Ferret Cardiac Muscle Research Articles

Adenosine affects the release of Ca2+ from the sarcoplasmic reticulum via A2A receptors in ferret skinned cardiac fibres

In this study, it was shown that adenosine potentiates caffeine-induced Ca2+ release. It was then proposed that the enhancement of the caffeine-induced Ca2+ release might occur by a direct effect on the ryanodine Ca2+ release channel or on other Ca2+ regulation mechanisms. Furthermore, A2A receptors may be functional on the ferret cardiac sarcoplasmic reticulum. Using chemically skinned fibres, experiments were conducted on ferret cardiac muscle to find out whether adenosine and the A1 and A2A adenosine receptor agonists (CCPA and CGS 21680) and antagonists (DPCPX and ZM 241385) affected caffeine-induced Ca2+ release and the Ca2+ sensitivity of contractile proteins. Changes in the caffeine-induced contracture brought about by adenosine and by adenosine-receptor agonists and antagonists were recorded in saponin-skinned fibres (50 microg ml(-1)). Tension-pCa relationships were then obtained by exposing Triton X-100-skinned fibres (1% v/v) sequentially to solutions of decreasing pCa. Adenosine (1-100 nm) and the specific A2A receptor agonist CGS 21680 (1-50 nm) produced a concentration-dependant potentiation of the caffeine-induced Ca2+ release from saponin-skinned fibres. The data plotted versus adenosine and CGS 21680 concentrations displayed sigmoid relationships (Hill relationship), with potentiation of Ca2+ release by 22.2 +/- 1.6 (n = 6) and 10.9 +/- 0.4% (n = 6), respectively. In addition, the potentiation of caffeine-induced Ca2+ release by adenosine (50 nm; 15.3 +/- 1.0%; n = 6) and by CGS 21680 (50 nm; 11.2 +/- 0.4%; n = 6) was reduced by the specific A2A receptor antagonist ZM 241385 (50 nm) to 8.0 +/- 1.4 (n = 4) and 5.4 +/- 1.2% (n = 4), respectively. The A1 receptor agonist CCPA (1-50 nm) and antagonist DPCPX (50 nm) had no significant effects on caffeine responses. In Triton X-100-skinned fibres, the maximal Ca(2+)-activated tension of the contractile proteins (41.3 +/- 4.1 mN mm(-2); n = 8), the Hill coefficient (nH = 2.2 +/- 0.1; n = 8) and the pCa50 (6.15 +/- 0.05; n = 8) were not significantly modified by adenosine (100 nm) or by CGS 21680 (50 nm).

Effects of volatile anesthetics on ryanodine-treated ferret cardiac muscle.

The volatile anesthetics halothane, isoflurane, and sevoflurane depress myocardial contractility by decreasing transsarcolemmal Ca2+ influx, Ca2+ release from the sarcoplasmic reticulum, Ca2+ sensitivity of the contractile proteins, and cross-bridge performance. The aim of this study is to assess and compare the effects of halothane, isoflurane, and sevoflurane on contractility in conditions in which sarcoplasmic reticulum Ca2+ release is abolished by pretreatment with ryanodine. Ferret right ventricular papillary muscles were exposed to ryanodine at 10(-6) M and then to incremental concentrations of halothane, isoflurane, or sevoflurane. In the presence of ryanodine, each anesthetic decreased isometric and isotonic contractility in a reversible, concentration-dependent manner with no differences between anesthetics and with little or no effect on time variables. It is likely that differences between anesthetic effects on contraction amplitude in isometric and isotonic twitches reside in their effects on the sarcoplasmic reticulum.

Effects of halothane and isoflurane on the intracellular Ca2+ transient in ferret cardiac muscle.

