Creatine kinase in regulation of heart function and metabolism. II. The effect of phosphocreatine on the rigor tension of EGTA-treated rat myocardial fibers.

Bundles of rat cardiac fibers were treated with EGTA to increase the permeability of the sarcolemma to ions and small molecules. In the medium without calcium, the EGTA-treated fibers developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and maximal tension at 0.1 mM MgATP in the medium. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. Phosphocreatine prevented rigor tension development in the absence of added MgATP when MgADP was added. In the presence of MgADP, phosphocreatine decreased rigor tension more rapidly and to a higher extent than added MgATP. At 5 mM MgADP, half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the Km value for phosphocreatine in the creatine-kinase reaction. These results demonstrate that the intact creatine kinase in the EGTA-treated fibers with increased sarcolemmal permeability is able to ensure rapid replenishment of MgATP in the myofibrillar compartment at the expense of phosphocreatine. The data obtained conform completely to the concept of adenine-nucleotide compartmentation in cardiac cells and of energy channelling by the phosphocreatine-creatine shuttle mechanism.

Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers.

Respiratory parameters of cardiac mitochondria were determined in the bundles of cardiac fibers skinned by using saponin that specifically removed sarcolemma, but left intracellular structures intact. In the assay medium which simulated the ion composition of cardiac cytoplasm maximal value of state 3 oxygen consumption per mol cytochromes aa3 was close to that value for isolated mitochondria. Ischemia and isopreterenol treatment were found to affect respiratory parameters of mitochondria in saponin-skinned fibers, among them creatine-stimulated respiration decreased most significantly, (3-4)-times under these conditions. The method described can be easily applied for determination of the mitochondrial respiratory parameters in small (5-10 mg) biopsy samples from human heart.

Rigor tension in single skinned rat cardiac cell: role of myofibrillar creatine kinase.

OBJECTIVE: To elucidate the role of bound creatine kinase in adenine nucleotide compartmentation in myofibrils, the effects of this enzyme's substrates and products on rigor tension were studied in using isolated skinned rat cardiomyocytes rather than fibers, to avoid restrictions due to concentration gradients within the multicellular preparations. METHODS: A new experimental set-up was built to allow continuous and stable measurements of force developed by cells. Triton X-100-treated cardiomyocytes were glued between a glass holder and the needle of a galvanometer. A feedback system allowed the precise measurement of force by recording the coil current necessary to prevent movement of the needle. RESULTS: At very low [Ca2+] (pCa 7), as MgATP level decreased, rigor tension appeared. In the absence of phosphocreatine (PCr), this tension started to rise at MgATP concentrations several times higher than in the presence of 12 mM PCr. In the absence of PCr, the pMgATP/tension curves of single cells usually had a complicated relationship which could not be analyzed by a simple Hill equation. In the absence of PCr, 250 microM MgADP strongly potentiated rigor tension development in the 1 mM-3 microM range of [MgATP]; at 100 microM MgATP, in the presence of MgADP, the tension was 4.6 times higher than in the absence of MgADP. Addition of 12 mM PCr immediately eliminated rigor. Finally, in the presence of 100 microM MgATP and 250 microM MgADP, a decrease in PCr resulted in rigor; the half-maximal contracture being recorded at 1 mM PCr. CONCLUSIONS: These results indicate a myofibrillar compartmentation of adenine nucleotides influenced by bound creatine kinase, since at equal MgATP concentrations in extramyofibrillar milieu the response of myofibrils strongly depends on the presence of PCr. Local accumulation of ADP in myofibrils due to a fall in cellular PCr and inability of myofibrillar creatine kinase to rephosphorylate ADP produced by myosin ATPase could be an important mechanism of diastolic tension rise in ischaemic conditions.

[Ability of a phosphocreatine-myofibrillar creatine kinase system to prevent the rigor tension of myocardial fibers]. / Sposobnost' sistemy fosfokreatin-miofibrilliarnaia kreatinkinaza predotvrashchat' rigornoe napriazhenie miokardial'nykh volokon.

