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

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

ESR spin trapping and NMR spectroscopy of the same heart shows correlation between energy depression and radical formation during postischemic reperfusion.

The relevance of radical formation in disturbances of energy metabolism in the postischemic heart is not clear. This study provides the first evidence of a significant correlation between the amount of oxy-radicals trapped in the effluent of isolated hearts upon reperfusion and the decreased myocardial content of phosphocreatine and ATP. This suggests that the loss of high-energy compounds might contribute to oxy-radical production during reperfusion. The application of ESR spin trapping and of NMR technique to the same heart is a new approach to investigate the pathobiochemical relevance of free radicals for the heart muscle.

Plasmin cleaves Abeta42 in vitro and prevents its aggregation into beta-pleated sheet structures.

The formation, aggregation and deposition of amyloid beta peptide (Abeta) is implicated in the aetiology of Alzheimer's disease. Impairment of proteolytic degradation of Abeta may be a key factor in the progression of the disease. We have used RP-HPLC and thioflavin T fluorescence to demonstrate that Abeta42 is rapidly cleaved by the protease plasmin and that cleavage prevented the aggregation of Abeta42, and its cleavage products, into beta-pleated sheet structures. Plasmin may fulfil a similar role in vivo.

Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP).

The formation of amyloid fibrils is considered to be an important step in the aetiology of Alzheimer's disease and other amyloidoses. Fibril formation in vitro has been shown to depend on many different factors including modifications to the amino acid profile of fibrillogenic peptides and interactions with both large and small molecules of physiological significance. How these factors might contribute to amyloid fibril formation in vivo is not clear as very little is known about the promotion of fibril formation in undersaturated solutions of amyloidogenic peptides. We have used thioflavin T fluorescence and reverse phase high performance liquid chromatography to show that ATP, and in particular AlATP, promoted the formation of thioflavin T-reactive fibrils of beta amyloid and, an unrelated amyloidogenic peptide, amylin. Evidence is presented that induction of fibril formation followed the complexation of AIATP by one or more monomers of the respective peptide. However, the complex formed could not be identified directly and it is suggested that AlATP might be acting as a chaperone in the assembly of amyloid fibrils. The effect of AlATP was not mimicked by either AlADP or AlAMP. However, it was blocked by suramin, a P2 ATP receptor antagonist, and this has prompted us to speculate that the precursor proteins to beta amyloid and amylin may be substrates or receptors for ATP in vivo.

Aspartate improves recovery of the recently infarcted rat heart after cardioplegic arrest.

BACKGROUND: We have previously shown that aspartate improves the tolerance of normal hearts to cardioplegia. The aim of this study was to investigate whether aspartate is also beneficial in the recently infarcted heart. METHODS: Myocardial infarction was produced in rats by left coronary artery ligation. Twenty hours later their hearts were perfused on an isolated working rat heart apparatus and underwent cardioplegic arrest for 30 min at 37 degrees C with or without 20 mM aspartate in the cardioplegic solution (n = 11 per group). Functional recovery and myocardial high energy phosphate levels were measured at the end of arrest and after 30 min of reperfusion. RESULTS: There was no difference in pre-arrest pump function between the untreated and aspartate-treated groups. However, after reperfusion the aspartate group generated more power (3.4 +/- 0.2 mJ/s per g) than the untreated group (2.5 +/- 0.3 mJ/s per g; P < 0.05) such that the percentage recovery of pre-arrest power in the aspartate group (67.7 +/- 3.5%) was greater than in the untreated group (53.6 +/- 4.9%; P < 0.05). The aspartate group also showed increases in aortic flow and myocardial oxygen consumption compared to the untreated group (P < 0.05). There were no between-group differences in high energy phosphate levels at the end of arrest or after reperfusion. CONCLUSION: Aspartate improves functional recovery of the recently infarcted heart during cardioplegic arrest, and therefore has potential as a useful adjunct to myocardial protection in patients with recent myocardial infarction undergoing cardiac surgery.

Evaluation of a lumped parameter model for isolated working rat hearts.

When a mechanical model of the rat aortic input impedance is perfused with a pulsatile pump, the computed values of the impedance components vary linearly with flow rate and are interactive. When the model is perfused by an isolated rat heart, the total load upon the left ventricle consists of the model and coronary impedances in parallel. Adenosine triphosphate induces changes in coronary impedance, and the redistribution of cardiac output from the model to the coronary circulation causes flow-related changes in the model impedance. Thus, the mechanical model does not provide a constant load for the isolated heart, because of variations in both the model and coronary impedances.

