Effects of terbutaline on force and intracellular calcium in slow-twitch skeletal muscle fibres of the rat.

1. The effect of the alpha2-adrenoceptor agonist, terbutaline, was investigated on simultaneously measured force and intracellular free calcium ([Ca2+]i) in intact rat soleus muscle fibres, and on contractile protein function and Ca2+ content of the sarcoplasmic reticulum (SR) in skinned fibres. 2. Terbutaline (10 microM) had no significant effect on either resting force or [Ca2+]i. Exposure to terbutaline increased both the integral of the indo-1 ratio transient and peak twitch force by 37%. 3. At sub-maximal (10 Hz) stimulation frequencies, terbutaline accelerated force relaxation but had highly variable effects on tetanic force amplitude. The corresponding indo-1 ratio transients were significantly larger, and faster to decay than the controls. 4. Terbutaline increased tetanic force at near maximal stimulation frequencies (50 Hz) by increasing tetanic [Ca2+]i. Force relaxation was accelerated at this frequency with no significant change in the indo-1 ratio transient decay rate. 5. All of terbutaline's effects on force and indo-1 ratio transients in intact fibres were completely blocked and reversed by ICI 118551 (1 microM). 6. Mechanically skinned fibres isolated from intact muscles pre-treated with terbutaline showed no significant changes in SR Ca2+ content, myofilament [Ca2+]i-sensitivity or maximum force generating capacity. 7. The results suggest that terbutaline primarily modulates force by altering the amplitude and decay rate of the [Ca2+]i transient via phosphorylation of both the ryanodine receptor (RR) and the SR pump regulatory protein, phospholamban (PLB). The high variability of responses of slow-twitch muscles to beta2-agonists probably reflects individual differences in basal phosphorylation levels of PLB relative to that of RR.     

Omecamtiv mecarbil: a new cardiac myosin activator for the treatment of heart failure

Introduction: Current available inotropic agents increase cardiac contractility, but are associated with myocardial ischemia, arrhythmias, and mortality. A novel selective cardiac myosin activator, omecamtiv mecarbil (CK-1827452/ AMG-423) is a small molecule that activates the sarcomere proteins directly, resulting in prolonged systolic ejection time and increased cardiac contractility.

Areas covered: This paper discusses the chemistry, pharmacokinetics, clinical efficacy and safety of omecamtiv mecarbil. Omecamtiv mecarbil represents a novel therapeutic approach to directly improve cardiac function and is therefore proposed as a potential new treatment of patients with systolic heart failure. The authors review results of previous studies investigating the effect of omecamtiv mecarbil in heart failure animal models, healthy volunteers, and patients with acute and chronic systolic heart failure.

Expert opinion: Results of phase I and phase II studies demonstrate that omecamtiv mecarbil is safe and well tolerated both as an intravenous and oral formulation. In healthy volunteers and chronic systolic heart failure patients, administration of omecamtiv mecarbil resulted in a concentration-dependent increase of left ventricular ejection time, ejection fraction, fractional shortening, and stroke volume. The first results of a double-blind, randomized, placebo-controlled phase IIb dose-finding study with the oral formulation of omecamtiv mecarbil demonstrated beneficial effects on cardiac function and N-terminal pro-brain natriuretic peptide levels. This study will provide essential dosing information for the requisite phase III trials which will investigate whether the beneficial effects of omecamtiv mecarbil translate into improved clinical outcomes.     

Diazepam increases calcium sensitivity of the skinned cardiac muscle fiber in guinea pig

Influences of diazepam, a benzodiazepine derivative, on the contractile response to calcium in skinned trabecular fibers of guinea pig heart were examined. Diazepam (100 microM) enhanced the contractile response of the skinned fiber to calcium and shifted the concentration-response curve to the left. The pCa50 values were 6.07+/-0.03 and 6.28+/-0.03 (P<0.05) in the absence and presence of diazepam, respectively. This result suggests that diazepam increases calcium sensitivity of contractile proteins in heart muscles.     

