The inotropic mechanism of the phosphodiesterase inhibitor OPC 3911 alone and in combination with rolipram, studied in papillary muscles of ferret and guinea-pig and isolated myocytes of guinea-pig ventricular muscle.

OPC 3911 is a potent inhibitor and PDE III is a specific inhibitor in cardiac muscle. The effects of the drug alone and in combination with the non-inotropic PDE IV inhibitor rolipram were analysed using hearts from guinea-pigs and ferrets. OPC 3911 had an EC50 value of 0.1 microM. At 0.1 microM peak force was increased by 50.7 +/- 7.6% (n = 6, P < 0.001), time to peak tension (TPT) reduced by 18.7 +/- 5.6% (n = 6, P < 0.05). Time to half relaxation (THR) was prolonged by 19 +/- 4.2% (n = 6, P < 0.001). After addition of rolipram (30 microM), there was a potentiation of peak force at all concentrations of OPC 3911. At 0.1 microM OPC rolipram increased peak force by 82.8 +/- 8.9% (n = 6, P < 0.001), reduced TPT by 73 +/- 6% (n = 6, P < 0.005) and increased THR by 27 +/- 5% (P < 0.01). OPC 3911 shortened action potential duration (APD) at 50% repolarization by 5.3 +/- 2.5% (n = 6, P < 0.05). Addition of rolipram prolonged APD by 3.7 +/- 2.5% (n = 6, P < 0.05). Second inward current (Isi) was increased at 3 microM OPC 3911 by 46 +/- 6% (n = 6, P < 0.05). The combination of OPC 3911 and rolipram intensified the Isi to 101 +/- 5% (n = 3). Rolipram slowed the rate of restitution and the onset of restitution was prolonged. Relative maximum post-extrasystolic potentiation was reduced in the presence of OPC 3911 from 67 +/- 5% to 45 +/- 6%. Adding rolipram caused potentiation of 55 +/- 6%. OPC 3911 increased the recirculation fraction of activator calcium from 0.36 to 0.42 (n = 10, P < 0.05). After addition of rolipram the recirculation fraction was 0.41 +/- 0.04 (n = 10, P < 0.05). The results suggest that rolipram exerts its potentiating effect on OPC 3911 via an increased Isi.

The contractile and electrophysiological effects of rolipram in guinea-pig papillary muscles and isolated ventricular myocytes.

Isometric force, action potentials and in voltage-clamp Isi (second inward current) and its current voltage relation were recorded in papillary muscles from guinea-pigs and from guinea-pig isolated ventricular myocytes (35-37 degrees C, 0.5-1 Hz). Rolipram (1-100 microM) had no significant effect on peak isometric twitch. The rate of rise of force and time to peak tension (TPT) was likewise unaffected. Time to half relaxation (THR) was increased in a dose-dependent manner and at 30 microM THR was prolonged by 25.3 +/- 6% (n = 10, P < 0.001). The effect of 30 microM rolipram on isometric force was frequency dependent. At 0.25 Hz peak force was increased by 6.3 +/- 3.1% (n = 7, P < 0.05). At 2 Hz rolipram exhibited a negative inotropic effect of 9.8 +/- 3.3% (n = 5, P < 0.02). Action potential duration at 90% repolarization was prolonged by 13 +/- 6 ms (n = 7, P < 0.05), and there was usually no effect on resting potential or action potential amplitude. Sometimes, however, a depressed plateau was recorded. Rolipram was without effect on Isi and its current-voltage relations. Time to full mechanical restitution after a test interval was not changed but the shape of the restitution curve was altered. The restitution process was much slower in the presence of rolipram. Hence, peak force was lower at test intervals shorter than 800 ms. Likewise, the shape of the curve relating postextrasystolic potentiation to test interval was altered by rolipram.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical and electrophysiological effects of milrinone on the force-frequency relationship in mammalian myocardium.

