and latencies of these potentials were measured and the variability (jitter) of latencies was calculated. Recordings after mipafox were also made with 3-Hz stimulation. Acetylcholinesterase activity was inhibited by mipafox (65% in the soleus; 76% in the EDL) and ecothiopate (59% in the soleus; 42% in the EDL). Mipafox and ecothiopate both increased postjunctional (muscle action potential) jitter in the soleus and EDL at 7 days after dosing. Organophosphates caused an increase in end-plate potential amplitudes in the soleus. Mipafox caused an increase in prejunctional (end-plate potential) jitter at 28 days after dosing in both muscles. A single dose of ecothiopate also caused an increase in prejunctional jitter at 28 days in the soleus. The OP-induced increase in jitter was different at different frequencies of stimulation. The results show that there are electrophysiological changes in both muscles after administration of organophosphorus compounds. The slow-twitch soleus appears more sensitive to prejunctional changes caused by OPs than the fast-twitch EDL.     

Abnormal mitochondrial function in myocardium of dogs with chronic heart failure

Chronic heart failure (HF) is associated with morphologic abnormalities of cardiac mitochondria that include hyperplasia, reduced organelle size and compromised structural integrity. In the present study, we examined mitochondrial respiration in myocardium of 10 normal dogs and 10 dogs with chronic HF (LV ejection fraction 24+/-2%) produced by intracoronary micro-embolizations. Mitochondrial respiratory rates were determined using a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Basal respiratory rate (VO), respiratory rate after addition of substrates, glutamate and malate (VSUB) and state 3 respiratory rate (VADP, after addition of ADP), were measured in tissue samples from the subendocardial and subepicardial LV free wall, interventricular septum and right-ventricular free wall. No differences were observed in basal respiratory rates between normal and HF tissue, while VSUB was significantly lower in HF compared to normal. VADP was 50-60% lower in HF compared to normal tissue (P<0.001). The results indicate abnormal mitochondrial respiratory activity in myocardium of dogs with chronic HF. These findings support the concept of low myocardial energy production in HF that can contribute to the global cardiac dysfunction.     

Early ischemia-induced alterations of the outer mitochondrial membrane and the intermembrane space: a potential cause for altered energy transfer in cardiac muscle?

Our aim was to carefully analyse the time-dependent changes that affect the mitochondrial function of myocardial cells during and after an ischemic episode. To this end, variables characterizing mitochondrial function have been evaluated on myocardial samples from isolated rat hearts subjected to different conditions of ischemia. The technique of permeabilized fibers was used in order to evaluate the mitochondrial function whilst retaining intracellular structure. The earliest alteration that could be detected was a decrease in the stimulatory effect of creatine on mitochondrial respiration. This alteration became more pronounced as the severity (or duration) of the ischemia increased. Afterwards, a significant decrease in the apparent Km of mitochondrial respiration for ADP also appeared, followed by a diminution of the maximal respiration rate which was partly restored by adding cytochrome c. Finally, for the most severe conditions of ischemia, the basal respiratory rate also increased. These observations are indicative of a sequence of alterations affecting first the intermembrane space, then the outer mitochondrial membrane, and finally the inner membrane. The discussion is focused on the very early alterations, that could not be detected using the conventional techniques of isolated mitochondria. We postulate that these alterations to the intermembrane space and outer mitochondrial membrane can induce disturbances both in the channelling of energy from the mitochondria, and on the signalling towards the mitochondria. The potential consequences on the regulation of the production of energy (ATP, PC) by the mitochondria are evoked.     

Urban-rural difference in cereal consumption by people in Shandong Province, China

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10-100 microM) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2.5-50 microM) increased (two-fold at 20-50 microM) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0.5-5.0 mM), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2.5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 microM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies.     

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.     

Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/myofibril system in muscle

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters phi and psi that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible 'coupling' are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when phi or psi is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when psi or psi is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless phi = psi = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the 'adenine nucleotide flux' at the ends of the model into 'creatine flux' in the middle; the overall net flux is small, but only zero if phi = psi. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.     

On the involvement of calpains in the degradation of the tumor suppressor protein p53

A method for non-invasive, quantitative 31P NMR spectroscopic investigation of mitochondrial function in human skeletal muscle in situ is described. High time resolution 31P NMR measurements of phosphocreatine, inorganic phosphate and ATP resonances were conducted on human forearm flexor muscle during involuntary twitch contraction at eight different frequencies. Mitochondrial and glyco(geno)lytic ATP synthesis fluxes, and the cytosolic free energy of ATP hydrolysis (delta GP), were calculated at incremental steady-states of energy balance. The covariation of mitochondrial ATP synthesis flux, JPMOP, and delta GP was quasi-linear over the physiological range of free energy values. Curve-fit analysis of the covariation yielded a maximal sustainable JPMOP of 0.24 +/- 0.06 mmol ATP l-1.s-1 and a midpoint potential, (delta GP)0.5, of 58.1 +/- 1.2 kJ/mole in the muscle cells.