The effect of chelerythrine on depolarization-induced force responses in skinned fast skeletal muscle fibres of the rat

1 We examined the effect of the protein kinase C (PKC) inhibitor chelerythrine on depolarization-induced force responses (DIFRs) and sarcoplasmic reticulum (SR) function in single, mechanically skinned skeletal muscle fibres of the rat. 2 In this study, the DIFRs in the skinned fibres normally underwent an irreversible loss of excitation-contraction coupling (ECC) after 10-15 responses. Chelerythrine (12 micro M) was shown to restore ECC in these fibres. Restored force responses were similar in peak (control 50.8+/-6.4%, chelerythrine 56.9+/-12.4% of maximum force, P=0.42, n=21), but significantly broadened compared to initial control responses (full-width at half maximum, control; 3.7+/-0.3 s, chelerythrine; 13.3+/-1.1 s, P<0.001). Early exposure to chelerythrine prevented run-down of DIFRs. Chelerythrine also induced spontaneous force responses in some fibres. 3 The PKC inhibitors calphostin C and staurosporine did not restore ECC, and the PKC activator phorbol 12-myristate 13-acetate did not promote loss of ECC in the skinned fibres. 4 Chelerythrine significantly increased SR Ca(2+) loading by 8.4+/-1.7% (P=0.02, n=9) and SR Ca(2+) release by at least 14.1+/-2.7% (P=0.004, n=11) in the skinned fibres. 5 Chelerythrine had no significant effect on maximum force production or the [Ca(2+)] producing half maximal activation of the myofilaments. However, chelerythrine did have a small effect on the slope of the force-Ca(2+) relationship (P=0.02, n=10). 6 Chelerythrine reverses the use-dependent loss of excitation-contraction coupling in skinend skeletal muscle fibres by a PKC independent pathway. Chelerythrine may be an important pharmacological probe for examining the mechanisms of contraction-induced muscle injury.     

The effects of beta-adrenoceptor activation on contraction in isolated fast- and slow-twitch skeletal muscle fibres of the rat.

1. The aim of the experiments was to examined the effects of beta-adrenoceptor activation on twitch and tetanic contractions in fast- and slow-twitch mammalian skeletal muscle fibres. Isometric force was recorded from bundles of intact fibres isolated from the normal and denervated slow-twitch soleus and normal fast-twitch sternomastoid muscles of the rat. 2. Terbutaline (10 microM), a beta 2-adrenoceptor agonist, induced an average 15% potentiation of peak twitch and peak tetanic force in normal soleus fibres and abbreviated twitch and tetanic relaxation. In white- and red-sternomastoid fibres, 10 microM terbutaline potentiated peak twitch force by about 7% and slowed twitch relaxation. 3. The potentiation of twitches and tetani by terbutaline was quantitatively similar in normal and denervated soleus fibres. However, in contrast to the normal soleus, terbutaline slowed twitch relaxation and had no effect on tetanic relaxation in denervated soleus fibres. 4. Adrenaline (10 microM) increased peak tetanic force by about 7% in both normal and denervated soleus fibres. 5. Exposure to (+/-)-propranolol (0.1 microM), a general beta-adrenoceptor blocker, completely abolished the tetanus potentiation by terbutaline. 6. Dibutyryl-cyclic AMP (2 mM) mimicked the effects of 10 microM terbutaline on peak tetanic force and tetanic relaxation in normal and denervated soleus fibres. Dibutyryl-cyclic AMP also potentiated peak twitch force in denervated soleus fibres but only after a brief period of twitch depression: the twitch depression might be due to butyrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Levosimendan improves calcium sensitivity of diaphragm muscle fibres from a rat model of heart failure

BACKGROUND AND PURPOSE

Diaphragm muscle weakness occurs in patients with heart failure (HF) and is associated with exercise intolerance and increased mortality. Reduced sensitivity of diaphragm fibres to calcium contributes to diaphragm weakness in HF. Here we have investigated the ability of the calcium sensitizer levosimendan to restore the reduced calcium sensitivity of diaphragm fibres from rats with HF.