Isometric force, action potential and current-voltage relation were studied in guinea-pig and ferret papillary muscles. Milrinone (1 microM) increased peak twitch force by 40 +/- 4%, reduced time to peak tension (TPT) by 12.1 +/- 3% (n = 6, P < 0.01) and reduced time to half relaxation by 17.3 +/- 4.1% (n = 6, P < 0.01). The effect of milrinone was potentiated by rolipram, a RI-PDE inhibitor which in itself had no inotropic effect. After the addition of rolipram peak isometric force was increased by 104 +/- 8% (n = 6, P < 0.001), TPT was further reduced whereas time to half relaxation was slightly increased after the addition of rolipram. Action potential duration at 75% repolarization was decreased by 11 +/- 5 ms (n = 6, P < 0.05). Milrinone also potentiated the second inward current (Isi) by 21 +/- 3.2% (n = 6, P < 0.01). Peak twitch force in response to a test stimulus after an interval, i.e. mechanical restitution was increased at all intervals. The onset of restitution was faster and time to full restitution also shortened. Maximum postextrasystolic potentiation was greater in the presence of milrinone, whereas relative potentiation was smaller in presence of milrinone (46 +/- 7%) than in control (74 +/- 7%). The recirculation fraction of activator calcium was enhanced by milrinone from 0.35 +/- 0.04 to 0.48 +/- 0.07. The results support the view that the positive inotropic effect of milrinone is due to a greater inflow of calcium during the action potential and a more efficient intracellular calcium handling.(ABSTRACT TRUNCATED AT 250 WORDS)

Influences of stimulation frequency and temperature on interval-force relationships in guinea-pig papillary muscles.

Relationships between contractile force and the preceding and pre-preceding stimulation intervals were studied in papillary muscles by interposing variable test intervals during steady-state pacing. The strength of test contractions increased exponentially to a maximum as the preceding (test) interval was lengthened. Contractility decreased as an exponential function of pre-preceding interval. At 37 degrees C, the half times for these processes were unaffected by increasing the steady-state frequency from 1 to 3 Hz. At 27 degrees C, the force increase with preceding interval was accelerated and the decay with pre-preceding interval was retarded as the stimulation frequency was increased from 0.33 to 2 Hz. The time-courses of force increase and decay were similar to each other during stimulation at an optimum frequency characteristic for the temperature. Cooling from 37 to 27 degrees C prolonged the half times for force increase and decay by factors of 4.5 and 3 respectively. The slope of the linear relationship between the force of the contraction pre-preceded by the test interval and the immediately subsequent contraction (recirculation fraction) was also halved. These results suggest that high stimulation frequency and low temperature uncouples cellular processes underlying the interval dependence of cardiac contractility. The temperature sensitivities are consistent with these processes being enzymatic. The reduced recirculation fraction provides a mechanism for the lowered threshold frequency for sustained mechanical alternans at 27 degrees C.

Effects of nifedipine and low [Ca2+] on mechanical restitution during hypothermia in guinea pig papillary muscles.

Increasing the frequency of steady state stimulation increases the rate of mechanical restitution in hypothermic (27 degrees C) guinea pig papillary muscles. In this paper we have investigated the influences of the calcium antagonist nifedipine and of reduced extracellular calcium concentration on this phenomenon. We found that nifedipine abolished the frequency dependent increase in the restitution rate, which was also sensitive to extracellular [Ca2+]. These findings suggest that the level of intracellular [Ca2+] can influence the rate of restitution. It is implied that this effect is mediated via ICa, the inward calcium current, which makes a larger than normal contribution to direct contractile activation in hypothermic myocardium.

Effects of amrinone on shortening velocity and force development in skinned skeletal muscle fibres.

The effects of amrinone were studied on single skinned fibres isolated from rat hindlimb muscles. In each fibre a force-velocity relation was determined during maximal calcium activation (pCa = 4.45) in control conditions and in the presence of amrinone. The MgATP concentration was 3.93 mM, close to the physiological value. After the experiment the fibre was classified as fast or slow on the basis of its reactivity with anti-myosin monoclonal antibodies. In fast fibres amrinone (3 mM) potentiated isometric tension (Po) by 13.8 +/- 2.9% (n = 13), reduced maximum shortening velocity (Vmax) by 32.6 +/- 3.2% and the curvature of the force-velocity relation (a/Po) was increased by 98.9 +/- 46.0%. All these effects were less pronounced in slow fibres, where Vmax was reduced only by 11.4 +/- 3.6 (n = 16). The effects of amrinone (0.3-6 mM) on the ATPase activity of myofibrils and myosin prepared from fast (tibialis anterior) and slow (soleus) rat skeletal muscles were studied. Amrinone was found to depress Ca-Mg dependent ATPase activity of myofibrillar preparations of the tibialis anterior (up to 16.6 +/- 2%) and, to a lesser extent, of the soleus (up to 7.2 +/- 1.2%). On the contrary, Ca-stimulated myosin ATPase activity was significantly increased by amrinone in myosin preparations from the tibialis anterior. Experiments were carried out to test whether amrinone (3 mM) might affect the sensitivity of the contractile system to MgATP concentration ([MgATP]). The results obtained showed that (1) the [MgATP] value at which isometric tension reached its maximum was shifted by amrinone from 0.1 mM to 0.3 mM, (2) the slope of the negative relation between [MgATP] and a/Po was made more steep by amrinone, and (3) the Km of the hyperbolic relation between [MgATP] and Vmax was increased from 0.39 to 1.71 mM by amrinone, thus indicating a reduced affinity of myosin for MgATP. These results are in accordance with the hypothesis that amrinone exerts a direct effect on the contractile mechanism.

