Biomedical subjects

R J Solaro

Publications and source records attributed to R J Solaro.

At least 109 records · Page 6Linked to original sources

PubMed1988

Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts.

Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation.

Animals↗

PubMed1988

Calcium transport properties of cardiac sarcoplasmic reticulum from cardiomyopathic Syrian hamsters (BIO 53.58 and 14.6): evidence for a quantitative defect in dilated myopathic hearts not evident in hypertrophic hearts.

Calcium uptake was measured in homogenates and microsomal preparations enriched in sarcoplasmic reticulum vesicles isolated from hearts of hypertrophic (BIO 14.6) and dilated (BIO 53.58) cardiomyopathic as well as control (F1B) Syrian hamsters at 3, 7, 9, and 11 months of age. Calcium uptake studies were done using the Millipore filtration technique under conditions known to restrict transport to the sarcoplasmic reticulum. Steady-state calcium uptake capacity was used as a measure of the relative amounts of sarcoplasmic reticulum in homogenates prepared from individual hearts. At 3 months of age, there were no differences in calcium uptake in homogenates from control or myopathic hearts. However, by 9 months, although calcium uptake of homogenates from control and hypertrophic hearts was the same, calcium uptake by homogenates from dilated hearts was significantly depressed both in initial rate and capacity. Similar trends were seen in the microsomal vesicle preparations, but the decrease in calcium uptake in the dilated hearts was significantly lower by 3 months of age. The catalytic activity of the sarcoplasmic reticulum transport enzyme was estimated from the ratio of velocity to capacity, which provides a measure of the fractional rate of filling of the sarcoplasmic reticulum with calcium. The velocity-to-capacity ratios were not different at any of the ages in both the homogenate and microsomal preparations. The results of this study demonstrate that a major defect in the dilated cardiomyopathy may be due to a decrease in the volume or number of sarcoplasmic reticulum calcium transport sites rather than a decrease in specific activity of the transport enzyme.

Algorithms↗

PubMed1988

Effects of acidosis on ventricular muscle from adult and neonatal rats.

We compared the response of ventricular muscle from adult and neonatal rats to hypercapnic acidosis. In adult muscle, acidosis caused an initial rapid fall of developed tension to 30 +/- 5% of control (mean +/- SEM, n = 6). However, tension recovered slowly to a steady state that was 56 +/- 6% of control. In neonatal muscle, acidosis caused a significantly smaller initial fall in tension to 43 +/- 3% (n = 8, p less than 0.05), but the tension then showed a subsequent slower fall to a steady state that was 29 +/- 4% of control, significantly less than in the adult (p less than 0.01). We have attempted to identify the mechanisms underlying these differences in response. In detergent-skinned myofibrils, reducing the pH from 7.0 to 6.5 caused a reduction in the pCa50 of 0.61 units in the adult muscle, but only 0.27 units in the neonatal ventricular muscle. Myofibrillar Ca2+ sensitivity in neonatal ventricular muscle is thus less susceptible to the effects of acidic pH than that of adult muscle. Since intracellular pH decreases rapidly on application of increased external CO2, these results are consistent with the finding that, initially, developed tension in neonatal muscles is less sensitive to the effects of acidosis. Sodium dodecylsulfate gel electrophoresis of myofibrillar preparations from adult and neonatal rats demonstrated differences in thin filament proteins, including troponin I, which may underlie the observed differences in Ca2+ sensitivity. In adult rat ventricular muscles, the slow recovery of tension during acidosis is associated with an increase in the amplitude of the Ca2+ transients to 263 +/- 34% of control (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis, Respiratory↗

PubMed1988

Sensitization of dog and guinea pig heart myofilaments to Ca2+ activation and the inotropic effect of pimobendan: comparison with milrinone.