In the calcium-free medium the EGTA-treated rat myocardial fibres developed rigor tension dependent on the concentration of MgATP in the bathing solution: half-maximal tension was recorded at 2.5 mM MgATP and the maximal tension at 0.1 mM. However, in the presence of 15 mM phosphocreatine without added creatine kinase a decrease of MgATP concentration to 0.1 mM did not result in any development of rigor tension. In the presence of MgADP phosphocreatine decreased rigor tension more rapidly and to the higher extent than MgATP. At 5 mM MgADP half-maximal rigor tension was observed in the presence of 2 mM phosphocreatine which is close to the km value for phosphocreatine in the creatine kinase reaction. These results demonstrate that the native creatine kinase in the EGTA-treated fibres is able to create high local ATP concentration in the myofibrillar compartment at the expense of phosphocreatine under the conditions of deficiency or even absence of ATP. It appears that at the energy supply disturbances the myocardial contracture develops at least partially due to low activity of the myofibrillar creatine kinase because of phosphocreatine deficiency.

[Quantitative relationship between ischemic heart disease and parameters of energy metabolism]. / O kolichestvennoi sviazi mezhdu ishemicheskim porazheniem serdsa i parametrami énergeticheskogo metabolizma.

The relationship between coronary heart disease, postischemic work recovery and tissue ATP levels as well as mitochondrial respiration rates were studied. Respiration of mitochondria was assessed without their isolation by using a novel method applying skinned fibers in physiological saline. The maximal mitochondrial respiration rates were unchanged during 35 min of normothermic ischemia in St. Thomas Hospital cardioplegic solution in the subsequent 30 min aerobic reperfusion period. A reversible increase in the basal respiration and a decrease in creatine-stimulated oxygen uptake were observed. Thus, the combined determination of mitochondrial respiration in situ in skinned cardiac fibers and tissue ATP may be a useful approach to studies of the pathogenesis of cardiac diseases.

[pH changes and the K+ and Na+ concentration in the blood of the coronary vein in experimental myocardial infarct complicated and not complicated by ventricular fibrillation]. / Ob izmeneniiakh pH i kontsentratsii K+ i Na+ v krovi koronarnoi veny pri éksperimental'nom infarkte miokarda, oslozhennom i ne oslozhnennom fibrilliatsiei zheludochkov.

It was found that following occlusion of the coronary artery in dogs, the rate of increase in K+ concentration in blood plasma draining directly from the focus of ischemia is greater in cases complicated by ventricular fibrillation. Fibrillation always occurs against the background of a decrease in pH and an increase in the K+ level in blood plasma draining from the focus of ischemia. It is suggested that inhibition of the development of disorders of acid-base and ion equilibrium in the myocardium would be an effective means of preventing ventricular fibrillation in the acute stage of myocardial infarction.

[Characteristics of the changes in the intra- and extracellular K+ and Na+ concentrations and the intra- and extracellular pH in the area of cardiac ishemia in experimental myocardial infarct complicated by ventricular fibrillation]. / Ob osobennostiakh izmenenii vnutri- i vnekletochnykh kontsentratsii K+ i Na+, vnutri- i vnekletochnogo pH v zone ishemii serdtsa pri éksperimental'nom infarkte miokarda, oslozhnennom fibrilliatsiei zheludochkov.

Localized ischemia of the heart complicated by ventricular fibrillation is characterized by a tendency to a higher rate of decrease in intra- and extracellular K+ gradient and intracellular pH in the myocardium as compared to cases without fibrillation. The higher rate of K+ escape from the ischemic cells may be linked with a sharper intracellular oxidation, evidence of which is the correlative dependence between the severity of disorders of K+ balance and decrease in intracellular pH in the myocardium in ischemia.

[Functional state of mitochondria, myofibrils and creatine kinase associated with these organelles of the myocardium in hamsters with hereditary cardiomyopathy]. / Funktsiona'lnoe sostoianie mitokhondrii, miofibrill i sviazannoi s étimi organellami kreatinkinazy v miokarde khomiakov s nasled- stvennoi kardiomiopatiei.

Functional states of the cardiac contractile apparatus and mitochondria were studied in hamsters with hereditary cardiomyopathy using myocardial fibers with sarcolemma, which had been exposed to saponin. This provided an opportunity of examining the respiratory characteristics of a total mitochondrial population in the myocardium of the animals of two ages (75-100 and 175-200 days). A higher calcium sensitization of myofibrils was found in hamsters with cardiomyopathy. Examination of the rigor tension-MgATP relationship in the presence or absence of phosphocreatine revealed that the animals showed a slightly lower functional activity of myofibrillar creatine kinase. The findings indicate that the creatine kinase system of cardiomyocytes is involved in hereditary cardiomyopathy, mitochondria, in particular, exhibiting much more profound disturbances, in other respects, myofibrils and mitochondria retain their basic functional properties.

