Synchronous In Situ ATPase Activity, Mechanics, and Ca Sensitivity of Human and Porcine Myocardium

Flash-frozen myocardium samples provide a valuable means of correlating clinical cardiomyopathies with abnormalities in sarcomeric contractile and biochemical parameters. We examined flash-frozen left-ventricle human cardiomyocyte bundles from healthy donors to determine control parameters for isometric tension (P_o) development and Ca²⁺ sensitivity, while simultaneously measuring actomyosin ATPase activity in situ by a fluorimetric technique. P_o was 17 kN m⁻² and pCa_50% was 5.99 (28°C, I = 130 mM). ATPase activity increased linearly with tension to 132 μM s⁻¹. To determine the influence of flash-freezing, we compared the same parameters in both glycerinated and flash-frozen porcine left-ventricle trabeculae. P_o in glycerinated porcine myocardium was 25 kN m⁻², and maximum ATPase activity was 183 μM s⁻¹. In flash-frozen porcine myocardium, P_o was 16 kN m⁻² and maximum ATPase activity was 207 μM s⁻¹. pCa_50% was 5.77 in the glycerinated and 5.83 in the flash-frozen sample. Both passive and active stiffness of flash-frozen porcine myocardium were lower than for glycerinated tissue and similar to the human samples. Although lower stiffness and isometric tension development may indicate flash-freezing impairment of axial force transmission, we cannot exclude variability between samples as the cause. ATPase activity and pCa_50% were unaffected by flash-freezing. The lower ATPase activity measured in human tissue suggests a slower actomyosin turnover by the contractile proteins.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

Na+ and K+ binding by glycerinated muscle fibers with reciprocal cation concentrations in the medium

The binding of Na+ and K+ by glycerinated muscle fibres was observed at reserve concentrations of NaCl in the medium. Under external concentrations of Na+ of K+ up to 0.4-0.5 mM, a constant fraction (0.15-0.25 mmoles/kg dry weight of the fibres) bound by glycerinated fibres was revealed. With the increase of NaCl or KCl concentration in the medium up to 10 mM the concentration of bound cations increased too. The parameters of Na+ and K+ sorption by glycerinated models were calculated. The values of Na+ and K+ binding limits were 4.4 and 1.8 mmole/kg dry weight of the fibres and those of affinity, 3.2 and 4.1 kcal/mol, respectively. The binding of one cation took place in conditions when its concentration was 10,000-20,000 fold less than that of the other cation. This points to the fact that Na+ and K+ binding is highly specific and is carried out by different centres. It is suggested that myosin ATPase is a substratum binding Na+ and K+ in glycerinated muscle fibres at reverse ratio concentrations of these cations in the medium.     

ATP-ASE ACTIVITY IN ISOLATED FLAGELLA OF CHLAMYDOMONAS SNOWIAE

ATPase in isolated glycerinated flagella of Chlamydomonas snowiaeis shown to be inhibited by high ATP concentrations, by DNPat pH 6.0 and by salyrgan. The enzyme is activated by Mg⁺⁺ andthe activation is reversed by Ca⁺⁺. A number of other factorswhich can affect the phototactic behaviour of the cells do notaffect ATPase activity, indicating that the response of directionalmotility is under separate control in the intact cell. ¹ This work is based on part of an M. Sc. thesis of I.C.-K.     

Modification of the contractile properties of rabbit skeletal muscle by ethylene glycol

The effect of ethylene glycol on the contractile properties of skeletal muscles was studied using glycerinated rabbit psoas muscle fibers. Measurements were made at an ionic strength of 0.2 M, pH 7.0, and at 10 degrees C. Ethylene glycol reversibly reduced isometric tension, active stiffness, the tension-to-stiffness ratio, and the shortening velocity at zero load (Vo) in a dose-dependent fashion. Ethylene glycol also reduced the Ca sensitivity for contraction. The extent of the reduction in Vo by ethylene glycol was much larger than that in the actomyosin ATPase activity reported by Travers and Hillaire (Eur. J. Biochem. 98, 293-299 [1979]). Although ethylene glycol reduced tension and Vo, the MgATP concentration dependence of these two quantities was almost unaffected. These results suggest that in the presence of ethylene glycol, force produced by crossbridges in the principal force-producing state is reduced and/or the relative population of the attached crossbridges in the low-force state increases. The results also suggest that the reduction in Vo by ethylene glycol is caused not only by a reduction in the actomyosin ATPase activity but also by a reduction in the shortening distance per mole of ATP split.     