Effects of Amrinone on shortening velocity, force development and ATPase activity of demembranated preparations of rat ventricular myocardium.

This study analyses the effects of Amrinone (bipyridine derivative with phosphodiesterase inhibitor properties) on the myofibrillar apparatus of rat myocardium. Thin trabeculae were isolated from the right ventricle and chemically demembranated. Force development and shortening velocity were measured during maximal calcium activations (pCa = 4.45) in control conditions and in the presence of 1-3 mM Amrinone. Maximum shortening velocity was obtained both from extrapolation of the force-velocity curve and with the slack test method. Amrinone was found to significantly reduce maximum shortening velocity and force development. Myofibrils and myosin were prepared from rat ventricular myocardium and their ATPase activity was assessed in control conditions and in the presence of Amrinone (0.3-6 mM). Ca-Mg dependent myofibrillar ATPase activity which was determined at low ionic strength was depressed by Amrinone in a dose-dependent way. Ca-stimulated ATPase activity determined at high ionic strength in myofibril or myosin preparations was not affected. Furthermore, Amrinone did not influence the pCa-ATPase activity curve of the myofibrillar preparations. A comparison between the inhibitory effects of Amrinone on myofibrils prepared from euthyroid rats and myofibrils prepared from hypothyroid rats was carried out. The ATPase activity was significantly less depressed in myofibrils prepared from hypothyroid rats than in those prepared from euthyroid rats. These results provide the first evidence of an effect of Amrinone on ATP splitting and force generation in the myofilament system of cardiac muscle.

Force production in voltage-clamped human atrial muscle.

Human atrial muscle preparations obtained during open heart surgery were mounted in a sucrose gap. Force and membrane currents were recorded during voltage clamp. After a 20-s rest, 10 clamps from a holding potential of -40 to 0 mV at 1.0 Hz were given. This was followed by a test clamp (called 1) of a varied duration and amplitude and two more test clamps (called 2 and 3) as during the priming period. Peak force of contraction 1 (F1) was independent of clamp duration from 2s to about 100 ms but declined at shorter durations. Peak force of contraction 2 (F2) and 3 (F3) increased with the duration and became potentiated. Increasing the clamp amplitude raised F1 to an optimum value at about +10 mV and there was a decline at higher voltages. Both F2 and F3 increased at higher amplitudes. A conventional bell-shaped current-voltage relation for the second inward current was obtained during clamp 1 with maximum inward current around -10 mV. In control experiments on isolated human myocytes peak current was recorded at somewhat more positive potentials. The relation between F3 and F2 was linear both when duration and amplitude of clamp 1 was varied. The slope of the line, interpreted as a measure of recirculation of activator calcium, was 0.4. It is concluded that force during voltage clamp in human atrial muscle is similarly related to membrane voltage as previously reported for guinea pig and ferret preparations.

Myocardial force interval relationships: influence of external sodium and calcium, muscle length, muscle diameter and stimulation frequency.

Several inotropic interventions were studied in thin papillary muscles under dynamic conditions. The effects on mechanical restitution and postextrasystolic potentiation were analysed. The decay of postextrasystolic potentiation was taken as a measure of recirculation fraction of activator calcium. The mechanical restitution curve had a plateau phase on its rising phase which was abolished in low extracellular sodium but pronounced in increased extracellular calcium. The recirculation fraction (RF) in control was 0.35 +/- 0.03; lowering the extracellular sodium by 20% increased the RF to 0.46 +/- 0.04 (n = 10). A reduction of sodium by 40% increased the RF to 0.57 +/- 0.04, whereas increasing extracellular calcium to 4 mM gave an RF of 0.48 +/- 0.05 (n = 10 in all cases). There was no significant effect on RF of changing basic stimulation frequency or muscle preparation length. These findings support RF as a good index of myocardial contractility. Furthermore, at muscle diameters above 0.65 mm the RF was found to be reduced, suggesting this diameter as critical for muscle function. Also, postextrasystolic potentiation in relation to preceding steady state contraction was markedly increased at these diameters. In conclusion, this study shows that RF is independent of stimulation frequency and muscle length, and that it is increased when calcium extrusion by the sodium/calcium exchange is reduced. Furthermore, RF is critically dependent upon the diameter of the preparation and mechanical restitution is changed by altered extracellular sodium concentration.

