Biphasic action of aldosterone on Akt signaling in cardiomyocytes.

Both aldosterone and Akt signaling play pivotal roles in the pathogenesis of heart failure. However, little is known about the correlation between them. We herein investigated whether aldosterone interacts with Akt signaling in a coordinated manner in cardiomyocytes. Neonatal rat cardiomyocytes were stimulated with aldosterone for either a short (10-min) or long (24-h) time. The phosphorylation of Akt and its downstream effector, GSK3ß, were transiently increased after short-term stimulation, which was blocked by either PI3K or Na(+)/H(+) exchanger inhibitors, but not by the mineralocorticoid receptor antagonist, eplerenone. Long-term stimulation also significantly increased Akt-GSK3ß phosphorylation and this effect was reduced by eplerenone. Thus, these results suggest that aldosterone activates Akt signaling via a biphasic reaction that occurs through different cascades. To understand the significance of the rapid action of aldosterone, cardiomyocytes were exposed to hydrogen peroxide for from 10 to 60 min. A short-term aldosterone stimulation (for up to 30 min) significantly protected cardiomyocytes from oxidative stress-induced cellular damage. Eplerenone did not abrogate this beneficial effect, while a PI3K inhibitor did. Therefore, during the early phase, aldosterone has favorable effects on cardiomyocytes, partly by acute activation of a mineralocorticoid receptor-independent cascade through the Na(+)/H(+) exchanger, PI3K, and Akt. In contrast, its persistent activity produces pathological effects partly by chronic Akt activation in a mineralocorticoid receptor-dependent manner.

beta-adrenergic stimulation restores the Ca transient of ventricular myocytes lacking t-tubules.

beta-adrenergic stimulation helps to synchronize Ca release in myocytes from failing hearts. Transverse (t-) tubules, which synchronize Ca release in normal cells and contain many of the elements of the beta-adrenergic pathway, may be depleted in such cells. The objective of the present study was to determine whether beta-adrenergic stimulation could reverse the desynchronization of Ca release observed in detubulated ventricular myocytes. The effect of isoprenaline (0.5 microM) on control and detubulated rat ventricular myocytes was investigated. Ca transients were monitored using whole-cell fluorescence and confocal microscopy, and Ca current recorded using the patch-clamp technique. Immunocytochemistry was used to investigate phospholamban (PLB) phosphorylation. Detubulation reduces and slows the Ca transient; these effects were reversed by isoprenaline. This restoration was associated with partial reversal of the desynchronization of Ca release that occurs in detubulated cells. Sarcoplasmic reticulum Ca load increased by the same amount in normal and detubulated cells, but Ca current increased less in detubulated cells (64%) than in control cells (124%) in response to isoprenaline. The pattern and extent of cAMP-dependent protein kinase and CaMKII-induced phosphorylation of PLB in response to isoprenaline was the same in both cell types. Thus, the beta-adrenergic pathway is functional in the absence of t-tubules; such stimulation appears to increase the speed of propagation of Ca via Ca-induced Ca release between adjacent clusters of ryanodine receptors, which may be relevant in pathological conditions, such as heart failure, in which t-tubules are depleted. The data also suggest that the Ca current responds to local signaling pathways, which are better coupled to the channel in the t-tubules than at the surface membrane, whereas PLB responds to whole-cell signaling.

Na+-Ca2+ exchange activity is localized in the T-tubules of rat ventricular myocytes.

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca2+] across the cell width. After detubulation, [Ca2+] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca2+]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca2+] as Ca2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na+-Ca2+ exchange current, although the density of the fast Na+ current was unaltered. It is concluded that Na+-Ca2+ exchange function, and hence Ca2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca2+ on stimulation.

Compensatory role of CaMKII on ICa and SR function during acidosis in rat ventricular myocytes.

It has been suggested that the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) increases during acidosis in cardiac muscle. Thus we have investigated the role of CaMKII during acidosis by monitoring intracellular Ca2+ (using fura-2) and ICa (using the perforated patch clamp technique) during acidosis, in the absence and presence of the CaMKII inhibitor KN-93, in rat isolated ventricular myocytes. In the absence of KN-93, acidosis (pH 6.5) increased the amplitude of the fura-2 transient and prolonged its decay, but in the presence of KN-93 acidosis did not alter the amplitude and prolonged the decay more. In the absence of KN-93, acidosis increased the amplitude of the caffeine-induced fura-2 transient but did not alter its amplitude in the presence of KN-93. ICa did not change significantly during acidosis in the absence of KN-93 but decreased during acidosis in the presence of KN-93. These results suggest that activation of CaMKII during acidosis helps to compensate for the direct inhibitory effects of acidosis on sarcoplasmic reticular Ca2+ uptake and ICa.