Halothane and isoflurane depress myocardial contractility by decreasing transsarcolemmal Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum. Decreases in Ca2+ sensitivity of the contractile proteins have been shown in skinned cardiac fibers, but the relative importance of this effect in intact living myocardium is unknown. The aims of this study were to assess whether halothane and isoflurane decrease myofibrillar Ca2+ sensitivity in intact, living cardiac fibers and to quantify the relative importance of changes in myofibrillar Ca2+ sensitivity versus changes in myoplasmic Ca2+ availability caused by these anesthetics. The effects of halothane and isoflurane (0-1.5 times the minimum alveolar concentration (MAC) in three equal increments) on isometric and isotonic variables of contractility and on the intracellular calcium transient were assessed in isolated ferret right ventricular papillary muscle microinjected with the Ca2+-regulated photoprotein aequorin. The intracellular calcium transient was analyzed in the context of a multicompartment model of intracellular Ca2+ buffers in mammalian ventricular myocardium. Halothane and isoflurane decreased contractility, time-to-peak force, time to half-isometric relaxation, and intracellular Ca2+ transient in a reversible, concentration-dependent manner. Halothane, but not isoflurane, slowed the increase and the decrease of the intracellular Ca2+ transient. Increasing extracellular Ca2+ in the presence of anesthetic to produce peak force equal to control values increased intracellular Ca2+ to values higher than control values. Halothane decreases myoplasmic Ca2+ availability more than isoflurane; halothane and isoflurane decrease myofibrillar Ca2+ sensitivity to the same extent; in halothane at 0.5 MAC and isoflurane at 1.0 MAC, the decrease in Ca2+ sensitivity is already fully apparent; halothane decreases intracellular Ca2+ availability more than myofibrillar Ca2+ sensitivity; and isoflurane decreases myoplasmic Ca2+ availability and Ca2+ sensitivity to the same extent, except at 1.5 times the MAC, which decreases Ca2+ availability more.

The role of the sarcoplasmic reticulum in the response of isolated ferret cardiac muscle to beta‐adrenergic stimulation

beta-Adrenergic stimulation of cardiac muscle leads to an increase in the strength of contraction and an abbreviation of its time course. We have investigated the role of the sarcoplasmic reticulum in these changes by monitoring force and cytoplasmic [Ca2+] in ferret papillary muscles, and the Ca2+ current in isolated ferret myocytes, during the application of isoprenaline in the absence and presence of the sarcoplasmic reticulum inhibitor ryanodine (10(-6) mol/l). Isoprenaline (10(-6) mol/l) led to a marked increase in the size of both the twitch and Ca2+ transient, and a decrease in their duration. In the presence of ryanodine, application of isoprenaline had no significant effect on either the size or the time course of the twitch. However, the increase in the Ca2+ current in response to isoprenaline was the same in the absence and presence of ryanodine. Increasing bathing [Ca2+] led to a prolongation of both the twitch and the Ca2+ transient. In the presence of ryanodine, increasing bathing [Ca2+] still increased the size, but decreased the duration, of the twitch. These data provide direct evidence that both the inotropic and lusitropic effects of isoprenaline are mediated via the sarcoplasmic reticulum.

Effects of acidosis and hypoxia on the response of isolated ferret cardiac muscle to inotropic agents.

The aim was to study the effects of acidosis and hypoxia on the response of cardiac muscle to inotropic agents which (a) act predominantly by increasing intracellular [Ca2+] (raising extracellular [Ca2+], noradrenaline, isoprenaline) and (b) act partly (phenylephrine) or predominantly (EMD 57033) by increasing myofilament calcium sensitivity. The experiments were performed on isometrically contracting, isolated ferret papillary muscles (n = 45). For each intervention dose-response curves were performed in control solution (pH 7.35), in hypercapnic acidosis (pH 6.85), and in hypoxia (produced by replacing O2 with N2 in the superfusing solution). In some experiments, the photoprotein aequorin was microinjected into superficial cells of the preparation in order to measure intracellular [Ca2+] as well as force. The results were broadly similar for both classes of inotropic agent. Acidosis caused a shift of the pCa-tension curve to the right (desensitisation of the myofilaments to calcium), but had no significant effect on maximum force. A sufficient inotropic stimulus supplied by either class of inotropic agent could completely reverse the negative inotropic effects of acidosis. The main difference between the two inotropic mechanisms was that the enhanced force produced by calcium sensitisers was associated with a reduction in calcium transient amplitude, while the other inotropes increased the amplitude. The main effect of hypoxia was to decrease maximum force. All the inotropes tested were relatively ineffective in reversing the force depression due to hypoxia. The negative inotropic effects of acidosis can be reversed by a sufficiently large inotropic stimulus. Since calcium transient amplitude is already increased in acidosis, the results suggest that calcium sensitisers are likely to be less arrhythmogenic in this situation. The relative ineffectiveness of the inotropes in hypoxia indicates that the main mechanisms causing reduced force in this situation lie downstream of the mechanisms of action of the inotropic agents tested.