[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.

Calcium binding in chemically skinned fibers of rat myocardium during force development.

The amount of bound calcium and force were measured in chemically skinned rat myocardial fibers during isometric contraction at different concentrations of free Ca2+. The data obtained suggest that calcium binding by cardiac myofibrils is cooperative and probably depends on mechanical tension.

[Calcium binding by chemically stripped fibers of the rat myocardium during force development]. / Sviazyvanie kal'tsiia khimicheski skinirovannymi voloknami miokarda krysy vo vremia razvitiia sily.

The aim of the study was to measure calcium binding by chemically skinned fibers of rat myocardium during force development at different concentrations of free Ca2+. Fiber fascicles were incubated in the solution with EGTA and 1% X-100 triton for 48 hours to achieve the maximal possible withdrawal of the sarcolemma and intracellular membrane structures. Ca2+ binding was determined using two markers. The fibers started developing a considerable isometric force at pCa 6.2-6.0, the maximal force was registered at pCa 5.0-4.8. At pCa 5.0 the fibers bound 3.59 +/- 0.21 nM of Ca/mg of protein, while at pCa greater than 6 less than one third of this amount was bound. When [Ca2+] was increased to over 1 microM the curve of Ca2+ binding became much steeper and coincided with the curve of force development. The results suggest that Ca2+ binding by myofibrils is evidently cooperative and depends on mechanic tension.

[The effect of alpha 1-stimulation and lithium on the calcium sensitivity of hyperpermeable myocardial fibers in the rat]. / Vliianie al'fa 1-stimuliatsii i litiia na kal'tsievuiu chuvstvitel'nost' giperpronitsaemykh volokon miokarda krysy.

Effects of alpha 1-adrenoagonist phenylephrine (PhE) and lithium, an inhibitor of inositolmonophosphatase, on calcium sensitivity of hyperpermeable rat myocardial fibers were studied. In the presence of propranolol, PhE reduced calcium sensitivity, and this effect was enhanced as calcium concentration was being increased during stimulation with PhE. This appears to relate to an activation of protein kinase C by calcium. LiCl also decreased calcium sensitivity of fibers. This result partially can be explained by a direct effect of Li+ on myofibrils. A combination of PhE and LiCl decreased calcium sensitivity even more, but the effect of PhE in this case was less expressed than in the absence of LiCl. Prasozin, an alpha 1-adrenoblocker, prevented from the effect of PhE both in the presence and in the absence of lithium, thus indicating that stimulation of alpha 1-adrenoceptors was involved in the effects of PhE and lithium observed in this study.

Creatine kinase and mechanical and mitochondrial functions in hereditary and diabetic cardiomyopathies.

To determine whether the development of cardiomyopathies is associated with alterations in creatine kinase function, the functional properties of cardiac contractile apparatus and mitochondria were studied in two different models of cardiomyopathies, the Syrian hamster (hereditary dilated cardiomyopathy, strain UM-X7.1, 200 days old) and the diabetic rat (4-6 weeks after injection of streptozotocin) using ventricular skinned fibers. After Triton X-100 treatment, the hereditary cardiomyopathic fibers demonstrated decreased maximal calcium-activated tension and unchanged calcium sensitivity, whereas fibers from diabetic hearts exhibited unchanged maximal tension and increased calcium sensitivity, when compared with their respective controls. In both cases myofibrillar creatine kinase appeared unchanged. The functional properties of total tissue mitochondria were evaluated using saponin-skinned fibers. Coupling between oxidation and phosphorylation was not altered in cardiomyopathies. Respiration rate (per unit of tissue dry weight) was normal in hereditary cardiomyopathy but was considerably lower in diabetic fibers compared with control fibers. In both models of cardiomyopathies, creatine-stimulated respiration was significantly lower than in controls, thus indicating the depression of functional activity of mitochondrial creatine kinase.