[Functional significance of 2 pathways of energy transport in cardiomyocytes]. / Funktsional'noe znachenie dvukh putei transporta énergii v kardiomiotsitakh.

To study the relative roles of creatine kinase (CK) and adenylate systems in cardiac energy turnover, the effect of CK inhibitor, iodoacetamide- (IAA, 0.5 mM), and 2-deoxyglucose-(DOG, 2 mM) induced) 65% depletion of adenine nucleotides at slightly decreased CK flux was determined in isolated rat heart. Both substances did not substantially affect contractile parameters of the isovolumic heart. However, an augmentation of cardiac work induced by isoproterenol addition was feeble and transient in IAA-treated hearts while the response of DOG-treated hearts was well preserved. The cardiac failure after IAA treatment was associated with irreversible fall in myocardial ATP content as evidenced by 31P-NMR technique. Furthermore, these hearts were unable to perform cardiac pump function due to insufficient cardiac filling and distensibility. The DOG-treated hearts exhibited 50% reduction in the pump function and were able to increase their work in elevated resistance. The results suggest that CK pathway is extremely important for both full cardiac relaxation and maximal contractile function.

[Adrenergic stimulation of the heart during inhibition of phosphocreatine or adenylate pathways of energy transfer in cardiomyocytes]. / Adrenergicheskaia stimuliatsiia serdtsa pri ingibirovanii fosfokreatinovogo ili adenilatnogo puti transporta énergii v kardiomiotsitakh.

Functional and metabolic response of an isovolumically perfused heart of a rat to isoproterenol (0.1 microM) has been studied. A heart with the normal content of adenine nucleotides (AN) and phosphocreatine (PCr) as well as that with the 5-fold reduced AN content (with 2-deoxyglucose treatment) significantly increased cardiac work index (PRP), maximal contraction rate (MCR) and maximal relaxation rate (MRR) (by 50, 30-40 and 100-150%, respectively). The effect was preserved for all the period of the hormone action (30 min) and was followed by a temporary decrease in the PCr content. The heart with an inhibited unidirectional flux of metabolites through creative kinase (CK) and normal level of AN responded to the hormone by the slower and decelerated growth of the function and in the heart with almost completely iodoacetamide (IAAm)-blocked CK the functional response was minimal and transient. In the latter a significant and irreversible decline in PCr and ATP content and a concomitant rise of inorganic phosphate took place. Both basal and isoproterenol-stimulated adenylate cyclase activity remained unchanged after IAAm treatment. An increase in PRP correlated with the elevation of the cytosolic ADP concentration, however, correlation was not uniform for different experimental groups. These data show significance of the creatine kinase system not only for maintenance of maximal work but also for a rapid functional response to the catecholamine stimulation.

Regulation of cardiac energy turnover by coronary flow: a 31P-NMR study.

The response of cytosolic phosphates ([ATP], [PCr], [Pi] and [ADP]) in rat hearts retrogradely perfused with different oxidizable substrates to increased workload induced by elevated coronary flow (CF) or by addition of inotropic agents has been investigated. Hearts were perfused with glucose (11 mM), pyruvate (5 mM), lactate (3 mM) or a combination of glucose (5.5 mM) and acetate (5 mM), octanoate (0.1 mM) or beta-hydroxybutyrate (5 mM). The initial [ATP]/[ADP] ratio was highest in pyruvate and lactate perfused hearts. Increasing the coronary flow 1.7-fold (from c. 56 to 96 ml/min x g dry wt) resulted in an increase in pressure-rate product (PRP) by 36-52% without significant changes in cytosolic phosphates. Dichloroacetate (1 mM), ruthenium red (2.5 micrograms/ml), or pre-treatment with theophylline (1 mM, 30 min) had no effect either functional or metabolic response to elevated CF in glucose-perfused hearts. Isoproterenol (Iso, 0.1 microM) infusion at maximal coronary flows lead to further elevation of PRP value by 36-88% and the ratio of the maximal rate of relaxation to LV developed pressure ((-dP/dt)m/LVDP) increased two-fold. Simultaneously, [PCr] decreased by 18-30%, [Pi] increased two-fold and ADP increased by 20-90% resulting in reduction of [ATP]/[ADP] by half and ATP affinity (A(ATP) = -delta G(ATP)) by 2.4-3.8 kJ/mol. In hearts perfused with acetate, octanoate and hydroxybutyrate in the presence of glucose, Iso addition resulted in intracellular pH decrease by 0.03-0.07 U and increase in lactate extrusion 1.5-2 times. In hearts perfused with glucose alone, decrease in PRP induced by perfusate Ca2+ reduction was associated with increase in PCr and decrease in Pi levels. These data show that coordinated regulation of energy supply and demand exerted by coronary flow/perfusion pressure does not depend on the availability of reducing equivalents but is rather controlled by oxygen supply and stretch-activated factors.