Estimates of beat to beat handling of activator calcium using measurements of [Ca2+]i in aequorin loaded ferret cardiac muscle.

The aims were (1) to measure simultaneously and on a beat to beat basis intracellular calcium concentration ([Ca2+]i) transients and force transients in isolated ferret cardiac trabeculae; (2) to obtain and compare independent estimates of the recirculating fraction of Ca2+ using the [Ca2+]i data and the force data (recirculating fraction is the fraction of activator Ca2+ taken up by the sarcoplasmic reticulum in each beat and, in the steady state twitch, the fraction of activator Ca2+ released by the sarcoplasmic reticulum); and (3) to estimate the amount of Ca2+ that returns to the sarcoplasmic reticulum and the amount that, during steady state contractions, enters the cytosol, presumably from the extracellular compartment, with each beat. Eight trabeculae were mounted in the myograph. The servo-controlled muscle length was 98% of the length at which developed force was maximal. A modified technique was used for chemical loading of aequorin, and a new method for computer controlled low level photon counting, storage, calibration, and analysis. [Ca2+]i transients and force transients were simultaneously recorded during potentiated beats, together with their respective decays toward control steady state [Ca2+]i transients and force transients. A modified test of postextrasystolic potentiation achieved with a brief train of rapid pacing followed by a pause was used to evoke the potentiated beats. At 2.0 mM extracellular Ca2+ ([Ca2+]o), resting [Ca2+]i was 283(SD 77) nM. The resting tension was 1.6(0.3) g.mm-2. The steady state [Ca2+]i transient and the peak potentiated [Ca2+]i transient averaged 992(165) and 1290(154) nM respectively. The corresponding tensions were 4.0(1.9) and 8.7(3.1) g.mm-2 respectively. The recirculating fraction of Ca2+ calculated from the dissipation of the potentiated [Ca2+]i transient averaged 45(4)%. This recirculating fraction was indistinguishable from the one calculated with another method from the decay of the force potentiation. This is the first study to estimate the recirculating fraction of activator Ca2+ using measurements of [Ca2+]i. The results indicate that over a wide range of [Ca2+]i and tensions the Ca(2+)-force relationship is well approximated by a straight line. At 2.0 mM [Ca2+]o it appears that some 450 nM of Ca2+ recirculates and that a similar amount per steady state beat enters the cytosol, probably from the extracellular compartment.

Beat-to-beat measurements of [Ca2+]i and force in ferret cardiac muscle after chemical loading of aequorin

This communication reports the development of a modified procedure for chemical loading of aequorin in small multicellular cardiac preparations, with special emphasis directed toward the implementation of a new method for computer-controlled low-photon counting and digital processing and analysis of the data to obtain intracellular Ca2+ concentration ([Ca2+]i). In eight ferret right ventricular trabeculae, we measured the mechanical performance and found that, at 1.25 mM extracellular Ca2+ concentration ([Ca2+]o), resting tension, developed tension, and time to peak tension were unchanged by the loading procedure. Estimated resting and peak systolic [Ca2+]i were 299 +/- 65 and 766 +/- 131 nM, respectively. Thirty minutes after raising the [Ca2+]o to 5 mM, there was a robust increase in mechanical performance, with peak systolic [Ca2+]i averaging 1,218 +/- 222 nM. The diastolic [Ca2+]i remained unchanged. In four other trabeculae, exposure to a low-Na(+)-containing superfusate demonstrated a remarkable beat-to-beat correspondence of increases in diastolic [Ca2+]i and resting tensions. The same beat-to-beat concordance was also observed between the rapidly changing amplitudes of peak [Ca2+]i and developed tension. In additional experiments, simultaneous recordings of [Ca2+]i and force transients were obtained during rapid pace pause maneuvers. These studies showed distinct and quantifiable fluctuations of [Ca2+]i in a 1:1 relation to the mechanical record to a frequency of at approximately 300 beats/min. These results demonstrate that beat-to-beat measurements of [Ca2+]i and tension transients can be obtained with good resolution in multicellular cardiac preparations.

