Functional coupling as a basic mechanism of feedback regulation of cardiac energy metabolism.

In this review we analyze the concepts and the experimental data on the mechanisms of the regulation of energy metabolism in muscle cells. Muscular energetics is based on the force-length relationship, which in the whole heart is expressed as a Frank-Starling law, by which the alterations of left ventricle diastolic volume change linearly both the cardiac work and oxygen consumption. The second basic characteristics of the heart is the metabolic stability--almost constant levels of high energy phosphates, ATP and phosphocreatine, which are practically independent of the workload and the rate of oxygen consumption, in contrast to the fast-twitch skeletal muscle with no metabolic stability and rapid fatigue. Analysis of the literature shows that an increase in the rate of oxygen consumption by order of magnitude, due to Frank-Starling law, is observed without any significant changes in the intracellular calcium transients. Therefore, parallel activation of contraction and mitochondrial respiration by calcium ions may play only a minor role in regulation of respiration in the cells. The effective regulation of the respiration under the effect of Frank-Starling law and metabolic stability of the heart are explained by the mechanisms of functional coupling within supramolecular complexes in mitochondria, and at the subcellular level within the intracellular energetic units. Such a complex structural and functional organisation of heart energy metabolism can be described quantitatively by mathematical models.

Macrocompartmentation of total creatine in cardiomyocytes revisited.

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments--mitochondrial matrix space and cytoplasm--in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 microg/ml for 30 min or 600 microg/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2-5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 +/- 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment--cytoplasm.

Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice.

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.

Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells.

Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.

Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in vivo.

In saponin-skinned muscle fibers from adult rat heart and m. soleus the apparent affinity of the mitochondrial oxidative phosphorylation system for ADP (Km = 200-400 microM) is much lower than in isolated mitochondria (Km = 10-20 microM). This suggests a limited permeability of the outer mitochondrial membrane (OMM) to adenine nucleotides in slow-twitch muscle cells. We have studied the postnatal changes in the affinity of mitochondrial respiration for ADP, in relation to morphological alterations and expression of mitochondrial creatine kinase (mi-CK) in rat heart in vivo. Analysis of respiration of skinned fibers revealed a gradual decrease in the apparent affinity of mitochondria to ADP throughout 6 weeks post partum that indicates the development of mechanism which increasingly limits the access of ADP to mitochondria. The expression of mi-CK started between the 1st and 2nd weeks and reached the adult levels after 6 weeks. This process was associated with increases in creatine-activated respiration and affinity of oxidative phosphorylation to ADP thus reflecting the progressive coupling of mi-CK to adenine nucleotide translocase. Laser confocal microscopy revealed significant changes in rearrangement of mitochondria in cardiac cells: while the mitochondria of variable shape and size appeared to be random-clustered in the cardiomyocytes of 1 day old rat, they formed a fine network between the myofibrils by the age of 3 weeks. These results allow to conclude that in early period of development, i.e. within 2-3 weeks, the diffusion of ADP to mitochondria becomes progressively restricted, that appears to be related to significant structural rearrangements such as formation of the mitochondrial network. Later (after 3 weeks) the control shifts to mi-CK, which by coupling to adenine nucleotide translocase, allows to maximally activate the processes of oxidative phosphorylation despite limited access of ADP through the OMM.

Mechanisms of thyroid hormone control over sensitivity and maximal contractile responsiveness to beta-adrenergic agonists in atria.

