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.     

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.     

AZT decreases rat myocardial cytochrome oxidase activity and increases beta-myosin heavy chain content

AZT, a widely-utilized drug for the treatment of HIV infection, inhibits the polymerase responsible for mitochondrial DNA replication (mtDNA). The aim of this study was to assess myocardial alterations caused by this action. Ventricular muscle from rats treated for > or = 35 days with 1 mg/ml of AZT in their drinking water was analysed for cytochrome oxidase activity and the content of mRNAs for the nuclear-encoded cytochrome oxidase (COX) subunit VIc and the mitochondrial-encoded COX subunit III. In addition contractile protein expression was assessed by examining mRNA levels for alpha- and beta-myosin heavy chains (MHC). Changes in MHC mRNA levels were correlated with changes in alpha- and beta-MHC proteins and changes in myofibrillar ATPase activity. Results show that AZT caused a reduction in COX activity, COX subunit III mRNA, and mtDNA levels. There was no decrease in the COX subunit VIc mRNA. MHC expression was altered such that the relative content of beta-MHC protein and mRNA were increased. Accumulation of beta-MHC was reflected in the reduction of myofibrillar ATPase activity at pCa values of 5.875 and 6.125. These data demonstrate that AZT induces a reorganization of cardiac gene expression indicative of changes in cardiac contractile properties. The observed decreases in mtDNA levels along with mRNA for a mitochondrial-encoded protein and COX activity is consistent with the postulated mechanism whereby AZT induces a myopathy by diminishing mtDNA replication.     

Effect of creatine phosphate on the contractile activity in acutely failing rat heart

The hypothesis was tested that infusion of a solution containing creatine phosphate (CP) into rats with acutely failing hearts would enhance recovery of cardiac function. The acutely failing heart was produced by constricting the ascending aorta. This overload produced failure in approximately 25 min. At the point of failure the constriction was removed and solutions containing sterile physiological saline (PSS), PSS and CP, PSS and creatine, or PSS and creatine plus phosphate were infused. Cardiac function was assessed from systolic and diastolic blood pressure, +/- dp/dt, heart rate, and cardiac work. Ca2+ uptake by isolated sarcoplasmic reticulum and the concentrations of selected blood and tissue metabolites were measured. Normal cardiac function was restored in the PSS-CP infused rats whereas all other treatments did not restore cardiac function. Adenosine triphosphate and CP had declined in the myocardium of the failing hearts while lactate was elevated. The concentrations of these metabolites were normal in the PSS-CP infused animals. The glycogen concentration in the myocardium was reduced following the constriction. Ca2+ uptake by isolated sarcoplasmic reticulum was depressed in the failed hearts but normal in the hearts of CP-infused animals. These results demonstrate that the infusion of CP into animals with failing hearts can be effective in restoring cardiac function.     

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     

A deletion of mitochondrial DNA in murine doxorubicin-induced cardiotoxicity

A deletion of about 4 kb has been determined in the mutated mitochondrial DNA (mtDNA) in cardiomyocytes with chronic doxorubicin (DOX)-induced cardiotoxicity in mouse. The incidence of the mtDNA deletion increased with the dosage and with the duration of the DOX administration. Coenzyme Q10 administration prevented the mtDNA deletion and decreased the thiobarbituric acid reactive substance content in the heart mitochondria, suggesting some free radical involvement in this mtDNA deletion. This mtDNA deletion may be involved in cardiomyopathy, which is known to be dosage-dependently induced by DOX administration.     