EXPERIMENTAL APPROACH

Coronary artery ligation in rats was used as an animal model for HF. Sham-operated rats served as controls. Fifteen weeks after induction of HF or sham operations animals were killed and muscle fibres were isolated from the diaphragm. Diaphragm fibres were skinned and activated with solutions containing incremental calcium concentrations and 10 µM levosimendan or vehicle (0.02% DMSO). Developed force was measured at each calcium concentration, and force–calcium concentration relationships were plotted.

KEY RESULTS

Calcium sensitivity of force generation was reduced in diaphragm muscle fibres from HF rats, compared with fibres from control rats (P < 0.01). Maximal force generation was ∼25% lower in HF diaphragm fibres than in control fibres (P < 0.05). Levosimendan significantly increased calcium sensitivity of force generation in diaphragm fibres from HF and control rats, without affecting maximal force generation.

CONCLUSIONS AND IMPLICATIONS

Levosimendan enhanced the force generating capacity of diaphragm fibres from HF rats by increasing the sensitivity of force generation to calcium concentration. These results provide strong support for testing the effect of calcium sensitizers on diaphragm muscle weakness in patients with HF.     

Hydrophobic properties of novel dihydronaphthyridine calcium antagonists and biological activity in porcine isolated cardiac and vascular smooth muscle

Summary Dihydronaphthyridine calcium antagonists are structurally related to the well known dihydropyridines and act in a similar manner. In order to establish the vascular selectivity of these compounds contractile force was evaluated in porcine isolated ventricular trabeculae and right coronary arteries. The dihydropyridine derivatives nisoldipine and nitrendipine as well as the structurally different compounds gallopamil and flunarizine were included for comparison purpose.All compounds studied exhibited dose-dependent negative inotropic and vasodilator activities. The negative inotropic potency of all dihydronaphthyridines, especially of the highly lipophilic bulky ester-variated derivatives (Goe 5584-A, Goe 5806-A) was comparable to that of flunarizine but was considerably less pronounced than that of nisoldipine, nitrendipine or gallopamil. By contrast, half-maximal vasodilator responses of the dihydronaphthyridines studied in coronary arteries being in the nanomolar concentration range were comparable to the dihydropyridines nisoldipine and nitrendipine, whereas the activity of gallopamil and flunarizine was less marked. On the other side the dihydronaphthyridines, especially the more hydrophilic 3-ethylester-4-(2-cyanophenyl) derivative Goe 5606, exerted an obvious biphasic concentration-response behaviour in coronary arteries leading to a high affinity relaxant process in subnanomolar concentrations, whereas the low affinity response could be observed at rather high concentrations (mostly > 1 µM). Mainly due to their relative uneffectiveness in cardiac muscle, vascular selectivity of the dihydronaphthyridines was considerably higher than that of the structurally related dihydropyridines nisoldipine resp. nitrendipine increasing in the following order: nitrendipine < nisoldipine < Goe 5606 < Goe 5438< Goe 5584-A < Goe 5806-A. By contrast, gallopamil exerted a slight cardiac preference, whereas the vascular selectivity of flunarizine was also pronounced. Negative inotropic potency of the dihydronaphthyridines decreased with increasing lipophilicity. However, an inverse relationship was found regarding the vasodilator activity of these compounds.Thus, modulation of the molecular structure, thereby affecting the physicochemical properties, may improve the vascular selectivity of the dihydronaphthyridines studied, when compared to dihydropyridine calcium antagonists.Parts of the results of this paper were presented on the spring meeting of the German Society of Pharmacology and Toxicology in Mainz 1989 (Werner et al. 1989) Send offprint requests to G. Werner at the above address     