This paper discusses the mechanisms of two basic effects of thyroid hormones on atrial responses to beta-adrenergic agonists, i.e. increased inotropic sensitivity and decreased maximal contractile responsiveness. The increased sensitivity of atria to beta-adrenergic agonists under thyroid hormones appears to be related to increases in beta-adrenoceptor density and Gs/Gi protein ratio, leading to activation of Gs-mediated pathway, but suppression of Gi-mediated pathway of adenylate cyclase regulation. Therefore, the i/c concentrations of cAMP and corresponding inotropic responses achieve their maximums at lower doses of beta-adrenergic agonist. Thyroid hormones also decrease the expression of phospholamban, but increase the expression of sarcoplasmic reticulum Ca2+-pump. As a result, the basal activity of sarcoplasmic reticulum Ca2+-pump increases, but its beta-adrenergic activation through phosphorylation of phospholamban decreases. It is suggested that these changes are causal for decreased maximal inotropic and lusitropic responses of atria to beta-adrenergic agonists.

Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles.

The present study was undertaken to compare the effects of hypothyroidism and hyperthyroidism on sarcoplasmic reticulum (SR) Ca(2+)-pump activity, together with assessment of the functional role of SR in providing activator Ca2+ under these altered thyroid states. In response to a shift from hypothyroid to hyperthyroid state, a 10 fold and 2 fold increase in SR Ca(2+)-pump activity in atria and ventricles, respectively, were observed. This was associated with the 8-9 fold increases in atrial contractility (+dT/dt) and relaxation (-dT/dt), but only with a 3-4 fold increase in their ventricular counterparts. Also, the recirculation fraction of activator Ca2+ (RFA) increased to a far greater extent in atria (4 fold) than in papillary muscles, and the relative increment in inhibition of developed tension by ryanodine became 3 times larger in atria than in papillary muscles. A positive force-frequency relationship (FFR) was observed in hypothyroid atria, whereas the hyperthyroid atria, hypothyroid and hyperthyroid papillary muscles showed a negative FFR. These results suggest the greater role of transsarcolemmal (SL) Ca2+ and smaller role of SR Ca2+ in activating contraction in hypothyroid atria compared to other preparations. Thyroid hormones decrease the contribution of SL and increase that of SR in providing activator Ca2+ to the greater extent in atria than in ventricles. This effect of thyroid hormones is based on larger stimulation of SR Ca(2+)-pump in atria compared to ventricles.

Thyroid hormones increase the contractility but suppress the effects of beta-adrenergic agonist by decreasing phospholamban expression in rat atria.

OBJECTIVE: The aim of the present study was to characterize the relationships between the thyroid-hormone-dependent changes in sarcoplasmic reticulum (SR) Ca2+ handling and contractile performance in atria. METHODS: Hypothyroidism in rats was induced by adding 0.05% 6-n-propyl-2-thiouracil to their drinking water for 6 weeks. Hyperthyroidism was induced by daily subcutaneous injections of L-thyroxine (1 microgram/g body weight) to euthyroid rats for 1 week. Left atria from the hearts with different thyroid states were examined by means of contractile measurements, SR oxalate-supported Ca(2+)-uptake, and Western blot of SR proteins. RESULTS: The tissue level of SR Ca(2+)-pump protein decreased in hypothyroid (46 +/- 6%) atria, but remained unchanged in hyperthyroid (110 +/- 8%) atria as compared with euthyroid atria. Hypothyroidism was associated with increased phospholamban expression (141 +/- 25%), whereas it was drastically downregulated under hyperthyroidism (21 +/- 4%). The rate of SR Ca(2+)-uptake, measured in the presence of the protein kinase A inhibitor, H-89, was higher in hyperthyroid atria and lower in hypothyroid atria than in euthyroid atria (397 +/- 40, 55 +/- 6 and 194 +/- 17 nmol Ca2+/g protein/min, respectively). However, the stimulation of SR Ca(2+)-uptake by the catalytic subunit of protein kinase A was relatively weaker in hyperthyroid (130 +/- 20% over control level without catalytic subunit) and stronger in hypothyroid (640 +/- 60%) than in euthyroid atria (280 +/- 40%). The rates of inotropic contraction (+dT/dt) were higher in the hyperthyroid atria (133 +/- 10 mN/s), but lower in hypothyroid atria (15 +/- 3 mN/s) than in their euthyroid counterparts (95 +/- 13 mN/s). Inversely, hypothyroid atria responded to isoproterenol with much larger increases in contractility (883 +/- 164% over the control values for the same muscle before addition of isoproterenol) and hyperthyroid with smaller increases (25 +/- 9%) than euthyroid preparations (207 +/- 17%) CONCLUSIONS: Thyroid hormones increase the contractility, but decrease the inotropic response to isoproterenol through decreasing the phospholamban/SR Ca(2+)-pump ratio in rat atria.