Effects of pH on contraction of rabbit fast and slow skeletal muscle fibers.

We have investigated (a) effects of varying proton concentration on force and shortening velocity of glycerinated muscle fibers, (b) differences between these effects on fibers from psoas (fast) and soleus (slow) muscles, possibly due to differences in the actomyosin ATPase kinetic cycles, and (c) whether changes in intracellular pH explain altered contractility typically associated with prolonged excitation of fast, glycolytic muscle. The pH range was chosen to cover the physiological pH range (6.0-7.5) as well as pH 8.0, which has often been used for in vitro measurements of myosin ATPase activity. Steady-state isometric force increased monotonically (by about threefold) as pH was increased from pH 6.0; force in soleus (slow) fibers was less affected by pH than in psoas (fast) fibers. For both fiber types, the velocity of unloaded shortening was maximum near resting intracellular pH in vivo and was decreased at acid pH (by about one-half). At pH 6.0, force increased when the pH buffer concentration was decreased from 100 mM, as predicted by inadequate pH buffering and pH heterogeneity in the fiber. This heterogeneity was modeled by net proton consumption within the fiber, due to production by the actomyosin ATPase coupled to consumption by the creatine kinase reaction, with replenishment by diffusion of protons in equilibrium with a mobile buffer. Lactate anion had little mechanical effect. Inorganic phosphate (15 mM total) had an additive effect of depressing force that was similar at pH 7.1 and 6.0. By directly affecting the actomyosin interaction, decreased pH is at least partly responsible for the observed decreases in force and velocity in stimulated muscle with sufficient glycolytic capacity to decrease pH.     

Ca++ controlled contraction-relaxation cycle in glycerinated amoeboid cells

Summary Contraction and relaxation in glycerinated amoeba models are controlled by the same factors which are known to control streaming in demembranated, living cytoplasm of amoeba. Threshold calcium concentration for the transition from relaxed to contracted state of glycerinated model was found to be about 10^–6 M.     

The chemo-mechanical coupling relation in the oscillatory contraction-relaxation cycles of insect fibrillar muscle.

The mechanical properties and the activity of the myofibrillar ATPase have been investigated at 21 degrees C on glycerinated back muscle from the water-bug Lethocerus colossicus. When the fibres were held under isometric conditions after stretching them by 0.5--4%, the ATPase required to maintain a given tension increases from 19 to 39 p-moles ATP split for each mg of tension developed as the Ca2+ level is increased from 10(-7) to up to 10(-5) M. The mechanical properties and the ATPase activity have been determined for Ca2+-activated fibres using sinusoidal frequencies of 1--30 HZ and oscillatory amplitudes of 0.5--6% peak-to-peak. In this way the R.M.S. velocity of sinusoidal movement was varied between 0.1-10 mm/sec. The rate of ATP splitting associated with oscillatory tension development, the dynamic tension cost, increases both with Ca2+ and with frequency of oscillation (at 1% peak-to-peak amplitude), becoming as high as four times the isometric value. The oscillatory power output which can be obtained is increased when the Ca2+ level is raised from 10(-7) to 10(-5) M or towards higher amplitudes of oscillation. The chemo-mechanical coupling efficiency increases proportionally with the R.M.S. velocity of muscle movement. In presence of 10(-5) M Ca2+ optimal efficiencies of 5.5--6.2 kcal work per mole ATP split are obtained at R.M.S. velocities of 1.3--2 muscle lengths/sec. The ability of the muscle fibres to perform osciillatory work at the higher frequencies was much reduced at lower Ca2+ levels of 10(-6) or 10(-7) M and the maximal efficiencies never exceeded 2.2 kcal/mole.     

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.     