The effect of acidosis on the ECG of the rat heart.

We have investigated the effect of acidosis on the ECG in isolated rat heart to determine whether acidosis has marked effects on the ECG, and have used pharmacological agents to investigate possible mechanisms whereby acidosis alters the ECG. Acidosis produced a marked decrease in heart rate and an increase in P-R interval with little apparent effect on the duration of the QRS complex. The effects of acidosis did not appear to be due to acidosis-induced changes in transmitter release from severed autonomic nerve terminals within the heart. Experimental Physiology (2001) 86.1, 27-31.

Inhibition of the K+ channel kv1.4 by acidosis: protonation of an extracellular histidine slows the recovery from N-type inactivation.

1. Acidosis alters the transient outward current, ito, in the heart. We have studied the mechanism underlying the effect of acidosis on one of the K+ channels, Kv1.4 (heterologously expressed in Xenopus laevis oocytes), known to underlie ito. 2. At pH 6.5, wild-type Kv1.4 current was inhibited during repetitive pulsing, in part as a result of a slowing of recovery from N-type inactivation. 3. Acidosis still caused slowing of recovery after deletion of just one (either the first or second) of the N-terminal inactivation ball domains. However, deletion of both the N-terminal inactivation ball domains greatly reduced the inhibition. 4. As well as the N-terminus, other parts of the channel are also required for the effect of acidosis, because, whereas the transfer of the N-terminus of Kv1.4 to Kv1.2 conferred N-type inactivation, it did not confer acidosis sensitivity. 5. Replacement of an extracellular histidine with a glutamine residue (H508Q) abolished the slowing of recovery by acidosis. Reduction of C-type inactivation by raising the bathing K+ concentration or by the mutation K532Y also abolished the slowing. 6. It is concluded that binding of protons to H508 enhances C-type inactivation and this causes a slowing of recovery from N-type inactivation and, thus, an inhibition of current during repetitive pulsing.

Hybrid logistic characterization of isometric twitch force curve of isolated ferret right ventricular papillary muscle.

We previously found that the isovolumic pressure curve of the left ventricle and the isometric twitch force curve of the right ventricular in situ papillary muscle, both of the blood-perfused canine heart, were precisely fitted by our newly proposed hybrid logistic function. This function describes the difference between the two S-shaped logistic functions for the rising and falling components: A/[1+exp{-(4B/A)(t-C)}]-D/[1+exp{-(4E/D)(t-F)}]+G. This function characterizes comprehensively both ventricular and in situ papillary muscle contraction and relaxation. In the present study, we hypothesized that this function could also characterize the force curve of the most popular, standard-type, isolated and Tyrode-superfused papillary muscle preparation. To test this hypothesis, we investigated how precisely the hybrid logistic function could fit 112 isometric twitch force curves observed in eight isolated and Tyrode-superfused ferret right ventricular papillary muscles at different muscle lengths and extracellular Ca2+ concentrations. We always obtained a precise curve fitting with a correlation coefficient above 0.9987. This fitting was much more precise than sinusoidal and polynomial exponential function curve fittings. These results supported the present hypothesis. We conclude that our hybrid logistic function reasonably characterizes the force curve of the isolated myocardial preparation. This result broadens the generality of the hybrid logistic characterization of ventricular isovolumic pressure and myocardial isometric twitch force generation. The hybrid logistic characterization seems to be an integrative expression of contractile processes in myocardial twitch contraction.

Dynamic relations among length, tension, and intracellular Ca2+ in activated ferret papillary muscles.