Low particulate type IV phosphodiesterase activity in hypothyroid rat atria.

Rats were made hypothyroid by adding propylthiouracil (PTU) to their drinking water. Some of the PTU-treated rats were given thyroid hormone injections for 5 days. Both soluble and particulate cAMP-phosphodiesterase activities of adipose and ventricular tissues were increased by 25-60% in hypothyroidism. In left atria, soluble cAMP-phosphodiesterase activity was not significantly altered in hypothyroidism, while total particulate cAMP-phosphodiesterase activity was lowered by 30%. This lowering was due to diminished isoenzyme IV activity, as studied with the isoenzyme-specific inhibitors rolipram and SK&F 94836. In conclusion, the present results show decreased particulate type IV cAMP-phosphodiesterase activity in hypothyroid rat atria. This may explain the increased responsiveness to isoproterenol in hypothyroid atria.

Thyroid hormones and the creatine kinase system in cardiac cells.

The paper reviews the current evidence on the role of thyroid hormones in regulating the creatine kinase energy transfer system at multiple structures in cardiac cells. 1) Thyroid hormones modulate the overall synthesis of phosphocreatine (PCr) by increasing the rate of mitochondrial oxidative phosphorylation. 2) Thyroid hormones regulate the total activity of creatine kinase and its isoenzyme distribution. In comparison with normal thyroid state (euthyroidism), hypothyroidism is characterized by decreased total creatine kinase activity owing to diminished fraction of creatine kinase. On the other hand, hyperthyroidism, while causing no change in total creatine kinase activity, leads to increased fractions of neonatal isoforms of creatine kinase, and, in case of prolonged hyperthyroidism, to decreased fraction of mitochondrial creatine kinase. The latter change is associated with partial uncoupling between mitochondrial creatine kinase and adenine nucleotide translocase reflected by decreased PCr/O ratio. 3) Hyperthyroidism leads to increased passive sarcolemmal permeability due to which the leakage of creatine along its concentration gradient occurs. As a result of (i) increased sarcolemmal permeability for creatine, (ii) uncoupling of mitochondrial PCr synthesis, and (iii) increased energy utilization rate the steady state intracellular PCr content decreases under hyperthyroidism which, in turn, increases the myocardial susceptibility to hypoxic damage. Thyroid state also modulates the protective effects of exogenous PCr on energetically depleted myocardium.

Enhanced negative inotropic effect of an adenosine A1-receptor agonist in rat left atria in hypothyroidism.

Left atria were isolated from rats made hypothyroid by adding propylthiouracil to their drinking water, such rats after saturating doses of thyroid hormones, and from control rats. Isoproterenol (ISO; 1 microM) increased the values of developed tension (DT), maximal rate of tension development (+dt/dt) and tension fall (-dT/dt). The effect was largest in hypothyroid and lowest in hyperthyroid atria. The adenosine A1-receptor agonist N6-(phenylisopropyl)-adenosine (PIA) had a powerful negative inotropic effect in ISO-stimulated atria. The effects of PIA on +dT/dt, -dT/dt and DT were enhanced in hypothyroidism. Adenosine receptor number was not decreased. The amount of total Gi-like proteins was estimated by pertussis toxin labeling. The amounts of Gi2 and Gi3 were estimated in Western blots using such antisera raised in rabbits against peptides corresponding to parts of their sequences, using purified recombinant alpha subunits as standards. The amounts of low and high molecular weight forms of Gs were estimated by cholera toxin labeling Gi2, Gi3 and pertussis toxin substrate concentrations were slightly lower in the hypothyroid animals, while the amounts of both forms of Gs per mg of protein were only half of those in euthyroid rat atria. The levels of Gi2 and Gi3 were greatly elevated as compared to Gs as membrane marker. These changes were reversed by treatment of the hypothyroid rats with thyroid hormones. In conclusion, the present results show an enhanced negative inotropic effect of an adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone.

