Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo.

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 microg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon--tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.

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.

Muscle creatine kinase-deficient mice. I. Alterations in myofibrillar function.

The regulation of contractile activity in mice bearing a null mutation of the M-isoform of creatine kinase gene, has been investigated in tissue extracts and Triton X-100-treated preparations of ventricular, soleus, and gastrocnemius muscles of control and transgenic mice. Skinned fiber experiments did not evidence any statistical difference in the maximal force or the calcium sensitivity of either muscle type. Rigor tension development at a low MgATP concentration was greatly influenced by phosphocreatine in control but not in transgenic mice as should be expected. In calcium-activated ventricular preparations, although the force developed by each cross-bridge was the same in control and transgenic animals, the rate constant of tension changes appeared to be markedly slowed in transgenic animals. As the ventricular isomyosin pattern was not altered, we suggested that, in transgenic animals, cross-bridge cycling was hindered by a local decrease in the MgATP to MgADP ratio, due to lack of a local MgATP regenerating system. Myokinase activity was not significantly changed while activities of pyruvate kinase or glyceraldehyde-3-phosphate dehydrogenase were found to be increased in transgenic animals. These results show that no fundamental remodelling occurs in myofibrils of transgenic animals but that important adaptations modify the bioenergetic pathways including glycolytic metabolism.

Muscle creatine kinase-deficient mice. II. Cardiac and skeletal muscles exhibit tissue-specific adaptation of the mitochondrial function.

Functional properties of in situ mitochondria and of mitochondrial creatine kinase were studied in saponin-skinned fibers taken from normal and M-creatine kinase-deficient mice. In control animals, apparent Km values of mitochondrial respiration for ADP in cardiac (ventricular) and slow-twitch (soleus) muscles (137 +/- 16 microM and 209 +/- 10 microM, respectively) were manyfold higher than that in fast-twitch (gastrocnemius) muscle (7.5 +/- 0.5 microM). Creatine substantially decreased the Km values only in cardiac and slow-twitch muscles (73 +/- 11 microM and 131 +/- 21 microM, respectively). As compared to control, in situ mitochondria in transgenic ventricular and slow-twitch muscles showed two times lower Km values for ADP, and the presence of creatine only slightly decreased the Km values. In mutant fast-twitch muscle, a decrease rather than increase in mitochondrial sensitivity to ADP occurred, but creatine still had no effect. Furthermore, in these muscles, relatively low oxidative capacity was considerably elevated. It is suggested that in the mutant mice, impairment of energy transport function in ventricular and slow-twitch muscles is compensated by a facilitation of adenine nucleotide transportation between mitochondria and cellular ATPases; in fast-twitch muscle, mainly energy buffering function is depressed, and that is overcome by an increase in energy-producing potential.

Myocardial adaptation to long-term action of substances associated with decreased intensity of cardiac function.

The effect of prolonged treatment of rats with 6-n-propyl-2-thiouracil (PTU), verapamil, or propranolol on cardiac pump function and the properties of myofibrils and mitochondria was studied. After 6-8 weeks of treatment, the heart rate and maximal cardiac output of the isolated heart of rats treated with verapamil or propranolol were higher than those in the control group. The PTU treatment was followed by lower heart rate and maximal work. Calcium sensitivity (pCa50 value) of skinned ventricular fibers was higher in all experimental groups compared to the control by 0.07-0.15 units. Myofibrillar Mg2+, Ca(2+)-ATPase activity measured in isolated Triton-skinned cardiomyocytes was considerably lower after PTU treatment than that in respective controls (0.128 +/- 0.013 vs 0.178 +/- 0.010 mumol Pi/min/mg protein). In contrast, long-term treatment with verapamil or propranolol was accompanied by increased activity to 0.223 +/- 0.018 and 0.254 +/- 0.015 mumol Pi/min/mg protein, respectively. Neither the basal mitochondrial respiration rate of saponin-skinned cardiac fibers nor its enhancement after addition of low ADP concentration or creatine was significantly altered in any experimental group. Also no difference between control and experimental groups was observed in the total activity of creatine kinase or relative percentage of its isoenzymes extracted from cardiac tissue. Thus the changes in cardiac pump function after prolonged treatment with agents decreasing cardiac function may be attributed to concomitant alterations of myofibrils while mitochondria remain relatively intact.

[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.

[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.

[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.

[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.

[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.

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.

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.