We compared the effects of the newer inotropic drugs, pimobendan (UD-CG 115 BS) and milrinone (Win 47203), on the electrical, mechanical and biochemical activity of intact and detergent-skinned preparations of cardiac muscle. Both of these agents increased contractile force of guinea pig papillary muscle preparations bathed under physiological conditions or depolarized with 25 mM K+o. The positive inotropic action was associated with potentiation of the Ca2+-dependent slow action potentials (APS). Contractile force developed in the presence of 25 mM [K]o and 1 microM isoproterenol was increased further by addition of 50 microM pimobendan with no effect on the slow action potential. Milrinone (50 microM) did not produce a further increase in the force or potentiate the slow APs. Pimobendan, in a dose-dependent manner, increased active tension developed by chemically-skinned dog heart muscle fibers at submaximally activating concentrations of Ca2+, whereas milrinone did not. At pCa 6.25, the half-maximal concentration of pimobendan for stimulation of force development was about 40 microM. At maximally activating levels of Ca2+ (pCa 4.5), pimobendan had little or no effect on force development. The effect of pimobendan on force was paralleled by changes in the Ca2+-activated Mg-ATPase activity of the isometric skinned fiber preparations. Moreover, the tension-cost (unit increase in ATPase rate/unit increase in force) was unchanged in the presence of pimobendan. Milrinone did not affect ATP hydrolysis by the skinned fiber preparations. Force-pCa and ATPase-pCa relations of skinned fiber preparations contracting isometrically were shifted to the left by 0.15-0.20 pCa units in the presence of 50 microM pimobendan. In contrast, there was no effect of pimobendan on the ATPase activity of unloaded myofibrillar preparations. The stimulation of force and ATPase activity of the skinned heart muscle fibers could be accounted for by an effect of pimobendan on the affinity of the regulatory (low affinity, Ca2+-specific) binding sites of cardiac troponin C. Ca2+ binding to the "structural" high affinity sites of troponin C was slightly inhibited. The results indicate that the positive inotropic actions of pimobendan, but not milrinone, may involve activation of the cardiac myofilaments by a direct effect involving an increased affinity of the regulatory site on troponin C for Ca2+.

Actin Cytoskeleton↗

PubMed1988

Differential effects of d- and l-pimobendan on cardiac myofilament calcium sensitivity.

The effects is of the optical isomers of pimobendan (UD-CG 115 BS), an inotropic agent, were studied on the electrical and mechanical activity of intact and detergent-skinned preparations of cardiac muscle from guinea pig and dog. Racemic pimobendan has been shown to increase contractile force and to potentiate slow action potentials (AP) induced by stimulation of papillary muscle partially depolarized with 25 mM [K]o. These effects are shown in this study to be mainly due to the l-optical isomer of pimobendan. When slow APs were maximally stimulated by 1 microM isoproterenol, addition of either the d- or l-isomer of pimobendan did not affect the slow AP parameters. However, under these conditions, contractile force was significantly increased to 124% of control by the d-isomer and to 184% of control by the l-isomer. These results suggest that pimobendan may have direct effects on the myofilaments and that these effects are dependent on the optical isomer of the compound. To test this directly, the effects of d- and l-pimobendan were compared on Ca++-activated force developed by detergent-skinned heart muscle fibers. Submaximal force developed at constant Ca++ was increased by both optical isomers, but the l-isomer had a significantly greater Ca++-sensitizing effect. For example at pCa 6.75 force was 270% of control in the presence of the d-isomer and 400% of control in the presence of the l-isomer. At pCa 5, there was no effect of either isomer on force developed by the skinned fiber preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1987

Calmidazolium, a calmodulin antagonist, stimulates calcium-troponin C and calcium-calmodulin-dependent activation of striated muscle myofilaments.

Although regulatory Ca2+-binding domains of calmodulin (CaM) and troponin C (TnC) are similar, it is interesting that agents that act as CaM antagonists appear to be TnC "agonists" in that they sensitize cardiac myofilaments to activation by Ca2+ (El-Saleh, S., and Solaro, R. J. (1987) Biophys. J. 51, 325 (abstr.). This indicates that the effects of agents that react with Ca2+-binding proteins may depend on protein-protein interactions involved in a particular Ca2+-dependent process. In experiments described here, we have explored this idea by testing effects of calmidazolium (CDZ), a potent calmodulin antagonist on striated muscle myofilaments regulated by cardiac TnC, skeletal TnC, and CaM. CDZ was shown to increase submaximal calcium activation of myofilament force and ATPase activity in both cardiac and skeletal muscle, but the effect was greater in the case of the cardiac preparations. In the presence of 10 microM CDZ, the free Ca2+ giving half-maximal activation was reduced to about 60% of the control value in the case of cardiac myofilaments. Analogous differential effects of CDZ were also seen in studies in which we measured direct effects of CDZ on Ca2+-dependent fluorescence changes of cardiac TnC and skeletal TnC labeled with probes reporting Ca2+ binding to the regulatory sites. Measurements were also done with myofibrillar preparations of psoas muscle in which the native skeletal TnC was removed and exchanged with cardiac TnC and CaM, both of which could substitute for skeletal TnC as a regulatory protein. CDZ was more effective in sensitizing Ca2+-dependent MgATPase activity of skeletal myofibrils containing CaM than in preparations containing the native TnC. However, CDZ was most effective in its Ca2+-sensitizing effect in the case of the preparations containing cardiac TnC. Our results indicate that effects of agents that bind to Ca2+-binding proteins depend not only on the particular variant, but also on the specific environment in which the Ca2+-binding proteins operate.