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca(2+)-channels, Na(+)-Ca2+ exchange and Ca(2+)-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca(2+)-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development +(dP/dt)max and pressure fall -(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and -(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca2+ and Ca2+ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca2+ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [3H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (Bmax) without any change in the dissociation constant for receptor-ligand complex (Kd) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca(2+)-stimulated ATPase, ATP-dependent Ca2+ uptake and ouabain-sensitive Na(+)-K+ ATPase were decreased whereas the Mg(2+)-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na(+)-K+ ATPase activity, whereas Ca(2+)-stimulated ATPase, ATP-dependent Ca2+ uptake, and Mg(2+)-ATPase activities were unchanged. The Vmax and Ka for Ca2+ of cardiac sarcolemmal Na(+)-Ca2+ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca(2+)-transport processes is regulated by thyroid hormones and the modification of Ca(2+)-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.

Thyroid control of contractile function and calcium handling in neonatal rat heart.

Newborn rats were rendered hyperthyroid (daily subcutaneous injections of L-triiodothyronine, 10 micrograms 100 g-1 body weight) or hypothyroid (0.05% 6-n-propyl-2-thiouracil in drinking water to nursing mothers) during the first 3 weeks of postnatal life. Compared with the euthyroid group, hyperthyroidism resulted in: (1) cardiac enlargement with right ventricular preponderance, (2) increased cardiac contractile function, (3) increased Ca2+ uptake by the sarcoplasmic reticulum (SR), (4) decreased sensitivity to the negative inotropic effect of verapamil and (5) greater inhibition of contractile function by ryanodine. Hypothyroidism generally resulted in opposite changes. The data suggest that the development of the heart and its contractile function during early postnatal life depends on the plasma level of thyroid hormones. In particular, the relative contribution of the SR and sarcolemmal Ca2+ transport to the control of cardiac contractility seems to be markedly affected by altered thyroid states. The postnatal maturation of the SR function is accelerated in hyperthyroidism but retarded in hypothyroidism. Consequently, hyperthyroid hearts appear to be less dependent and hypothyroid ones more dependent on trans-sarcolemmal Ca2+ fluxes when compared with age-matched euthyroid animals.

Thyroid control over membrane processes in rat heart.

We have studied the effects of hypo- and hyperthyroidism on sarcolemmal (SL) and sarcoplasmic reticular (SR) ion transport processes and mitochondrial energy production in rat heart. The following conclusions were derived. 1) Compared with euthyroid state, hyperthyroidism led to increased SR Ca(2+)-accumulation. In SL, the activities of Ca(2+)-stimulated adenosine triphosphatase (ATPase), ATP-dependent Ca2+ pumping, and Na(+)-Ca2+ exchanger were not affected; but ouabain-sensitive Na(+)-K(+)-ATPase activity was enhanced. 2) Hypothyroidism resulted in depressed activities of Ca2+ pumps both in SL and SR. In SL, the Na(+)-K(+)-ATPase activity was decreased, but Na(+)-Ca2+ exchange was unaltered. 3) Thus slower relaxation of the hypothyroid myocardium may be attributed to depressed functioning of Ca2+ pumps in SR and SL, whereas faster relaxation of the hyperthyroid heart may be based on increased Ca(2+)-pumping activity of SR. 4) Hyperthyroidism and hypothyroidism, respectively, led to enhanced and decreased rates of mitochondrial phosphocreatine synthesis. The thyroid state appears to control the functional coupling between mitochondrial creatine kinase and ATP-ADP translocase: the energy of oxidative phosphorylation was transformed into phosphocreatine more effectively in mitochondria from hypothyroid hearts than in those from hyperthyroid hearts.