On the role of sulfhydryl groups in the ATPase activity and pellet height response of Tetrahymena cilia

The effects of N-ethylmaleimide and p-hydroxymercuribenzoate on the ATPase activity of glycerinated Tetrahymena cilia, of 30 S dynein extracted from the cilia, and on the residual ATPase remaining after extraction were studied and correlated with the effects of these reagents on the pellet height response of these cilia. Simultaneous addition of N-ethylmaleimide and ATP to cilia caused a slight inhibition of ATPase activity. Preincubation of the cilia with low N-ethylmaleimide in the absence of ATP, however, enhanced the ATPase activity; the enhancement decreased with increasing time of preincubation. Preincubation of cilia with high N-ethylmaleimide caused increasing inhibition. p-Hydroxymercuribenzoate was more potent than N-ethylmaleimide, usually causing only an inhibition which increased if the cilia were preincubated with p-hydroxymercuribenzoate in the absence of ATP.The pellet height response of these cilia, which serves as a convenient assay of some events related to ciliary motility, was inhibited about 50% by high concentrations of N-ethylmaleimide in the presence of ATP. Preincubation of the cilia with low concentrations of N-ethylmaleimide led to complete loss of the pellet height response. p-Hydroxymercuribenzoate was a more potent inhibitor of the pellet height response than N-ethylmaleimide; complete inhibition was attained even in the presence of ATP, while preincubation with a low concentration of p-hydroxymercuri-benzoate caused a very rapid loss of pellet height response.The ATPase activity of the crude dynein obtained by extraction of cilia and removal of the axonemes was approximately doubled by preincubation with N-ethylmaleimide. 30 S Dynein, obtained from the crude dynein by sedimentation on a sucrose density gradient, was slightly inhibited by N-ethylmaleimide; p-hydroxy-mercuribenzoate was more potent. The residual ATPase activity remaining on the axonemes after two extractions was also only inhibited by N-ethylmaleimide and by p-hydroxymercuribenzoate.These results demonstrated that SH groups influence both the ATPase activity of dynein and the pellet height response of glycerinated cilia. The possible significance of the similarity in enhancing effect of N-ethylmaleimide on cilia ATPase and on myosin ATPase was discussed.     

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.     

Effect of polyamines on actomyosine-ATP-ase activity in smooth muscles of the uterus

The ability of the aliphatic polyamines to inhibit [figure: see text] the ATPase activity of smooth muscle actomyosine satisfies the succession: spermine > spermidine > putrescine that is correlated with magnitude of positive charge at physiological value of pH. The most effective inhibitor of the ATP hydrolysis process is the spermine, which highest inhibitory action is manifested at 10(-3) M concentration, in lesser concentration (10(-5) M) activates the actomyosine ATPase. While defining the kinetic parameters of the ATP hydrolysis reaction catalyzed by uterus myometrium the correlation between inhibiting the ATPase activity of myometrium contractile complex under introduction into the incubation medium of 10(-3) M spermine and decreasing the affinity of actomyosine for ATP was made; the activating effect of spermine on ATPase activity of actomyosine complex in the presence of 10(-5) M spermine correlated with the increase of actomyosine affinity for Mg2+.     

Cyclic AMP regulation of myosin isozymes in mammalian cardiac muscle

Hyperpermeable cells from rat heart contain a cAMP-dependent system that can increase the maximum Ca-activated force (contractility) of the contractile proteins. In two different conditions where the relative concentration of the myosin isozymes changes, i.e., hypothyroidism and aging, the size of the increase in contractility from activation of the cAMP-regulated system varies closely with the relative concentration of V1, the isozyme of myosin with the greatest Ca- and actin-activated ATPase activity. The existence of another system for the regulation of the slow isozyme V3 has been demonstrated, and it may be inhibited by beta-adrenergic activity. The possibility of cAMP-dependent myosin regulation of contraction in addition to Ca regulation of troponin is considered. Phosphorylation of the contractile proteins themselves is not required for the increased contractility.     

Effect of calcium on the temporal characteristics of muscle contraction

It is shown that the shape of mechanical relaxation time spectrum of single glycerinated rabbit fibre m. psoas changed with calcium concentration. When increasing Ca++ of the rate constant of delayed tension development increases several times in the absence of inorganic phosphate. Addition of 10 mM of inorganic phosphate increases this rate constant and sharply decreases its calcium dependence. These data are discussed in terms of possible connection between cross-bridges by means of ATPase reaction product-inorganic phosphate.     