Adrenergic stimulation of rat hearts with severely reduced cytosolic adenine nucleotide pool and [ATP]/[ADP]ratio.

The effect of severe reduction of cytosolic adenine nucleotide (AdN) pool and [ATP]/[ADP] ratio (by 2-deoxyglucose treatment) on functional and metabolic responses of isovolumic rat heart to increased energy demand induced by coronary flow (CF) rise and isoproterenol (Iso) addition has been investigated. AdN-depleted hearts had reduced phosphocreatine (PCr, by 80%), ATP (by 75%), [ATP]/[ADP] (24 times) and pressure-rate product (PRP, by 60%). An elevation of CF was followed by the increase in PRP in control and AdN-depleted hearts by 40-45% with unchanged metabolic parameters. At increased CF, Iso caused a further rise in PRP in both groups due to elevation of heart rate; however maximal levels of PRP in the AdN-depleted group still remained lower than that of control (by 40%). Only in control experiments was Iso addition accompanied by an increase in the difference between left-ventricular end- and minimal diastolic pressure, cytosolic [Pi] and [ADP], and some decrease in PCr and [ATP]/[ADP]. These data imply that severely reduced cytosolic [ATP]/[ADP] does not prevent acceleration of Ca2+ turnover by Iso in cardiomyocytes, it but restricts maximal force development affecting the myofibrils.

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

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

Substrate accessibility to cytosolic aspartate aminotransferase improves posthypoxic recovery of isolated rat heart.

The effects of aspartate (Asp) and 2-oxoglutarate (2-OG) on metabolism and function of isolated rat heart during hypoxia and reoxygenation were studied. Hearts were subjected to oxygenated perfusion with Krebs-Henseleit buffer supplied with 11 mM glucose (20 min) and anoxic perfusion with the buffer saturated with N2 (20 min), followed by reoxygenation (30 min). The substrate concentrations in the perfusate were 3.5 mM each. The additives had no effect on the energy metabolism and function of the oxygenated heart despite a two-fold rise in myocardial Asp and 2-OG. Substrate supplementation during anoxic perfusion resulted in reduced lactate dehydrogenase release and less depression of cardiac function. Prevention of Asp, glutamate, and 2-OG degradation in hypoxic myocardium was accompanied by relief of glycolytic flux and better preservation of ATP, phosphocreatine (PCr), and total creatine (Cr). Reoxygenation without the additives after supplemented anoxic perfusion failed to improve recovery of high-energy phosphates and cardiac function compared to control. However, during reoxygenation with the additives the treated hearts showed less cell membrane damage and enhanced recovery of contractile and pump function. These effects were associated with higher myocardial contents of ATP, PCr, and adenine nucleotides and a smaller Cr loss during reoxygenation. A more effective restoration of oxidative metabolism was related to promoted glucose oxidation due to replenishment of the malate-aspartate shuttle reactants. The results substantiate the use of substrates of cytosolic aspartate aminotransferase for myocardial protection against hypoxia/reoxygenation stress.

Control of cardiac energy turnover by cytoplasmic phosphates: 31P-NMR study.