Ca2+ and segment length dependence of isometric force kinetics in intact ferret cardiac muscle.

The influence of Ca2+ and sarcomere length on myocardial crossbridge kinetics was studied in ferret papillary muscle by measuring the rate of force redevelopment following a rapid length step that dropped the force to zero. Tetanic stimulation with 5 mumol/L ryanodine was used to obtain a steady-state contraction, and segment length was measured and controlled using a sense-coil technique that measures changes in the cross-sectional area of the central region of the muscle. The rate constant for the recovery of force (ktr) following a rapid length release was obtained by fitting the data with a single exponential function. Contrary to results from skinned skeletal fibers in which ktr increases almost 10-fold from low to maximal activation levels, ktr was found not to increase at higher activation levels in this study. Similarly, although force increased with segment length under all conditions, ktr never increased with length. Data presented here are consistent with a model of myocardial Ca2+ activation in which Ca2+ modulates the number of crossbridges interacting with the thin filament and are inconsistent with a model in which Ca2+ modulates the kinetics of transitions to force producing states within the actomyosin cycle. Differences in the activation dependence of the force redevelopment rate between cardiac and skeletal muscle suggest that there are fundamental differences in the mechanism of Ca2+ activation between these two muscle types.

Differential effects of the optical isomers of EMD 53998 on contraction and cytoplasmic Ca2+ in isolated ferret cardiac muscle.

EMD 53998 (a thiadiazinone) is a novel inotropic substance that increases the Ca2+ sensitivity of the myofilaments in skinned cardiac fibers and has been found to have similar effects in intact cardiac muscle. However, the compound also possesses the ability to inhibit phosphodiesterase III, indicating that its actions in intact cardiac muscle are likely to be complex. The present study was carried out to investigate the possibility that the optical isomers of EMD 53998--(+)EMD 57033 and (-)EMD 57439--which have recently been shown to possess a separation of sensitization and phosphodiesterase inhibition in subcellular preparations, might also demonstrate this separation of activities in intact cardiac muscle. The experiments were performed on isolated ferret papillary muscles, which were contracting isometrically. In some preparations, the photoprotein aequorin was injected into superficial cells to measure intracellular Ca2+ as well as force. (+)EMD 57033 caused a substantial positive inotropic effect that was associated with prolongation of the twitch, reduction in the amplitude of the Ca2+ transient, and abbreviation of the Ca2+ transient. This is the profile expected of a Ca(2+)-sensitizing compound. Conversely, (-)EMD 57439 caused a less marked positive inotropic effect that was associated with an abbreviation of the twitch, an increase in the amplitude of the Ca2+ transient, and an abbreviation of the Ca2+ transient. This is the profile expected of an agent producing its inotropic effect by increasing cAMP (e.g., phosphodiesterase inhibition). The results indicate that the optical isomers of EMD 53998 possess a remarkable separation of Ca(2+)-sensitizing and phosphodiesterase-inhibiting activities in intact cardiac muscle. These actions were additive and could account for the effects observed with EMD 53998. (+)EMD 57033 appears to be the first inotropic agent that acts predominantly by increasing myofilament calcium sensitivity.

The effect of acidosis on beta-adrenergic receptors in ferret cardiac muscle.