To study the effects of mechanical constraints on the Ca2+ affinity of cardiac troponin C, we analyzed the tension and aequorin light (AL) responses to sinusoidal length changes (5-10% of the initial muscle length) in aequorin-injected, tetanized cardiac muscles. The amplitude of the quasi-sinusoidal tension and AL responses decreased with increasing length-perturbation frequency from 0.5 to 1 Hz at 24 degreesC and from 1 to 3 Hz at 30 degreesC. The increase in AL corresponded well to the decrease in tension; likewise, the decrease in AL to the increase in tension and the tension response lagged behind the length change. A further increase in frequency (>1 Hz at 24 degreesC and >3 Hz at 30 degreesC) markedly increased the amplitude of the tension responses but decreased the amplitude of the AL responses. The increase in AL lagged behind the decrease in tension; likewise, the decrease in AL lagged behind the increase in tension, and the tension response led the length change. From previous mechanistic interpretations of the frequency dependence of the amplitude of tension response, we argue that the Ca2+ affinity of cardiac troponin C changes in parallel with the active tension (i.e., the number of active cross bridges) but not with the passive tension produced by the length perturbation-induced cross-bridge strain.

Left coronary artery-left ventricle fistula with right coronary artery spasm.

A 72-year-old woman was admitted to our hospital for evaluation of chest pain. Coronary angiography showed a left coronary artery-left ventricle fistula. An acetylcholine provocation test induced vasoconstriction of the right but not the left coronary artery. Her chest pain was not relieved by combined therapy with isosorbide dinitrate, diltiazem and nicorandil. Because of the coronary spasm, beta-blockers could not be used. However, her chest pain was relieved after the administration of a minor tranquilizer. Thus, the patient's chest pain was unlikely to be associated with either the fistula or the coronary spasm.

Effects of acidosis on Ca2+ sensitivity of contractile elements in intact ferret myocardium.

We investigated the effects of acidosis on the intracellular Ca2+ concentration ([Ca2+]i) and contractile properties of intact mammalian cardiac muscle during tetanic and twitch contractions. Aequorin was injected into ferret papillary muscles, and the [Ca2+]i and tension were simultaneously measured. Acidosis was attained by increasing the CO2 concentration in the bicarbonate (20 mM)-buffered Tyrode solution from 5% (pH 7.35, control) to 15% (pH 6.89, acidosis). Tetanic contraction was produced by repetitive stimulation of the preparation following treatment with 5 microM ryanodine. The relationship between [Ca2+]i and tension was measured 6 s after the onset of the stimulation and was fitted using the Hill equation. Acidosis decreased the maximal tension to 81 +/- 2% of the control and shifted the [Ca2+]i-tension relationship to the right by 0.18 +/- 0.01 pCa units. During twitch contraction, a quick shortening of muscle length from the length at which developed tension became maximal (Lmax) to 92% Lmax produced a transient change in the [Ca2+]i (extra Ca2+). The magnitude of the extra Ca2+ was dependent on the [Ca2+]i immediately before the length change, suggesting that the extra Ca2+ is related to the amount of troponin-Ca complex. Acidosis decreased the normalized extra Ca2+ to [Ca2+]i immediately before the length change, which indicates that the amount of Ca2+ bound to troponin C is less when [Ca2+]i is the same as in the control. The decrease in the Ca2+ binding to troponin C explains the decrease in tetanic and twitch contraction, and mechanical stress applied to the preparation induced less [Ca2+]i change in acidosis.

Length dependence of Ca(2+)-tension relationship in aequorin-injected ferret papillary muscles.

The possible contractile proteins, which are related to the length-dependent change in the relationship between intracellular Ca2+ concentration ([Ca2+]i) and tension, were investigated using aequorin-injected ferret papillary muscles. Tetanic contraction was produced by applying repetitive stimulation to the ryanodine-treated preparations, and the relationships between [Ca2+]i and tension were measured. When the muscle length was decreased from maximal length (Lmax), at which maximal tension is produced, to 95 and 90% Lmax, the maximal tension was significantly decreased. [Ca2+]i required for producing 50% of the maximal tension was significantly increased from 1.05 +/- 0.04 microM (Lmax) to 1.17 +/- 0.04 microM (95% Lmax) and to 1.22 +/- 0.04 microM (90% Lmax). Isoproterenol (Iso) accentuated the length-dependent change in the [Ca2+]i-tension relationship. The decrease in the Ca2+ sensitivity induced by Iso was larger at shorter muscle lengths compared with that at Lmax. It is, therefore, suggested that adenosine 3',5'-cyclic monophosphate-dependent phosphorylation of troponin I and/or C protein alters the length dependence of the [Ca2+]i-tension relationship and that troponin I and/or C protein might be involved in the length-tension-dependent change in the affinity of the contractile elements for Ca2+.

[Oscillatory changes in the intracellular Ca2+ concentration in cardiac muscles].