Effects of halothane on contractile properties of skinned fibers from cardiomyopathic animals.

The effects of clinical concentrations of halothane (1 and 2% v/v) on detergent treated cardiac fibers were studied in two different models of cardiomyopathic animals, the Syrian hamster UM-X7.1, and the streptozotocin-induced diabetic rat. The changes of contractile properties in cardiac muscle observed on cardiomyopathic animals, although of moderate importance, were different in these two models. The cardiomyopathic hamsters exhibited macroscopic structural changes in cardiac muscle responsible for a significant decrease in maximal activated tension, but myocardial calcium sensitivity was unchanged. On the other hand, in diabetic rats, maximal activated tension was unchanged, while a slight but significant increase in myocardial calcium sensitivity was observed. Addition of halothane produced a similar dose-dependent decrease in myocardial calcium sensitivity, in both the controls and the two groups of cardiomyopathic animals. Halothane exposure was also associated with a dose-dependent decrease in maximal calcium activated tension in all groups, an effect that was more pronounced in cardiomyopathic hamsters than in their control at the lowest anesthetic concentration. These results indicate that the negative inotropic effects of halothane are additive to the myocardial depression observed in these cardiomyopathies.

Cellular mechanisms of alterations in myocardial contractile function in experimental cardiomyopathies.

The high-energy-phosphate content, myocardial ultrastructure, left ventricular (LV) pressure, and pump function of isolated rat hearts were determined in four kinds of chronic myocardial damage induced by either autoimmunization, treatment of rats with adriamycin or noradrenaline in increasing doses, or infection with smallpox virus. Mild fibrosis, swollen mitochondria, and hyper-contracted and overdistended sarcomeres were typical ultrastructural alterations. The ATP and phosphocreatine content as well as cardiac output at standard load conditions were substantially lower in all four experimental groups. Mild bradycardia and increased LV diastolic pressure and stiffness occurred in all except the autoimmunized group. LV diastolic stiffness was inversely correlated with cardiac output and phosphocreatine content and directly correlated with LV systolic pressure. Both increased myofibrillar sensitivity to Ca2+ ions and energy deficiency within myofibrils may have contributed to increased myocardial stiffness. The increased stiffness limits LV filling but facilitates pressure development, and in this way partly compensates for the decreased contractility of cardiomyopathic hearts.

[Protective effect of 2,3-butanedione monooxime on ischemic myocardium of rats]. / Zashchitnoe deistvie 2,3-butandion monoksima na ishemicheskii miokardkrys.

Effects of 2,3-butanedione monoxime (BDM), an ATPase inhibitor, on ischemia-reperfusion myocardial injury were examined in isolated working rat hearts perfused in vitro. Following cardiac arrest induced by cardioplegic solution, global ischemia was produced for 30 min. In untreated hearts, reperfusion for 45 min resulted in an incomplete recovery of cardiac pump function. When BDM was added to the cardioplegic solution up to 20 mM, the recovery of cardiac function was significantly improved average by 19%. This BDM effect can, presumably, reduce ATP losses during ischemia and like that improve recovery of cardiac function during reperfusion.

Energy-linked functional alterations in experimental cardiomyopathies.

Changes in high-energy phosphate content and cardiac contractile function of isolated rat hearts as well as changes in Ca2+ sensitivity and mitochondrial respiration of myocardial skinned fibers were assessed in hereditary cardiomyopathies and in cardiomyopathies induced by chronic treatment with adriamycin or norepinephrine, by autoimmunization, by diabetes, or by creatine deficiency. The sum of ATP and phosphocreatine contents as well as cardiac output at standard load conditions was substantially lower in almost all groups. The common features of cardiac pump failure were mild bradycardia, elevated left ventricular (LV) diastolic pressure, and stiffness that limited cardiac contractile adaptation to volume or resistance loads. The LV diastolic stiffness at maximal functional load was inversely correlated with high-energy phosphate content. Increased myofibrillar sensitivity to Ca2+ and defective function of mitochondrial creatine kinase were found in skinned myocardial fibers. These results suggested that both increased myofibrillar Ca2+ sensitivity and energy deficiency within myofibrils may contribute to increased myocardial stiffness. Increased stiffness limits LV filling but facilitates pressure development, which partly compensates for decreased contractility of cardiomyopathic hearts.