The effect of DDT and dieldrin on skeletal muscle fibres

The effects of the chlorinated hydrocarbon insecticides, DDT and dieldrin, on the electrical activity of skeletal muscle fibres of the clawed toad, Xenopus laevis, were investigated by means of intracellular microelectrodes. Both compounds caused an increase in the amplitude of the negative afterpotential and a slowing-down of the falling phase of the action potential, without affecting the resting potential. Neither DDT nor dieldrin caused repetitive activity. It is concluded that the increase in the duration of the action potential, measured at a potential level of ?50 mV, may be responsible for part of the effects of these insecticides on the mechanical response of skeletal muscle.     

Effects of chlorpromazine on excitation-contraction coupling events in fast-twitch skeletal muscle fibres of the rat

1. Single mechanically skinned fibres from the rat extensor digitorum longus muscle, which allow access to intracellular compartments, were used to examine the effects of 0.5-100 microM chlorpromazine hydrochloride (CPZ) on the major steps of the excitation-contraction (E-C) coupling to elucidate the involvement of skeletal muscle in the neuroleptic malignant syndrome (NMS). 2. At 1 microM, CPZ caused a 20-30% increase in the force response induced by t-system depolarisation and a marked increase in the rate of caffeine-induced SR Ca(2+) release. At [CPZ]> or =2.5 microM, there was an initial increase followed by a marked decrease of the t-system depolarisation-induced force responses, while the potentiating effect on the caffeine-induced SR Ca(2+) release remained. These effects were reversible. 3. CPZ had no effect on the maximum Ca(2+)-activated force, but caused reversible, concentration-dependent increases in the Ca(2+) sensitivity of the contractile apparatus at [CPZ] > or =10 microM, with a 50% predicted shift of 0.11 pCa (-log [Ca(2+)]) units at 82.3 microM CPZ. 4. CPZ did not alter the rate of SR-Ca(2+) loading at 1 and 10 microM, but reversibly reduced it by approximately 40% at 100 microM by reducing the SR Ca(2+) pump. Nevertheless, the SR Ca(2+) content was greater when fibres became unresponsive to t-system-induced depolarisation in the presence than in the absence of 100 microM CPZ. 5. The results show that CPZ has concentration-dependent stimulatory and inhibitory effects on various steps of the E-C coupling, which can explain the involvement of skeletal muscle in NMS and reconcile previous divergent data on CPZ effects on muscle.     

A novel action of deltamethrin on membrane resistance in mammalian skeletal muscle and non-myelinated nerve fibres

The pyrethroids, deltamethrin and cismethrin, were assessed for their ability to change membrane conductance directly in skeletal muscle and indirectly in non-myelinated nerve fibre preparations from the rat. In diaphragm muscle fibres of the rat, input resistance was significantly increased (35%) by deltamethrin but not by cismethrin, compared with solvent alone. In perfused vagus nerve from the rat, the amplitude of the post-tetanic hyperpolarization was significantly increased (100%) by deltamethrin both in vitro and ex vivo but not by cismethrin or solvent. In both test systems the actions of deltamethrin were abolished by changing the perfusate to a low chloride solution. The enhancement of amplitude of post-tetanic hyperpolarization by deltamethrin was reversed by ivermectin, a compound known to increase the resting chloride flux in neuronal preparations. Depolarizing afterpotentials, indicative of a prolongation in sodium conductance, did not develop until 0.5-1.0 hr after the enhancement of the amplitude of post-tetanic hyperpolarization by deltamethrin in the vagus preparations. The amplitude of post-tetanic hyperpolarization was not enhanced by exposure of the vagus to veratrine. These observations reinforce the conclusion that the enhancement of post-tetanic hyperpolarization by deltamethrin is not the result of intracellular accumulation of sodium. In addition, the chloride-dependent nature of the effects of deltamethrin, in both muscle and non-myelinated nerve, suggests that they are in both cases due to a reduction in resting membrane chloride conductance. This novel action of deltamethrin would be expected to amplify the effect of prolonged sodium current and thus influence the actions on excitable membranes both directly and indirectly.     