Synchronization of cells in the S phase of the cell cycle by 3'-azido-3'-deoxythymidine: implications for cell cytotoxicity

The mechanism of synergy between 3'-azido-3'-deoxythymidine (AZT) and anticancer agents was investigated with emphasis on cell-cycle events. Exposure of exponentially growing WiDr human colon carcinoma cells to AZT resulted in synchronization of cells in the S phase of the cell cycle. Following treatment with AZT at 50 or 200 microM, 62% +/- 3% or 82% +/- 4% of the cells were in the S phase as compared with 36% +/- 2% in the control. Bromodeoxyuridine uptake studies revealed that the synchronized cells actively synthesized DNA. At concentrations of up to 200 microM, AZT produced a cytostatic rather than cytotoxic effect as indicated by viability and cell growth measurements. At 200 microM, AZT-induced synchronization was significant (P = < 0.001) after 12 h of drug exposure, reached a maximum at 24 h, and reversed to baseline levels by 72 h even in the continued presence of the drug. This indicates that AZT-induced cytostasis is a transient and reversible effect. The cell-cycle events seen with AZT in WiDr cells were also observed in eight of nine human tumor cell lines tested. Isobologram analysis of WiDr cells preexposed to AZT for 24 h and then exposed to either AZT-5-fluorouracil or AZT-methotrexate for a further 72 h revealed synergy between AZT and the anticancer agents, indicating that AZT-induced synchronization may have therapeutic benefits.     

Rat platelet aggregation by ATP. Aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen

This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets; it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase system caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometry of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalicylic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules.     

Diabetes-induced changes in lens antioxidant status, glucose utilization and energy metabolism: effect of DL-alpha-lipoic acid

The study was aimed at evaluating changes in lens antioxidant status, glucose utilization, redox state of free cytosolic NAD(P)-couples and adenine nucleotides in rats with 6-week streptozotocin-induced diabetes, and to assess a possibility of preventing them by DL-alpha-lipoic acid. Rats were divided into control and diabetic groups treated with and without DL-alpha-lipoic acid (100 mg x kg body weight(-1) x day(-1), i.p.). The concentrations of glucose, sorbitol, fructose, myo-inositol, oxidized glutathione, glycolytic intermediates, malate, alpha-glycerophosphate, and adenine nucleotides were assayed in individual lenses spectrofluorometrically by enzymatic methods, reduced glutathione and ascorbate--colorimetrically, and taurine by HPLC. Free cytosolic NAD+:NADH and NADP+:NADPH ratios were calculated from the lactate dehydrogenase and malic enzyme systems. Sorbitol pathway metabolites were found to increase, and antioxidant concentrations were reduced in diabetic rats compared with controls. The profile of glycolytic intermediates (increase in glucose 6-phosphate and fructose 6-phosphate, decrease in fructosel,6-diphosphate, increase in dihydroxyacetone phosphate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and no change in lactate), and 5.9-fold increase in alpha-glycerophosphate suggest diabetes-induced inhibition of glycolysis. Free cytosolic NAD+:NADH ratios, ATP levels, ATP/ADP x inorganic phosphate (Pi), and adenylate charge were reduced in diabetic rats while free cytosolic NADP+:NADPH ratios were elevated. Diabetes-induced changes in the concentrations of antioxidants, key glycolytic intermediates, free cytosolic NAD+:NADH ratios, and energy status were partially prevented by DL-alpha-lipoic acid, while sorbitol pathway metabolites and free cytosolic NADP+:NADPH ratios remained unaffected. In conclusion, diabetes-induced impairment of lens antioxidative defense, glucose intermediary metabolism via glycolysis, energy status and redox changes are partially prevented by DL-alpha-lipoic acid. The findings support the important role of oxidative stress in lens metabolic imbalances in diabetes.     