Contraction of cardiac muscle is regulated by intracellular Ca2+ concentration ([Ca2+]i) change. The Ca2+ increased by the action potential is removed by Ca2+ removal mechanisms (sarcoplasmic reticulum, Na-Ca exchanger and Ca pump of the surface membrane) and is also bound to troponin. However, under some specific conditions which induced an overload of Ca2+, oscillatory changes in [Ca2+]i, which are considered to be due to regenerative Ca2+ release from the sarcoplasmic reticulum, appear. In Ca2+ overload, a transient increase in [Ca2+]i is observed after the falling phase of the Ca2+ transient which is induced by electrical stimulation. In accordance with the transient increase in [Ca2+¿i, the membrane transiently depolarizes (delayed after-depolarization). This membrane depolarization is considered to be due to the inward currents through the Na-Ca exchanger and non-specific cation channels. If the depolarization is large enough to reach the threshold, the action potentials are triggered (triggered activity). Thus, an increase in [Ca2+]i is one of possible factors in triggering of arrhythmia.

Effect of developed tension on the time courses of Ca2+ transients and tension in twitch contraction in ferret myocardium.

OBJECTIVES: The aim of the study is to test the hypothesis that tension-dependent change in the affinity of cardiac troponin-C influences the time courses of Ca2+ transients and tension in twitch contraction. METHODS: The Ca(2+)-sensitive photoprotein, aequorin, was microinjected into superficial cells of ferret papillary muscles and the Ca2+ transients and tension were simultaneously measured. The peak of developed tension was altered by changing the extracellular Ca2+ concentration, initial muscle length, and the application of 2,3-butanedione monoxime. RESULTS: In each maneuver, the decay time of Ca2+ transients was prolonged and the tension relaxation time was shortened when the peak of developed tension was decreased. In contrast, when the peak of developed tension was increased, the decay time of Ca2+ transients was shortened and the tension relaxation time was prolonged. The decay time of Ca2+ transients measured with different maneuvers was negatively correlated with the peak tension and the tension relaxation time was positively correlated with the tension peak. CONCLUSIONS: The changes in the decay time of Ca2+ transients and the tension relaxation time indicate that developed tension modulates the affinity of troponin-C for Ca2+ in normal twitch contraction.

Mechanisms of the inotropic effects of UD-CG 212 Cl, an active metabolite of pimobendan, on ferret papillary muscles.

We investigated the effects of an active metabolite of pimobendan, UD-CG 212 Cl, on Ca(2+) transients and tension using the aequorin method. When extracellular [Ca(2+)] ([Ca2+]o) in the Tyrode's solution was 2 mM, UD-CG 212 C1 (10(-7)-10(-4)M) increased the peak of Ca(2+) transients, accompanying a slight increase in peak tension. When [Ca(2+)]o was decreased to 0.5 mM, the twitch-potentiating effect of UD-CG 212 C1 was more remarkable, but the increase in the Ca(2+) transients at low concentrations of UD-CG 212 C1 (10(-7)-10(-6)M) was not significant as it was at 2 mM [Ca2+]o. The effects of UD-CG 212 Cl on the time courses of Ca(2+) transients and tension were evaluated at 0.5 mM [Ca2+]o. UD-CG 212 Cl shortened the decay time of Ca(2+) transients and the time to peak tension. However, the relaxation time was not significantly altered. UD-CG 212 C1 (10(-6)M) did not significantly change the relation between [Ca(2+)]i and tension in tetanic contraction. Therefore, the twitch-potentiating effect of UD-CG 212 Cl might not be due to an increase in the Ca(2+) sensitivity of the contractile elements. The slight increase in cyclic AMP due to the inhibition of phosphodiesterase type III by UD-CG 212 Cl could explain the twitch-potentiating effect and the faster time courses of Ca(2+) transients and tension.

Cross-bridge-dependent changes in the intracellular Ca2+ concentration in mammalian cardiac muscles.

A change in muscle length significantly alters the developed tension in mammalian cardiac muscle compared to that in skeletal muscle fibers. The intracellular mechanisms related to the length-dependent change in developed tension have been studied using Ca2+ indicators in intact preparations; a cross-bridge-dependent change in the affinity of troponin-C for Ca2+ is a possible mechanism. This hypothesis is further supported by the measurement of Ca2+ bound to troponin-C in skinned preparations. The molecular mechanism of the cross-bridge-dependent change in the affinity of troponin-C for Ca2+ is not fully understood although the studies which employ the substitution of troponin-C in skinned preparations, transgenic animals and in an animal model with heart disease have been performed. We reviewed the current studies by analyzing the intracellular mechanism responsible for the length-dependent change in tension development in mammalian cardiac muscle.