Acidosis decreases the force of contraction of cardiac muscle in response to noradrenaline. The role of beta-adrenergic receptors in this response to acidosis was investigated. Radioligand techniques were used to determine beta-adrenergic receptor number and the degree of G-protein coupling, and to see whether these were altered in tissues subject to acidosis. The effect of pH on agonist and antagonist binding to these receptors was also investigated. Tissue pre-exposure to acidic conditions had no effect on numbers of beta-adrenergic receptors and no effect on the affinity of [125I]iodocyanopindolol (ICYP) for the receptors. Agonist competition experiments indicated that there was no change in the affinity of isoprenaline for these receptors, and there was no change in the relative proportions of high and low affinity binding sites. When radioligand experiments were performed under acidic conditions, however, the total number of beta-adrenergic receptors increased, and the affinity of these receptors for isoprenaline increased. This increase in agonist affinity might, therefore, minimize the shift to the right seen in the dose-response curve to noradrenaline during acidosis.

The effect of acidosis on the relationship between Ca2+ and force in isolated ferret cardiac muscle.

1. The relationship between force and intracellular [Ca2+] (monitored using the protein aequorin) has been investigated in papillary muscles isolated from ferret hearts, under control conditions (superfusate pH (pHo) 7.3) and during acidosis (pHo 6.8). 2. At pHo 7.3, increasing bathing [Ca2+] from 0.5 mmol l-1 to 8 mmol l-1 led to an increase in the size of the intracellular calcium transient. At the lower [Ca2+] this was accompanied by an increase in developed force; however, at the higher bathing [Ca2+] developed force reached a plateau. 3. Acidosis (produced by increasing the [CO2] of the gas with which the muscle superfusate was equilibrated) decreased maximum force and shifted the curve relating peak developed force to peak intracellular [Ca2+] to the right. 4. The mechanisms underlying the apparent decrease in the sensitivity of the contractile proteins to Ca2+ were investigated by applying rapid length changes to papillary muscles at control pHo, during acidosis, and after bathing [Ca2+] had been increased to match force during acidosis to that in control. 5. Acidosis decreased the change in force produced in response to a given length change (i.e. decreased muscle stiffness) but when bathing [Ca2+] was increased during acidosis, muscle stiffness returned to control. 6. Acidosis had no effect on muscle stiffness after the induction of rigor in the muscle (produced by metabolic inhibition). 7. It is suggested that in intact cardiac muscle the major effect of a mild acidosis is to decrease the sensitivity of the contractile proteins to Ca2+, hence decreasing the number of bound cross-bridges.

Effects of 8-Bromo-cyclic GMP on contraction and on inotropic response of ferret cardiac muscle

The effects of guanosine 3′:5′-cyclic monophosphate (cGMP) on cardiac contraction are not established. Using isolated electrically-stimulated ferret papillary muscle at 29°C, 2.0 m m calcium we investigated the effects of 8-Bromo-cGMP on (a) basal contraction for comparison with the effects of reduction in extracellular calcium or of reduction in resting muscle length; (b) contraction of preparations stimulated by isoprenaline, the dihydropyridine calcium agonist Bay K8644 or post-extrasystolic potentiation. 8-Bromo-cGMP (0.1 m m) induced a small significant reduction in isometric twitch tension (TT) (7%), isotonic shortening (PS) (6%) and in twitch duration, but had no effect on maximum unloaded shortening velocity ( V max) or rate of tension development ( + dτ dt ). Reduction in muscle length induced a similar immediate effect on contraction. Reduction of extracellular calcium (2.0 m m to 1.25 m m) reduced TT by 24% and PS by 14% as well as V max (19%) and + dτ dt (29%), but did not alter twitch duration. Bay K8644 (0.01 to 10 μ m) produced increases in TT, + dτ dt , PS and twitch duration each of which was significantly reduced in the presence of 8-Bromo-cGMP (0.1 m m). 8-Bromo-cGMP had no effect on the responses to isoprenaline 1 n m to 100 μ m—which increased TT, + dτ dt and PS but markedly reduced twitch duration—nor on post-extrasystolic potentiation which increased TT and + dτ dt but slightly reduced twitch duration. These results show that 8-Bromo-cGMP induces changes similar to the immediate effects of reduction in resting muscle length, and reduces the positive inotropic effects of Bay K8644 but not those of isoprenaline or post-extrasystolic potentiation.