Taurine blocks ATP-sensitive potassium channels of rat skeletal muscle fibres interfering with the sulphonylurea receptor

Taurine is a sulphonic aminoacid present in high amounts in various tissues including cardiac and skeletal muscles showing different properties such as antioxidative, antimyotonic and anti-schaemic effects. The cellular mechanism of action of taurine is under investigation and appears to involve the interaction of the sulphonic aminoacid with several ion channels. Using the patch-clamp technique we studied the effects of taurine in rat skeletal muscle fibres on ATP-sensitive K(+) channel (K(ATP)) immediately after excision and on channels that underwent rundown. The cytoplasmic application of 20 mM of taurine reduced the K(ATP) current; this effect was reverted by washout of the drug solution. In this experimental condition the IC(50) was 20.1 mM. After rundown, taurine inhibited the K(ATP) current with similar efficacy. Competition experiments showed that taurine shifted the dose-response inhibition curve of glybenclamide to the left on the log-dose axis without significantly affecting those of ATP or Ca(2+) ion. Single channel recording revealed that taurine affects the close state of the channel prolonging it and reducing the bursts duration. Our data indicate that taurine inhibits the muscular K(ATP) channel interfering with the glybenclamide site on the sulphonylurea receptor of the channel or on the site allosterically coupled to it. During ischaemia and hypoxia, the skeletal and heart muscles undergo several changes; for example, the activation of K(ATP) channels and loss of the intracellular taurine content. The depletion of taurine during ischaemia would contribute to the early activation of K(ATP) channels and salvage the intracellular ATP content.     

Effect of calcium channel antagonists on calcium uptake and release by isolated rat cardiac mitochondria

The effects of calcium channel antagonists on Ca2+ uptake and Na+-induced Ca2+ release were studied in isolated rat cardiac mitochondria. Diltiazem, nitrendipine and nimodipine were more effective inhibitors of Na+-induced Ca2+ release (IC50 = 19-100 microM) than of Ca2+ uptake (IC50 = 0.2-1 mM). Nitrendipine and nimodipine had virtually identical IC50 values for inhibiting Ca2+ uptake, but nitrendipine was 3-4 times more potent than nimodipine at inhibiting Na+-induced Ca2+ release. If these calcium channel antagonists achieve intracellular concentrations in the range of 10(-5)-10(-4) M, our results suggest that calcium channel antagonists would preferentially inhibit mitochondrial calcium release more than mitochondrial calcium uptake.     

The action of caffeine in promoting ultrastructural damage in frog skeletal muscle fibres

1. Caffeine at concentrations above 5 mM was shown to cause rapidly extensive ultrastructural damage to the myofibrils of frog skeletal muscle. 2. The effect was promoted at lower temperatures, whereas the myofibrils were protected by prior exposure to procaine. 3. It is argued that caffeine causes a Ca2+-induced release of Ca2+ (the CROC) from the S.R. and that the consequent rise in [Ca2+]i promotes the ultrastructural damage observed. 4. Myofibril digradation is also produced by treatment of the muscle with the divalent cation ionophore A23187; this effect is not protected by either procaine or Dantrolene sodium. 5. It is suggested that A23187 causes the release of Ca2+ from the S.R. by a mechanism that differs from both excitation and the CROC; the resultant rise in [Ca2+]i again causes myofibril degradation. 6. The ways in which a marked rise in [Ca2+]i could cause muscle damage and the possible relevance of these findings to the sequence of events in the development of myopathies of human skeletal muscle are discussed.     