Actin Cytoskeleton↗

PubMed1987

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers.

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.

Animals↗

PubMed1986

Alteration of actin-tropomyosin interaction in 2,4-pentanedione-treated rabbit skeletal myofibrils.

In previous work, we (El-Saleh, S., Theiret, R., Johnson, P., and Potter, J. D. (1984) J. Biol. Chem. 259, 11014-11021) presented evidence that Ca2+ activation of skeletal myofilaments depends on a specific actin domain. We showed that rabbit skeletal thin filaments reconstituted with actin modified at Lys-237 activate heavy meromyosin X Mg2+-ATPase activity independently of the Ca2+ ion concentration. The modification, which apparently blocks the inhibitory effects of troponin-tropomyosin (Tn X Tm), on acto-heavy meromyosin X Mg2+-ATPase activity, consisted of conversion of Lys-237 to an enamine by reaction of purified actin with 2,4-pentanedione (PD). In experiments reported here, we have treated myofibrils with PD with the idea of altering actin in its native state within the myofilament lattice. Preparations of native and Tn X Tm free ("desensitized") myofibrils were incubated with PD (100 mol/mol of actin lysine) under rigorous conditions (10 mM 4-morpholinepropanesulfonic acid, pH 7.0, 2.0 nM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.4 mM dithiothreitol, and 0.15 mM NaN3). Actin isolated from PD X myofibrils contained 0.5 mol of enamine/mol. In the presence of Ca2+, the Mg2+-ATPase activity of PD-treated myofibrils was 110-120% of the maximum Ca2+-stimulated Mg2+-ATPase activity of untreated control myofibrils. In low free Ca2+ (pCa greater than 8), the Mg2+-ATPase activity of the PD-treated myofibrils was not suppressed and remained at 100-106% of the maximum activity of the control myofibrils. Ca2+ sensitivity of the PD-treated myofibrils was restored following treatment with hydroxylamine, which hydrolyzes enamine's products. Preparations of desensitized myofibrils reconstituted with PD-modified or unmodified Tn X Tm demonstrated the same Ca2+-sensitive ATPase activities. On the other hand, preparations reconstituted with unmodified or PD-modified Tn X Tm and PD-modified desensitized myofibrils were insensitive to Ca2+ ion concentration. The Mg2+-ATPase activity of preparations of myosin treated with PD was not activated by modified or unmodified actin. Our results indicate that is is possible to produce an active state(s) of the myofibrils in the absence and presence of Ca2+ by specific alteration of the actin X Tm interaction following modification of myofibrillar actin most likely at Lys-237.

Actins↗

PubMed1986

Effects of oligomycin and acidosis on rates of ATP depletion in ischemic heart muscle.

The perfusion of canine cardiac muscle with 10 microM oligomycin produced a nearly 90% slowing of the net rate of tissue ATP depletion from 0.200 to 0.025 mumol X min-1 X g wet wt-1 of tissue during a subsequent myocardial autolytic interval during which tissue pH was held constant. Moreover, lowering the tissue pH during the autolytic process by 0.6 unit from approximately 6.8 to approximately 6.2 produced a nearly 60% slowing of the net rate of tissue ATP depletion from 0.200 to 0.087 mumol X min-1 X g wet wt-1. The pH dependence of the net rate of tissue ATP depletion (by an oligomycin-sensitive process) was that predicted from the mitochondrial ATPase pH-inhibition profiles reported earlier (J. Biol. Chem. 258: 9657-9661, 1983). When taken together with our observation that the mitochondrial ATPase comprises approximately 90% of the total of all of the ATP hydrolyzing activities present in cardiac muscle cells, data reported here suggest that the protonic inhibition of the mitochondrial ATPase plays a major role in regulating the rate of tissue ATP depletion during myocardial ischemia.