[High functional activity of mitochondrial creatine kinase of the myocardium before ischemia is tied with the worst restoration of contractility after reperfusion]. / Vysokaia funktsional'naia aktivnost' mitochondrial'noi kreatinkinazy miokarda do ishemii sochetaetsia s khudshim vosstanovleniem sokratimosti posle reperfuzii.

Isolated Langendorff-perfused guinea pig hearts were arrested with a cardioplegic solution containing 10 mM phosphocreatine + 15 mM glutamate (PG group) or not containing them (control group). Total normothermic ischemia lasted 45 min followed by 30 min reperfusion. Mitochondrial respiration in the absence and presence of different concentrations of ADP and creatine was studied in biopsy samples after saponin treatment. The samples were taken before and after ischemia as well as after the reperfusion period. A slightly better relative recovery of developed pressure (RRDP) in PG group was associated with higher mitochondrial acceptor control ratio after reperfusion. When results in both groups were taken together, marked negative correlations between the preischemic mitochondrial indices (particularly, those related to creatine kinase activity) and RRDP were revealed. Relative changes in these indices after ischemia demonstrated tight positive correlations with RRDP. Thus, the hearts having higher functional activity of mitochondrial creatine kinase are more sensitive to ischemia, other conditions being equal.

Mitochondrial respiration in myocardial biopsy samples as a criterion of postischemic recovery of the cardiac contractility.

Isolated perfused guinea pig hearts were arrested by a high K+ cardioplegic solution containing (PG group) or lacking (control group) 10 mM phosphocreatine + 15 mM glutamate. Total normothermic ischemia lasted 45 min followed by 30 min reperfusion. Mitochondrial respiration in the absence and presence of different concentrations of ADP and creatine was studied in biopsy samples (6-8 mg) after saponin treatment. The samples were taken before and after ischemia, as well as after the reperfusion period. A slightly better relative recovery of developed pressure (RRDP) in PG group was associated with higher mitochondrial acceptor control ratio after reperfusion (5.74 +/- 0.32 vs. 4.54 +/- 0.21 in PG and control groups, resp., p less than 0.01). When the results obtained in both groups were treated together, tight correlations between the pre- or postischemic mitochondrial state and RRDP were revealed. Higher values of RRDP were found for the hearts with lower preischemic values of (low ADP + creatine)-stimulation of mitochondrial respiration (r = -0.57, p less than 0.01). Relative changes in this mitochondrial parameter during ischemic period were in a good correlation with the RRDP (r = 0.82, p less than 0.001). The data suggest that the study of the mitochondrial function in myocardial biopsy samples before ischemia and reperfusion could provide a useful information for the prognosis of cardiac function recovery.

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.

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.

[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.

Quantitative evaluation of relationship between cardiac energy metabolism and post-ischemic recovery of contractile function.

Quantitative Evaluation of Relationship between Cardiac Energy Metabolism and Post-ischemic Recovery of Contractile Function. Mechanisms of ischemic damage were studied by defining the relationships between post-ischemic work recovery and tissue ATP levels in isolated rat hearts as well as mitochondrial respiration rates in skinned myofibrils. Pre-ischemic levels of ATP were reduced by 2-deoxyglucose treatment and assessed using 31P-NMR. A 70% fall of ATP was not associated with decreased functional recovery. Mitochondrial respiration was assessed without mitochondrial isolation in skinned cardiac fibers in physiological salt solution using a novel method developed by Veksler et al. Maximal rates of mitochondrial respiration were not changed after 35 min of normothermic ischemia using St. Thomas's Hospital cardioplegic solution followed by 30 min of aerobic reperfusion. Only a reversible increase in the rate of basal respiration and a decrease in creatine-stimulated oxygen uptake were observed. Thus, mitochondrial oxidative phosphorylation, as assessed in skinned myofibrils, was tolerant to an ischemic period which induced permanent depression of contractile function along with alterations in metabolite distribution. As a result, tissue level of ATP and rates of mitochondrial respiration provided an estimate of ischemic damage only in cases where damage reached a very severe extent.

[Recording of the respiration of skinned cardiac fibers--a method that permits an evaluation of the functional parameters of mitochondria without their isolation from the tissue]. / Registratsiia dykhaniia skinirovannykh volokon serdtsa--metod, pozvoliaiushchii otsenivat' funktsional'nye parametry mitokhondrii bez ikh vydeleniia iz tkani.

Respiratory parameters of cardiac mitochondria were determined in the bundles of cardiac fibers skinned by saponin that specifically removed sarcolemma but left intracellular structures intact. The described method can be used for determination of the mitochondrial respiratory parameters in small biopsy samples from the human heart.