Tension-dependent changes of the intracellular Ca2+ transients in ferret ventricular muscles.

1. We measured the change in intracellular Ca2+ transients, using aequorin, in response to muscle length change during twitch contraction in ferret ventricular muscles. 2. Intracellular Ca2+ concentration ([Ca2+]i) was transiently increased when the muscle length was quickly shortened to 92% of maximum length (Lmax) at various times after stimulation (this increase in [Ca2+] is termed extra-Ca2+). The magnitude of extra-Ca2+, measured at different extracellular Ca2+ concentrations ([Ca2+]o), showed a dependence upon the magnitude of tension reduction and upon [Ca2+]i immediately before the length change. 3. In the presence of caffeine (5 mM), the difference between the Ca2+ transient at Lmax and at shorter lengths showed a time course similar to the difference between the developed tension at both lengths. A quick release in the caffeine-treated preparation produced the extra-Ca2+ with a slower time course compared with that observed in the absence of caffeine. Stretching the muscle from 96% Lmax to Lmax produced more active tension and decreased [Ca2+]i. 4. These results indicate that the affinity of troponin-C, a major Ca2+ binding protein, which controls contraction, is influenced by developed tension i.e. cross-bridge attachment and detachment.

Effects of thapsigargin on aequorin-injected and skinned preparations of ferret ventricular muscles.

OBJECTIVE: We investigated the effects of thapsigargin (TG) (0.1-1 microM) on the relation between intracellular Ca2+ concentration and tension in ferret papillary muscles using aequorin-injected and skinned preparations. METHODS: Aequorin was injected into the superficial cells of ferret papillary muscles; the Ca2+ signals of aequorin and tension in twitch and those with the application of 15 mM caffeine were simultaneously measured. The alteration of Ca2+ sensitivity of the contractile elements was examined by measuring the pCa-tension relation in Triton-X-treated skinned preparations. RESULTS: TG decreased the peak of the Ca2+ signal accompanied by a prolonged decay time. However, the tension was scarcely altered even at 1 microM TG. TG inhibited the caffeine-induced Ca2+ signal. Prolongation of decay of the Ca2+ signal by TG in twitch was further enhanced by isoprenaline (10 nM). The pCa-tension relation of the skinned preparation was slightly but significantly shifted to the right by TG. CONCLUSIONS: The apparent dissociation of the effects of TG on the Ca2+ signal and tension in intact preparations is not a result of alteration of the Ca2+ sensitivity of the myofilaments. The effects of TG in multicellular preparations are probably limited to the outer layer of the preparation. The slower time course of the Ca2+ signal induced by TG is due to the inhibition of Ca2+ uptake by sarcoplasmic reticulum, which is more significantly observed when the intracellular Ca2+ transient is increased by isoprenaline.

Effects of adenosine on Ca2+ transients and tension in aequorin-injected ferret papillary muscles.

The effects of adenosine on Ca2+ transients and tension in ferret papillary muscles were investigated using the aequorin method. Adenosine (0.01-1 mM) reduced the peak of Ca2+ transients and caused a slight concentration-dependent decrease in tension. Adenosine prolonged the decay time of Ca2+ transients but did not alter the time course of tension. In isoproterenol (0.1 microM)-treated preparations, adenosine decreased the peak of Ca2+ transients but did not alter the peak of tension. Adenosine prolonged the isoproterenol-shortened decay time of Ca2+ transients. The effects of adenosine on Ca2+ transients were antagonized by the selective A1 receptor antagonist 8-cyclopentyl-1,3,-dipropylxanthine. In the presence of isoproterenol, adenosine (0.1 mM) shifted the intracellular [Ca2+]/tension relation to the left. These results can be explained by the hypothesis that adenosine inhibits the activity of adenylate cyclase via stimulation of the A1 receptor, other mechanisms however cannot be overlooked. The prolongation of the decay time of Ca2+ transients and the increase in the Ca2+ sensitivity of the contractile elements are the underlying mechanisms of adenosine which maintain developed tension in twitch response, although adenosine decreases the peak of Ca2+ transients.