Effect of phosphorylation of porcine cardiac troponin I by 3':5'-cyclic AMP-dependent protein kinase on the actomyosin ATPase activity

1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of Pi per mol of troponin I. 2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5 x 10(-8) M-2.4 x 10(-5) M). Half-maximum activation of ATPase was obtained with a concentration of 7 x 10(-7) M Ca2+ (unphosphorylated) and 1.3 x 10(-6) M Ca2+ (phosphorylated), respectively. Maximum ATPase activity was reached with 3 x 10(-6) M Ca2+ (unphosphorylated) and 1.0 x 10(-5) M Ca2+ (phosphorylated). 3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect of ATPase activity. 4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.     

Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA-ATPase that enhanced the superprecipitation of actomyosin

1. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, activated the superprecipitation of myosin B (natural actomyosin) from rabbit skeletal muscle. The maximum change in the turbidity increased with increasing purealin concentrations and was three times the control value in the presence of 50 microM purealin. 2. The ATPase activity of myosin B was also elevated to 160% of the control value by 10 microM purealin. On the other hand, purealin inhibited the myosin ATPase in the presence of 10 mM CaCl2 and 0.5 M KCl (Ca2+-ATPase), and the concentration for the half inhibition was 4 microM. 3. On the other hand, purealin activated the myosin ATPase in the presence of 5 mM EDTA and 0.5 M KCl (EDTA-ATPase). The maximum activation by 10 microM purealin was 160% of the control value. 4. Furthermore, similar results concerning the modification of ATPase activities by purealin were obtained in myosin subfragment-1 instead of myosin. 5. These results suggest that purealin activates the superprecipitation of myosin B by affecting the myosin heads directly. It is also an interesting observation that there is a correlation between the activities of the myosin EDTA-ATPase and actomyosin ATPase of myosin B.     

Calcium requirements of cardiac myofibril ATPase activity following exhaustive exercise

Myocardial contractility is reduced in rats following strenuous activity. Thus, the purpose of this study was to determine some of the cellular mechanisms that may contribute to the depressed contractile function. Myofibril ATPase activity was determined with varying free calcium and monomeric vanadate (Vi) concentrations. The Mg2+ stimulated myofibril ATPase activities were significantly reduced in the activity group (E). Myofibril ATPase activity from control animals increased from 0.056 +/- 0.021 to 0.216 +/- 0.030 mumol X Pi X mg-1 X min-1 with 0.1-10.0 microM Ca2+. The addition of 15.0 microM Vi resulted in a 37% decrease in ATPase activity of C animals. With regard to the experimental group, the myofibril ATPase activity at 0.1 and 1.0 microM Ca2+ were depressed (P less than 0.05) with the values at 5.0 and 10.0 microM Ca2+ being similar to the control group (P greater than 0.05). Incubations with Vi resulted in an enhanced myofibril ATPase activity for E compared to C animals. The ATPase activities were increased by 17, 10, 10 and 15% at 3.0, 5.0, 10.0 and 15.0 microM Vi. The results suggest that the exhaustive exercise raises the CA2+ requirement for half-maximal activation of cardiac myofibril ATPase activity and that the contracto-regulatory mechanism of cardiac muscle is similarly altered.     

Mg-ATPase and CA2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts – effects of calcium sensitizing agents MCI-154, DPI 201–106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca²⁺ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca²⁺ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10^–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca²⁺ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca²⁺ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca²⁺ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.     

Thyroxine-induced cardiomegaly: assessment of nucleic acid, protein content and myosin ATPase of rat heart.

1 mg/kg L-thyroxine was administered to rats for 14 days to evaluate the potential of the hyperthyroid state to induce heart hypertrophy and its effect on myosin adenosine-triphosphatase (ATPase) activity. Evidence of hyperthyroidism such as weight loss, elevation of rectal temperature, increased heart rate and oxygen consumption, was observed in all treated rats. Cardiac enlargement was determined by comparison of wet and dry ventricle weights, myocardial RNA, DNA and protein content. Wet and dry ventricle weights and the level of cardiac RNA and protein were augmented by thyroxine treatment. ATPase activity of cardiac myosin was stimulated as the Ca2+ concentration in the incubation medium increased. No difference was found in Ca2+-activation, salt sensitivity or ATPase activity of unreacted and sulphydrylmodified cardiac myosins from euthyroid or hyperthyroid groups. The results showed that in hyperthyroid rats, in contrast to some other species, the biochemical mechanism responsible for the enhancement of cardiac contractility is not an increased myosin ATPase.