Chronic exposure of rats to hypoxic environment alters the mechanism of energy transfer in myocardium

We have investigated the effect of chronic exposure of rats to an hypoxic environment (10% O2; 3 weeks), on the first step of the intracellular energy transfer process in the myocardium, i.e. the transfer at mitochondrial level of high energy bonds from ATP to creatine. In the left ventricles from rats adapted to normobaric hypoxia, we observed, using the permeabilized fiber technique, that the stimulatory effect of creatine on the mitochondrial respiration in presence of a low ADP concentration (0.1 mM) was attenuated when compared to control. Furthermore, the creatine-induced decrease of the apparent K(m) for ADP of the mitochondrial respiration, which is observed in control, was significantly reduced. Both the basal and maximal respiratory rates of the fibers were unchanged by the hypoxic exposure of the rats. A significant decrease of the total creatine kinase activity from 755 to 630 IU/g wet weight (for control and hypoxic rats, respectively) was detected and was accompanied by a 25% decrease in mitochondrial isoform activity (mitoCK) and in the mitoCK/citrate synthase ratio. In the right ventricles, identical alterations in the effect of creatine on apparent K(m) for ADP were observed while we did not detect any changes in CK activity. The decrease in mitoCK activity and the fall in the reactivity of respiration to creatine could be interpreted as a mechanism for downregulating oxygen demand during chronic hypoxia. The consequences of such alterations on energy metabolism of cardiomyocytes under conditions of reduced oxygen supply are discussed.     

Phosphotransfer reactions in the regulation of ATP-sensitive K+ channels

ATP-sensitive K+ (K(ATP)) channels are nucleotide-gated channels that couple the metabolic status of a cell with membrane excitability and regulate a number of cellular functions, including hormone secretion and cardioprotection. Although intracellular ATP is the endogenous inhibitor of K(ATP) channels and ADP serves as the channel activator, it is still a matter of debate whether changes in the intracellular concentrations of ATP, ADP, and/or in the ATP/ADP ratio could account for the transition from the ATP-liganded to the ADP-liganded channel state. Here, we overview evidence for the role of cellular phosphotransfer cascades in the regulation of K(ATP) channels. The microenvironment of the K(ATP) channel harbors several phosphotransfer enzymes, including adenylate, creatine, and pyruvate kinases, as well as other glycolytic enzymes that are able to transfer phosphoryls between ATP and ADP in the absence of major changes in cytosolic levels of adenine nucleotides. These phosphotransfer reactions are governed by the metabolic status of a cell, and their phosphotransfer rate closely correlates with K(ATP) channel activity. Adenylate kinase catalysis accelerates the transition from ATP to ADP, leading to K(ATP) channel opening, while phosphotransfers driven by creatine and pyruvate kinases promote ADP to ATP transition and channel closure. Thus, through delivery and removal of adenine nucleotides at the channel site, phosphotransfer reactions could regulate ATP/ADP balance in the immediate vicinity of the channel and thereby the probability of K(ATP) channel opening. In this way, phosphotransfer reactions could provide a transduction mechanism coupling cellular metabolic signals with K(ATP) channel-associated functions.     

Increased oxidative and delayed glycogenolytic ATP synthesis in exercising skeletal muscle of obese (insulin-resistant) Zucker rats

1. To examine metabolic correlates of insulin resistance in skeletal muscle, we used 31P magnetic resonance spectroscopy to study glycogenolytic and oxidative ATP synthesis in leg muscle of lean and obese Zucker rats in vivo during 6 min sciatic nerve stimulation at 2 Hz. 2. The water content of resting muscle was reduced by 21 +/- 7% in obese (insulin-resistant) animals compared with lean animals, whereas the lipid content was increased by 140 +/- 70%. These results suggest that intracellular water content was reduced by 17% in obese animals. 3. During exercise, although twitch tensions were not significantly different in the two groups, rates of total ATP synthesis (expressed per litre of intracellular water) were 48 +/- 20% higher in obese animals, suggesting a 50 +/- 8% reduction in intrinsic "metabolic efficiency'. Changes in phosphocreatine and ADP concentration were significantly greater in obese animals than in lean animals, whereas changes in intracellular pH did not differ. 4. These results imply that oxidative ATP synthesis during exercise is activated earlier in obese animals than in lean animals. This difference was not fully accounted for by the greater increase in the concentration of the mitochondrial activating signal ADP. Neither the post-exercise recovery kinetics of phosphocreatine nor the muscle content of the mitochondrial marker enzyme citrate synthase was significantly different in the two groups. The increased oxidative ATP synthesis in exercise must therefore be due to altered kinetics of mitochondrial activation by signals other than ADP. 5. Thus, the insulin-resistant muscle of obese animals may compensate for its decreased efficiency (and consequent increased need for ATP) by increased reliance on oxidative ATP synthesis.     