Acidosis↗

PubMed1986

Differential effects of pH on calcium activation of myofilaments of adult and perinatal dog hearts. Evidence for developmental differences in thin filament regulation.

Our results show that calcium activation of myofilament preparations of dog heart in the perinatal period is unaffected by a reduction in pH from 7.0 to 6.5, which, in adult heart myofilaments, induces a 0.4 pCa unit (-log molar free calcium concentration) rightward shift in the relation between pCa and myofibrillar adenosine triphosphatase activity. Acidic pH also had no effect on calcium binding to myofibrillar troponin C of perinatal hearts. The stoichiometry of troponin C bound calcium at full myofilament activation (about 3 mol calcium/mol troponin C) was the same for adult and perinatal heart myofibrils, as was their myofibrillar troponin C content. Moreover, there were no differences in isoelectric pH of troponin C from adult and perinatal hearts. We tested whether variants of myofilament proteins other than troponin C could account for the differential effects of acidic pH. In adult and perinatal dog heart preparations, myosin heavy chain isoenzymes appeared the same as measured, using native pyrophosphate gel electrophoresis. No evidence for thick filament-related calcium regulation in the perinatal heart myofilaments was obtained, when tested in studies in which native thin filaments were displaced with a 10-fold molar excess of pure actin. In preparations in which native thick filaments were displaced with a 10-fold molar excess of pure skeletal muscle myosin, the effects of acidic pH on calcium activation were the same as in native adult and perinatal preparations. Our major conclusion from these results in that the perinatal heart myofilaments are likely to possess variations in thin filament activity and structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMed1986

Myocardial actions of milrinone: characterization of its mechanism of action.

The effect of milrinone on hemodynamic, pharmacologic, and key metabolic processes involved in regulating myocardial cellular calcium was evaluated in an attempt to characterize the mechanism of action of this compound. Data from open-chest dog preparations and isolated guinea pig and rat heart preparations showed that milrinone causes a significant increase in contractility (+ dP/dt of left ventricular pressure) and coronary blood flow. Based on evidence from the Langendorff heart preparations, this positive inotropic effect of milrinone may be related in part to the drug's significant effect on positive chronotropy. However, the coronary-dilating action by milrinone appears to be direct and independent of positive chronotropy. Biochemical studies demonstrated that milrinone appears to have little effect on cardiac subcellular Ca++ transport systems. Preliminary findings indicate, however, that milrinone may play a role in initiating an earlier spontaneous release of Ca++ from the sarcoplasmic reticulum. The implications of these and other results in relation to the drug's mechanism of action are discussed.

1-Methyl-3-isobutylxanthine↗

PubMed1986

Stimulation of cardiac myofilament force, ATPase activity and troponin C Ca++ binding by bepridil.

We report that bepridil, a Ca++ channel blocker and calmodulin antagonist, which has been shown to enter myocytes, stimulates the mechanical and biochemical activity of cardiac myofilaments. Bepridil increased significantly the level of Ca++-dependent actomyosin Mg++-ATPase activity of myofibrils and the submaximal force developed by chemically skinned trabeculae of pig heart. In the range of concentrations (10-100 microM) in which bepridil showed this stimulatory activity, diltiazem and verapamil were without effect. The effect of bepridil on myofilament force and ATPase activity was higher at relatively low free Ca++ concentrations, and myofibrils lacking troponin-tropomyosin were not affected by bepridil. Associated with the stimulation of force and ATPase activity by bepridil was an increase in the amounts of Ca++ bound to troponin C (TnC). That bepridil stimulates TnC Ca++ binding was also shown in experiments using pure TnC labeled with 2-(4'-iodoacetamidoanilo)naphthalene-6-sulfonic acid, a fluorescent probe that reports Ca++ bound to the single "regulatory" site. Effects of bepridil on the fluorescence of a felodipine-cardiac TnC complex indicate that bepridil binds to TnC over the same range of doses where it affects myofilament activity. Our results indicate that the inotropic action of bepridil may result from a net response of heart cells to influences on the delivery of Ca++ to the myofilaments and their response to Ca++.