The action of 2,3-butane-dionemonoxime on the inotropic state in guinea-pig myocardium.

Isolated papillary muscles from guinea-pig right ventricles were used (temperature 33 degrees C, stimulation frequency 0.5 Hz). Isometric twitch and action potentials were recorded. Upon addition of 2,3-butane-dionemonoxime (BDM) (2 mM) the peak twitch force was reduced from 4.17 +/- 0.4 mN/mm2 to 1.68 +/- 0.3 mN/mm2 (n = 9, P less than 0.001). The time course of the isometric twitch was slightly altered. Time to peak tension (TPT) was reduced by 12.0 +/- 3% (n = 9, P less than 0.001) whereas time to half relaxation (THR) was left unaffected. The rate of rise of force was reduced by 35 +/- 3.2 mN/mm2s i.e. 46 +/- 3%. The action potential duration and amplitude was not significantly changed by the drug. The shape of the curve relating peak twitch force of an extra beat to the preceding test interval, i.e. mechanical restitution, was affected by 2 mM 2,3-butane-dionemonoxime. The curve reached its maximum faster after addition of the drug. Maximum postextrasystolic potentiation (force in response to the prepreceding test interval) was 3.2 +/- 0.4 mN/mm2 in 2 mM 2,3-butane-dionemonoxine and 7.6 +/- 0.7 mN/mm2 in control (n = 6). However the percentage potentiation was very similar in control (82%) and in presence of 2,3-butane-dionemonoxime (91%). Peak twitch force in relation to peak force of the preceding potentiated contraction during decay of postextrasystolic potentiation was analysed. There was a linear relation between the variables, the slope being 0.34 +/- 0.04 in control and 0.30 +/- 0.02 in 2,3-butane-dionemonoxime. This suggests that the drug is without an action on the fraction of calcium recirculating within the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Inotropic mechanisms of amrinone in papillary muscles from guinea-pig hearts.

Isometric force and action potentials were recorded in papillary muscles from guinea-pigs (temperature 33 degrees C, stimulation frequency 0.5 Hz). Amrinone (1 mM) increased peak twitch force (to 220% of control, n = 12) and rate of rise of force (to 221% of control, n = 12), while time from peak to half-relaxation was markedly reduced (to 70% of control). The time to peak force was not significantly changed. Action potential at 50% repolarization was shortened (93.3% of control, n = 8), whereas plateau voltage became more positive. Peak twitch force in response to a test stimulus after a varied interval, i.e. mechanical restitution, was increased at all intervals by the drug. However, the time to full mechanical restitution (1.5 s) was not affected. Forces in response to the test interval preceding the previous contraction (post-extrasystolic potentiation) were analysed. Maximum potentiation was 16.0 mN mm-2 (2.7 +/- 0.4%) before and 22.6 mN mm-2 (1.7 +/- 0.1%) after addition of the drug, i.e., the relative potentiation was diminished in the presence of the drug. The test interval for optimum potentiation was shortened from 370 to 320 ms (P less than 0.05, n = 12) in 1 mM amrinone. During decay of post-extrasystolic potentiation peak force of the post-potentiated contraction was linearly related to force of the potentiated contraction. The slope of this line (which is believed to monitor recirculation of activator calcium) was increased by amrinone from 0.37 to 0.50 (P less than 0.01, n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of amrinone on the electromechanical coupling in frog skeletal muscle fibres.

The contractile effects of 1.1 mM amrinone were studied on isolated skeletal muscle fibres of the frog (2-5 degrees C, slack length). Amrinone potentiated the twitch amplitude and the maximum tetanic tension and also enhanced the maximum contracture response elicited by 117.5 mM K+ (mean increase 6.3 +/- 1.8%, n = 6, P less than 0.02). The time to half-relaxation of the potassium contracture was slightly increased (mean change 13.8 +/- 3.5%, n = 7, P less than 0.01). Amrinone furthermore shifted the S-shaped curve relating contracture tension to log K+ to the left, and thus reduced the threshold depolarization (the mechanical threshold) needed to elicit a contracture (mean reduction 11 +/- 1 mV). The duration of the action potential at the -25 mV level was slightly increased by amrinone, whereas the resting membrane potential was unaffected.