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.     

Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations

Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudy et al. (1995) PNAS, 92, 729-733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60-88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+-ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+-ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2-20-fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.     

Fatty acids as natural uncouplers preventing generation of O2.- and H2O2 by mitochondria in the resting state

Both natural (laurate) and artificial (m-chlorocarbonylcyanide phenylhydrazone; CCCP) uncouplers strongly inhibit O2.- and H2O2 formation by rat heart mitochondria oxidizing succinate. Carboxyatractylate, an ATP/ADP antiporter inhibitor, abolishes the laurate inhibition, the CCCP inhibition being unaffected. Atractylate partially releases the inhibition by laurate and decelerates the releasing effect of carboxyatractylate. GDP is much less effective than carboxyatractylate in releasing the laurate inhibition of reactive oxygen species (ROS) formation. Micromolar laurate concentrations arresting the ROS formation cause strong inhibition of reverse electron transfer from succinate to NAD+, whereas State 4 respiration and the transmembrane electric potential difference (delta psi) level are affected only slightly. It is suggested that (i) free fatty acids operate as natural 'mild uncouplers' preventing the transmembrane electrochemical H+ potential difference (delta muH+) from being above a threshold critical for ROS formation by complex I and, to a lesser degree, by complex III of the respiratory chain, and (ii) it is the ATP/ADP-antiporter, rather than uncoupling protein 2, that is mainly involved in this antioxidant mechanism of heart muscle mitochondria.     

Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy

An arginine to glutamine missense mutation at position 403 of the beta-cardiac myosin heavy chain causes familial hypertrophic cardiomyopathy. Here we study mice which have this same missense mutation (alphaMHC403/+) using an isolated, isovolumic heart preparation where cardiac performance is measured simultaneously with cardiac energetics using 31P nuclear magnetic resonance spectroscopy. We observed three major alterations in the physiology and bioenergetics of the alphaMHC403/+ mouse hearts. First, while there was no evidence of systolic dysfunction, diastolic function was impaired during inotropic stimulation. Diastolic dysfunction was manifest as both a decreased rate of left ventricular relaxation and an increase in end-diastolic pressure. Second, under baseline conditions alphaMHC403/+ hearts had lower phosphocreatine and increased inorganic phosphate contents resulting in a decrease in the calculated value for the free energy released from ATP hydrolysis. Third, hearts from alphaMHC403/+ hearts that were studied unpaced responded to increased perfusate calcium by decreasing heart rate approximately twice as much as wild types. We conclude that hearts from alphaMHC403/+ mice demonstrate work load-dependent diastolic dysfunction resembling the human form of familial hypertrophic cardiomyopathy. Changes in high-energy phosphate content suggest that an energy-requiring process may contribute to the observed diastolic dysfunction.     

Attenuation by creatine of myocardial metabolic stress in Brattleboro rats caused by chronic inhibition of nitric oxide synthase