The effects of thyroid state on sarcomere dynamics of ventricular cells and contraction of papillary muscles in the rat heart.

Diastolic sarcomere length, amplitude of maximal sarcomere shortening, maximal rate of sarcomere shortening, maximal rate of sarcomere re-lengthening, time to peak sarcomere shortening and trans-sarcolemmal ion currents were measured in isolated ventricular cells from euthyroid, hypothyroid and hyperthyroid rats. The data were compared with the developed tension and time to peak tension of papillary muscles. The diastolic sarcomere length was not affected by the changes in thyroid state. Hypothyroidism led to an increased time to peak sarcomere shortening, an increased time to peak tension of the papillary muscle, and a depression of the maximal rates of shortening and elongation of the sarcomeres. Changes in dynamics of the sarcomere and contraction of papillary muscle did not occur in parallel under the influence of hyperthyroidism. In comparison with the euthyroid state, the time to peak tension was shortened and amplitude of shortening of the sarcomere was increased. The time to peak shortening of the sarcomere and developed tension of papillary muscle remained unaltered. In cardiac cells, hypothyroidism was associated with a decreased slow Ca2+ current and hyperthyroidism with an increased slow Ca2+ current. In contrast to euthyroid and hypothyroid cardiac cells, the hyperthyroid cardiomyocytes exhibited a trans-sarcolemmal transient inward current after repolarization. Both hypothyroidism and hyperthyroidism resulted in a depressed potentiation of sarcomere shortening and myocardial developed tension after resting.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of stimulation frequency responses and calcium dependency of functional parameters in hyperthyroid rat ventricular papillary muscles.

The effects of stimulation frequency (0.2-1.5 Hz) and extracellular calcium concentration ([Ca2+]o) (0.6-15.0 mM) on the contractile function of thin papillary muscles of euthyroid and hyperthyroid rats were studied. Hyperthyroidism led to a decrease in developed tension (DT) and time to peak tension (TPT), but it exhibited no influence on the maximal rates of contraction (+dT/dt) and relaxation (-dT/dt). Also, the mean rates of contraction were similar in euthyroid and hyperthyroid muscle groups. The increase in stimulation frequency brought about a marked decrease in DT, +dT/dt, and -dT/dt of euthyroid papillary muscles at lower frequencies in comparison to papillary muscles in the hyperthyroid group. At stimulation frequencies above 1.0 Hz, the absolute and relative levels of DT and -dT/dt of hyperthyroid myocardium were elevated over euthyroid preparations. At the same time, TPT was unchanged in any of the muscle groups. Hyperthyroidism modulated the relationships between contractile parameters and [Ca2+]o. At a [Ca2+]o of 1.0-4.0 mM, the DT of hyperthyroid papillary muscles was lower than in euthyroid muscle. At 4.0 and 8.0 mM of [Ca2+]o, the equal values of maximal DT were registered for euthyroid and hyperthyroid papillary muscles, respectively. An increase in the [Ca2+]o in the range of 1.0-15.0 mM was accompanied by an increase in TPT of both muscle groups, but to a greater extent in hyperthyroid myocardium. In conclusion, the myocardium of hyperthyroid rat appeared to exhibit decreased sensitivity to calcium as well as to the negative inotropic effect of enhanced stimulation frequency. Alterations of the processes of transsarcolemmal movement and intracellular recycling of Ca2 may be implicated.

[Synthesis of phosphocreatine in heart mitochondria of rats with hyperthyroidism]. / Sintez fosfokreatina v mitokhondriiakh gipertireoidnogo miokarda krysy.