Dissociation of changes in apparent myofibrillar Ca2+ sensitivity and twitch relaxation induced by adrenergic and cholinergic stimulation in isolated ferret cardiac muscle.

In isolated, aequorin-injected ferret cardiac muscle we measured the apparent myofilament Ca2+ sensitivity and its relationship to twitch relaxation time in the presence of autonomic perturbations. The Ca2+-tension relation was determined from the peak aequorin luminescence and peak twitch tension measured in muscles across a broad range of bathing [Ca2+] in the presence and absence of acetylcholine (ACh) (1 microM) or isoproterenol (ISN) (1 microM), or both drugs. ACh shifted the relationship of peak tension to (peak) aequorin light leftward, which suggests an increase in myofilament Ca2+ sensitivity, but it did not alter relaxation, which was measured as the time for peak tension to decay by 50% (t 1/2 R). ISN produced its previously documented effects, i.e., a rightward shift of the relationship of peak tension to peak aequorin light and a decrease in t1/2R. ACh abolished the ISN effect on the peak tension-aequorin light relationship but did not reverse the effect of ISN to decrease t1/2R. The effects of ACh and ISN of modulating the apparent myofilament Ca2+ sensitivity in intact muscles, corroborate findings of previous studies in isolated myofibrillar preparations. However, these perturbations of myofilament Ca2+ sensitivity in the intact muscle do not relate to twitch relaxation, measured as t1/2R, since (a) ACh affects the former but not the later and (b) the effect of ISN on the Ca2+-tension relationship is abolished by ACh, while the relaxant effect persists.

Use-dependence of ryanodine effects on postrest contraction in ferret cardiac muscle.

During an investigation of the effect of ryanodine on contractions in cardiac muscle, it was found that long rest periods removed all or most of the drug's effect. Therefore, we studied the kinetics of block development and recovery from block produced by low concentrations of ryanodine (1-100 pM) on the postrest contractions of ferret papillary muscle. At 100 pM, ryanodine depressed steady-state contraction amplitude slightly (4.2 +/- 1.1% mean +/- SEM, n = 10) but strongly inhibited (40-80%) the first contraction (postrest contraction) elicited on restimulation of the preparation after rest periods of 1 second to 5 minutes. Under control conditions, the nearly maximal potentiation of the twitch occurring after a standard test rest period (30 seconds of rest) was not affected by a preceding conditioning rest of up to 20 minutes. In the presence of 100 pM ryanodine, a conditioning rest increased the amplitude of the twitch elicited after a test rest, and the test rest contraction recovered toward control (drug-free) amplitude monoexponentially (time constant, 582 +/- 105 seconds). Block of postrest contraction could be reinduced by stimulation and occurred faster when higher rates were used (time constants, 758 seconds at 1 Hz and 107 +/- 26 seconds at 3 Hz). Since rest potentiation of twitch tension is believed to be mostly dependent on extra calcium released from the sarcoplasmic reticulum, the results suggest that the ryanodine-induced blockade of calcium release from the sarcoplasmic reticulum is use-dependent and recovers during diastole.(ABSTRACT TRUNCATED AT 250 WORDS)

Concealed positive force-frequency relationships in rat and mouse cardiac muscle revealed by ryanodine.

Increases in stimulation frequency between 0.3 and 2 Hz decrease developed tension in rat, seemingly inconsistent with the explanation that an elevation of intracellular Na+-ion concentration is responsible for the positive force-frequency relationships observed in most species. Thus the force-frequency relationships were reevaluated in isolated atrial muscle of rat and mouse heart that show negative relationships, comparing them with ferret and guinea pig that show positive relationships. Ryanodine (2 nM, 45-min exposure) markedly reduced potentiated postrest contractions in all four species and caused a marked negative inotropic effect especially at low stimulation frequencies in rat and mouse, a moderate effect in ferret, but only a modest effect in guinea pig. In the presence of ryanodine, all four species showed positive force-frequency relationships. These results indicate that activator calcium in rat, mouse, and ferret cardiac muscle has a large ryanodine-sensitive component that shows a negative force-frequency relationship, masking a component for positive force-frequency relationships that exists in all species.