Actin Cytoskeleton↗

PubMed1985

Phosphorylation and functional modifications of sarcoplasmic reticulum and myofibrils in isolated rabbit hearts stimulated with isoprenaline.

Isoprenaline stimulation of perfused rabbit hearts was associated with simultaneous phosphorylation of proteins in the myofilaments and phospholamban in the sarcoplasmic reticulum (SR). Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi, freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline, and myofibrils and SR were prepared from the same hearts. Stimulation of 32P incorporation in troponin I (TnI) and C-protein by isoprenaline was associated with a decrease in Ca2+-sensitivity of the myofibrillar Mg2+-dependent ATPase activity. Stimulation of 32P incorporation in SR by isoprenaline was associated with an increase in the initial rates of oxalate-facilitated Ca2+ transport, assayed with SR vesicles in either microsomal fractions or homogenates from the perfused hearts. These findings provide evidence that phosphorylation of TnI, C-protein and phospholamban in the intact cell is associated with functional alterations of the myofibrils and SR which may be responsible in part for the effects of catecholamines on the mammalian myocardium.

Animals↗

PubMed1985

Effects of diacetyl monoxime on cardiac excitation-contraction coupling.

Diacetyl monoxime (DAM) is a negative inotropic agent. To identify the mechanism of its actions, electrical and mechanical studies with various cardiac tissues were carried out. DAM (0.2-20 mM) inhibited the contractile force in both normal and 22 mM KCl-depolarized (in presence of 10(-6) M isoproterenol) guinea-pig papillary muscles in a concentration-dependent manner. In general, there was a lack of major effects of DAM on sarcolemmal electrical properties. The fast action potentials were somewhat depressed and the slow action potentials were slightly enhanced. In chemically skinned pig ventricular muscles, the myofibrillar contraction induced in 6.25 pCa was inhibited by DAM in a similar concentration range. DAM also produced an apparent decrease in sensitivity toward Ca++ in this preparation. Myofibrillar adenosine triphosphatase assay showed similar results as in the skinned muscles. All DAM effects were reversible upon washout and could be partially antagonized by raising [Ca++]. Taken together, the negative inotropic effect of DAM cannot be ascribed to an inhibitory effect on the slow inward current, as suggested previously. An inhibitory effect at the myofibril level is a distinct possibility. Additional effects of DAM on the sarcoplasmic reticulum cannot be ruled out.

Action Potentials↗

PubMed1984

The effect of acidic pH on the ATPase activity and troponin Ca2+ binding of rabbit skeletal myofilaments.

We compared the effect of reductions in pH from 7.0 to 6.5 and to 6.2 on Mg-ATPase activity and Ca2+ binding of rabbit psoas myofilaments. The amounts of Ca2+ bound attributable to myofilament whole troponin complexes (Tn) were determined from studies on Tn free (desensitized) myofibrils and from measurements of the myofilament Ca2+ binding subunit of Tn content (0.55 +/- 0.10 nmol/mg of myofilament protein). The myofibrillar Mg-ATPase activity was half-maximal at the -log molar free Ca2+ concentration (pCa) 5.83 at pH 7.0, 5.54 at pH 6.5, and 5.43 at pH 6.2 Maximal ATPase activity was the same at the three pH values as was the ATPase activity of desensitized myofibrils. The amounts of Ca2+ bound to myofibrils or chemically skinned fibers as a function of pCa decreased as pH was lowered from 7.0 to 6.5 and to 6.2 in a manner predicted from the pH-induced shifts in Mg-ATPase activity as a function of pCa. The alterations in myofibrillar ATPase activity and Ca2+ binding associated with the acidic pH values occurred whether or not EGTA was used to buffer the free Ca2+ concentration. Moreover, lowering the pH from 7.0 to 6.2 reduced Ca2+ binding to myofilament Tn whether or not the fibers and myofibrils were in rigor. Our data indicate that the effects of acidic pH on myofilament ATPase activity and Ca2+-binding are related to a reduction in the affinity of Ca2+-binding sites on Tn.

Adenosine Triphosphatases↗

PubMed1984

The hill coefficient for the Ca2+-activation of striated muscle contraction.