The mechanisms of the phosphocreatine/creatine ratio decrease in female Wistar rats with hyperthyroidism were studied. L-Thyroxin was injected to animals in doses of 50 and 100 micrograms/100 g of body weight, daily for 1 and 2 weeks. Oxidative phosphorylation and the rate of phosphocreatine synthesis were studied in isolated rat heart mitochondria. It was found that hyperthyroidism caused an increase in the ADP-activated mitochondrial respiration, whereas the coupling between electron transport and ADP phosphorylated remained at a constant level. Besides oxidative phosphorylation, activation, hyperthyroidism increased the rate of phosphocreatine synthesis at high values of the phosphocreatine/oxygen ratio. Thus, hyperthyroidism is unaccompanied by and significant changes in the coupling of mitochondrial creatine kinase with oxidative phosphorylation.

Characteristics of Ca2(+)-stimulated ATPase in rat heart sarcolemma in the presence of dithiothreitol and alamethicin.

We have studied the activities of Ca2(+)-stimulated ATPase in rat heart sarcolemma upon modulating the redox state of membrane thiol groups with dithiothreitol (DTT). The suitability of alamethicin to unmask the latent activity of this enzyme was also investigated. The Ca2(+)-stimulated ATPase in sarcolemma exhibited two activation sites--one with low affinity (Km = 0.70 +/- 0.2 mM; Vmax = 10.0 +/- 2.2 mumol Pi/mg/h) and the other with high affinity (Km = 0.16 +/- 0.7 mM; Vmax = 4.6 +/- 0.8 mumol Pi/mg/h) for Mg2+ ATP. Alamethicin at a ratio of 1:1 with the sarcolemmal protein caused a 3-fold activation of Ca2(+)-stimulated ATPase without affecting its sensitivity to Ca2+ or Mg2+ ATP. Treatment of sarcolemma with deoxycholate or sodium dodecyl sulfate resulted in a total loss of the enzyme activity; high concentrations of alamethicin also showed a detergent-like action on the sarcolemmal vesicles. DTT at 5-10 mM concentrations caused a 4-5 fold activation of Ca2(+)-stimulated ATPase in sarcolemma and this effect was observed to be dependent on the concentration of Mg2+ ATP. DTT increased the affinity of the enzyme to Mg2+ ATP at the high affinit site and enhanced the Vmax at the low affinity site in addition to increasing the sensitivity of Ca2(+)-stimulated ATPase to Ca2+. DTT protected the Ca2(+)-stimulated ATPase against deterioration by detergents and restored the enzyme activity after treatment with N-ethylmaleimide. The mechanism of action of DTT on Ca2(+)-stimulated ATPase may involve the reduction of essential thiols at the active site of the enzyme or its interaction with specific DTT-dependent inhibitor protein. No changes in the sensitivity of sarcolemmal Ca2(+)-stimulated ATPase to orthovanadate was evident in the absence or presence of DTT and alamethicin. The results suggest the use of both DTT and alamethicin for the determination of Ca2(+)-stimulated ATPase activity in sarcolemmal preparations.

[Characteristics of the frequency dependence of the contraction force in the hyperthyroid rat myocardium]. / Osobennosti chastotnoi zavisimosti sily sokrashcheniia gipertireoidnogo miokarda krysy.

We have studied the effect of increased contraction frequency (from 0.2 to 1.5 Hz) on developed tension (delta T) in thin papillary muscles of eu- and hyperthyroid rats. The results show that while increasing the contraction frequency, the delta T of euthyroid papillary muscles decreased at lower frequencies than in hyperthyroid group. Also, at the contraction frequencies above 1.0 Hz the absolute and relative levels of delta T of hyperthyroid myocardium were less decreased than in euthyroid preparations. In conclusion, the myocardium of hyperthyroid rat is characterized by a decreased sensitivity to negative inotropic effect of enhanced contraction frequency. In is probably due to the acceleration of the processes of intracellular Ca2+ recycling during diastole under the influence of hyperthyroidism.

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.