Energy flux, estimated from the cardiac work index (pressure-rate product) and the rate of oxygen consumption, was varied in different ways; and the free concentrations of cytosolic phosphates were detected by the 31P-nuclear magnetic resonance method. A reversible decrease in phosphocreatine (PCr) and concomitant increase in [ADP] at nearly constant Pi were induced by 2-deoxyglucose (2-DG) treatment and its subsequent washout and were followed by a reversible suppression of pressure-rate product and elevation of end-diastolic pressure. 2-DG treatment also resulted in an irreversible and severe reduction of the cytosolic adenine nucleotide pool (to approximately one-third of control value) that did not recover during 2-DG washout. Reduction of the energy turnover rate, either by suppression of the PCr shuttle with iodoacetamide or by inhibition of the respiratory chain with amytal, was associated with a drop of PCr level, an elevation of both [ADP] and [Pi], and a rise of end-diastolic pressure. In contrast, a decrease in energy flux by reduction of perfusate Ca2+ led to a PCr rise and a fall in [ADP] and [Pi]. Most of these experimental groups were exposed to two types of loads, isoproterenol stimulation and coronary flow (CF) elevation. Iodoacetamide-treated hearts showed a poor mechanical response to both types of loads compared with other groups. The metabolic response to isoproterenol was uniform in all groups and was associated with some decrease in PCr and increase of [ADP] and [Pi], implying limitations in the respiratory chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of pre-ischaemic disturbances in energy metabolism with postischaemic dysfunction of the rat isolated working heart.

1. Metabolic and functional effects of two protocols of preconditioning were compared in rat isolated hearts subjected to 20 min global ischaemia (37 degrees C) and reperfusion (30 min Langendorff + 15 min working). Prior to the ischaemic period, hearts were perfused according to Langendorff (control group) or were preconditioned by three 5 min cycles or two 10 min cycles of ischaemia and reperfusion (PC-I and PC-II groups, respectively). 2. There was no difference in the contractile function between the two preconditioned groups at the onset of sustained ischaemia, although the PC-II group showed enhanced release of adenosine (Ado), inosine, hypoxanthine and xanthine into the interstitium accompanied by losses of tissue adenine nucleotides (sigmaAN = ATP + ADP + AMP), total creatine (sigmaCr = phosphocreatine + creatine) and activation of glycolysis following the preconditioning period. During reperfusion, the PC-I group showed enhanced functional recovery, higher contents of sigmaAN and sigmaCr, and the smallest lactate dehydrogenase release compared with these indices in the control and PC-II groups. Postischaemic myocardial dysfunction was similar in the control and PC-II groups. 3. Functional recovery of hearts in both preconditioned groups was positively correlated with myocardial contents of ATP, sigmaAN and sigmaCr at the end of reperfusion, but not with pre-ischaemic Ado release into the interstitium. The results suggest that pre-ischaemic disturbances of energy metabolism, rather than activation of Ado receptors or stunning, may contribute to efficacy of multiple preconditioning in the rat isolated heart.

[Metabolic and functional consequences of complete inhibition of creatine kinase by iodoacetamide in the perfused heart]. / Metabolicheskie if funktsional'nye posledstviia polnogo ingibirovaniia kreatinkinazy iodatsetamidom v perfuziruemom serdtse.

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

The creatine kinase system and cardiomyopathy.

Changes in the creatine kinase system, cellular energetics, regulation of respiration and alterations in parameters of contractility in experimental animals (myopathic hamsters), and in patients with dilated cardiomyopathy were studied. 31P-NMR methods were used to show that cardiomyopathic hearts are characterized by decreased work index, lower tissue ATP, phosphocreatine, and total creatine contents and diminished creatine kinase activity and energy fluxes. In isolated mitochondria, only the creatine kinase activity was decreased. Both in cardiomyopathic hamsters and human hearts a share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to 18% and that of BB elevated from 5% in control to 20%, at an unchanged relative level of MM. In saponins-skinned cardiac fibers on cardiomyocytes creatine (Cr, 25 mM) decreased Km for ADP in regulation of respiration from 133 +/- 20 to 20 +/- 4 microM due to activation of coupled mitochondrial creatine kinase-oxidative phosphorylation reactions in control hamster hearts. In the case of cardiomyopathy it decreased Km for ADP only to 81 +/- 13 microM. In endocardial biopsy samples from the hearts of patients with dilated cardiomyopathy taken during angiography, creatine stimulated respiration was decreased by 36% of control value, which correlated well with increase of end-diastolic pressure and fall in ejection fraction. Thus, changes in mitochondrial creatine kinase expression diminished the efficiency of cellular regulation of respiration in cardiomyopathic hearts that may have functional consequences for hemodynamics or may be adaptive alterations in response to decreased contractility.