The following arguments are presented for the observation that curves relating free Ca2+ and force development of thin filament regulated myofilaments of skinned muscle fibers have Hill coefficient (n) greater than 4, which is the number of Ca2+ binding sites on troponin: Activation of the myofilaments is a process relaxing to a nonequilibrium steady state or stationary state. Systems operating at nonequilibrium stationary states are known to display Hill coefficients greater than the number of interacting sites and similar results have been obtained for Ca2+ activation of myofilament isometric force. The size of the basic subunit of thin filament regulated muscle may be the entire thin filament rather than seven actins, one tropomyosin, and one troponin. In this case the number of interacting sites may be on the order of hundreds. Hysteresis in the Ca2+ activation of isometric force might result from multiple stationary states and also might give rise to Hill coefficients greater than 4.

Animals↗

PubMed1984

Coordination of cardiac myofibrillar and sarcotubular activities in rats exercised by swimming.

We measured the functional activity of both myofibrils and fragmented sarcoplasmic reticulum (FSR) in microsomal fractions and homogenates of hearts of sedentary rats and rats exercised by swimming 75 min twice daily for 8 wk. Ca2+ transport was measured under conditions that restricted uptake of Ca2+ to the SR vesicles in the homogenates or microsomal fractions. There was a significant increase in myosin Ca2+-ATPase activity of myofibrils prepared from hearts of swimmers, indicating that a "training effect" had occurred. The mean rate of Ca2+ transport and mean storage capacity were the same for SR vesicles in the homogenates and microsomal fractions from hearts of controls and swimmers. At the same free Ca2+ concentration, the velocity of Ca2+ transport by FSR in homogenate preparations was inversely related to the myofibrillar ATPase activity in a series of preparations from hearts of swimmers, but there was little correlation between the same activities measured in preparations from a series of sedentary rats. Our results suggest that the increase in the rate of relaxation of hearts from exercised rats is not due to an increase in the rate of Ca2+ transport by the SR but may be due to other factors, which include an increase in the rate of cross-bridge cycling or an alteration in the relation and coordination between cross-bridge cycling and SR Ca2+ transport activity.

Adaptation, Physiological↗

PubMed1984

Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH.

The aim of experiments described here was to test whether deactivation of cardiac myofibrils in acidic pH is associated with decreases in amounts of calcium bound to myofilament troponin. We determined the amounts of myofibrillar bound calcium attributable to troponin, from measurements of calcium binding to myofibrils and to myosin and from determination of the troponin C content of the myofibrillar preparations (0.40 nmol troponin C/mg protein). In measurements done at 2 mM free magnesium, 2 mM (magnesium-adenosine triphosphate, ionic strength 0.12, 22 degrees C, the pCa50 (-log of the half maximally activating molar free calcium) for myofibrillar magnesium-adenosine triphosphatase activity was 5.87 at pH 7.0, 5.49 at pH 6.5, and 5.04 at pH 6.2. This change in calcium sensitivity of myofibrillar magnesium-adenosine triphosphatase activity was present whether or not ethyleneglycol-bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, was used to buffer the free calcium and whether or not myofibrillar troponin I had been phosphorylated by cyclic adenosine 3',5'-monophosphate-dependent protein kinase. However, the change in pCa50 of myofibrillar adenosine triphosphatase activity induced by acidic pH, was greater when free magnesium was reduced from 2.0 to 0.05 mM, and less when free magnesium was increased from 2.0 mM to 10 and 15 mM. The change in pCa50 with acidic pH was less if the ionic strength was reduced from 0.12 to 0.035 M. The magnesium-adenosine triphosphatase activity of troponin/tropomyosin-free myofibrils was independent of pCa and unaffected by a reduction of pH from 7.0 to 6.5. The affinity of myofibrillar troponin C for calcium decreased as pH was reduced from 7.0 to 6.5 and to 6.2 with and without ethyleneglycolbis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and in a manner predicted from the effect of acidic pH on pCa50 for myofibrillar activation. Our results are consistent with the idea that at least part of the mechanism responsible for deactivation of the adenosine triphosphatase activity of cardiac myofilaments in acidic pH is a reduction in the affinity of myofibrillar troponin C for calcium.

Adenosine Triphosphatases↗