1. The present experiment was undertaken to investigate: (a) the effect of nitric oxide synthase (NOS) inhibition, mediated by oral supplementation of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on measures of myocardial energy metabolism and function: (b) the effect of oral creatine supplementation on these variables, in the absence and presence of L-NAME. 2. In one series of experiments, 4 weeks oral administration of L-NAME (0.05 mg ml-1 day-1 in the drinking water) to Brattleboro rats caused significant reductions in myocardial ATP, creatine, and total creatine concentrations and an accumulation of tissue lactate when compared with control animals. Administration of creatine (0.63 mg ml-1 day-1 in the drinking water) for 4 weeks elevated myocardial creatine and total creatine concentrations and reduced lactate accumulation, but did not significantly affect ATP or phosphocreatine (PCr). Concurrent treatment with creatine and L-NAME prevented the reduction in creatine and total creatine concentrations, and significantly attenuated the accumulation of lactate and the reduction in ATP seen with L-NAME alone. 3. In a second series of experiments, 4 weeks treatment with L-NAME and creatine plus L-NAME increased mean arterial blood pressure in conscious Brattleboro rats. Hearts isolated from these animals showed decreased coronary flow and left ventricular developed pressure (LVDP), and total mechanical performance. Treatment with creatine alone had no measurable effect on either mean arterial blood pressure or coronary flow in isolated hearts. However, there was an increase in LVDP, but not in total mechanical performance, because there was a bradycardia. 4. These results indicate that creatine supplementation can attenuate the metabolic stress associated with L-NAME administration and that this effect occurs as a consequence of the action of creatine on myocardial energy metabolism.     

Identification of two novel 5'' noncoding exons in human MNB/DYRK gene and alternatively spliced transcripts

OBJECTIVES: This study was performed to determine the degree and time course over 6 years of cardiomyocyte hypertrophy and myocardial fibrosis of the cardiac allograft in transplanted patients. BACKGROUND: Diastolic dysfunction and to a certain extent systolic dysfunction are common cardiac findings after heart transplantation. The development of posttransplant cardiomyocyte hypertrophy and myocardial fibrosis likely contributes to these derangements. METHODS: Cardiomyocyte diameter and percent fibrosis were determined in serial endomyocardial biopsy specimens obtained from 1 month up to 6 years following heart transplantation in 50 patients. Endomyocardial biopsy specimens from 40 patients with primary dilated cardiomyopathy and 11 normal subjects were similarly analyzed for control data. Analyses were performed in a blinded format using a validated computerized image analysis system (Optimas 5.2). RESULTS: Early (1 month) cardiomyocyte enlargement decreased to the smallest diameter 6 months posttransplant, but thereafter progressively increased by 10% to 20% over the subsequent 5- to 6-year period. Although not statistically established, principal stimuli may include a discrepancy in body size (recipient > donor), coronary allograft vasculopathy and posttransplant systemic hypertension. Percent myocardial fibrosis rose early (1 to 2 months) posttransplant and thereafter remained at the same modest level of severity. CONCLUSIONS: Cardiomyocyte diameter of the transplanted heart gradually increases over time, while percent myocardial fibrosis rises early and remains in a modestly elevated plateau after 2 months posttransplant. These histostructural changes likely contribute to the hemodynamic and cardiac functional alterations commonly observed posttransplant.     

Alterations in energy metabolism of hypertrophied rat cardiomyocytes: influence of propionyl-L-carnitine

Alterations in energy metabolism, reduced fatty acid oxidation, and cardiac carnitine content have been implicated in the evolution from compensated to decompensated cardiac hypertrophy. We determined high-energy nucleotide levels in hypertrophied quiescent cardiomyocytes isolated from rat hearts 4 weeks after banding of abdominal aorta. In hypertrophied quiescent cardiomyocytes, a decrease in ATP content (p = 0.03), and ratios of ATP/total adenine nucleotides and of ATP/ADP were observed, together with an increase in ADP. In addition, palmitate, but not glucose oxidation, was markedly reduced in hypertrophied myocytes. In the presence of 25 microM propionyl-L-carnitine (PLC) or L-carnitine (LC), palmitate oxidation was significantly stimulated in hypertrophied myocytes. The ATP/ADP ratio was significantly increased only with PLC. This effect was not due to an enhanced PLC uptake, since total PLC uptake was 50% lower than that of LC. Changes in the energy generating system of quiescent myocytes occur early in pressure overload hypertrophy, and these alterations can be attenuated by PLC.