Calcium sensitivity and the effect of the calcium sensitizing drug pimobendan in the alcoholic isolated rat atrium

We compared the effect of inotropic interventions (isoproterenol and pimobendan) and the relation between Ca2+ and isometric twitch force in atrial muscle from control rats and rats that had consumed alcohol for 2 months. At extracellular Ca2+ concentrations of 1-4 mM, alcohol atria developed less force than the controls. The median effective concentration (EC50) for extracellular Ca2+ was 3.2 +/- 0.01 mM for the alcohol group and 2.8 +/- 0.001 mM for the control group, whereas at maximal Ca2+, developed force was the same in both groups. To test whether the myofilament response to Ca2+ is altered with chronic alcohol consumption, we measured the relation between Ca2+ and force of atrial fiber bundle preparations extracted with Triton X-100. The Ca2+-force relation of alcohol atria (EC50 = 2.4 +/- 0.001 microM) was significantly shifted to the right of that of the control atria (EC50 = 1.94 +/- 0.001 microM Ca2+). Compared with controls, the alcohol atria demonstrated a significant depression in the inotropic effect of the beta-adrenergic agonist isoproterenol over a broad concentration range (10(-9)-10(-6) M). We also tested the effect of pimobendan, an inotropic agent with both phosphodiesterase-inhibiting and myofilament Ca2+-sensitizing actions. Developed force at concentrations of pimobendan <75 microM was similar between groups. However, at concentrations of pimobendan >75 microM, the developed force in alcohol atria was significantly less than control. Our results indicate that 2 months of alcohol consumption is associated with decreases in myofilament Ca2+ sensitivity and altered responsiveness to different inotropic agents.     

Tissue selectivity of the novel calcium antagonist sesamodil fumarate in isolated smooth muscles and cardiac muscles

The effects of the novel Ca2+ antagonist sesamodil fumarate (JAN), (+)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[(3,4- methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo-2H- 1,4-benzothiazine hydrogen fumarate (SD-3211), on isolated smooth muscles and cardiac muscles were investigated and compared with those of diltiazem, verapamil, nifedipine and nicardipine. Ca2+ antagonistic activity of SD-3211 (pA2 = 8.42) was more potent than that of diltiazem and verapamil, but less potent than that of nifedipine and nicardipine in isolated pig coronary artery. The inhibition of Ca2(+)-induced contraction by SD-3211 was not reversed by drug washout, whereas that by diltiazem was easily reversed by drug washout. SD-3211 produced a concentration-dependent relaxation (EC50 = 5.7 x 10(-8) mol/l) of KCl-contracted pig coronary artery. The order of potency of the various compounds correlated with their respective Ca2+ antagonistic activities. SD-3211 antagonized KCl-induced contraction, but not that induced by A23187, in the rabbit aorta. On the other hand, negative inotropic and chronotropic effects of SD-3211 on the guinea pig right atria approximated those of diltiazem and verapamil. These results suggest that SD-3211 exerts a potent and long-lasting Ca2+ antagonistic effect on isolated arteries, possessing pharmacological properties diverse from those of known Ca2+ antagonists with respect to tissue selectivity, i.e., it is more vasoselective than diltiazem and verapamil, and more cardioselective than nifedipine and nicardipine.     

阿司咪唑对大鼠离体心肌电生理特性的影响

目的：通过观察阿司咪唑对大鼠离体心肌自律性、兴奋性和有效不应期的作用。了解阿司咪唑对心肌电生理特性的影响，以初步探索其心脏作用机制。方法：将心房肌和心室乳头肌标本置于盛有Tyrode’s液的恒温器官浴槽中，向浴槽中累积加入阿司咪唑。记录右心房一窦房结的自发频率，测定左心房与乳头肌收缩的阈刺激电压值和有效不应期。结果：阿司咪唑能减慢窦房结自发频率，降低心肌自律性；能增加引起心房肌和心室乳头肌收缩的阈刺激电压值，降低心肌兴奋性；能使心房肌和心室乳头肌的有效不应期明显延长。结论：阿司咪唑能干扰心肌正常电生理特性，对心肌电生理活动具有抑制作用。