Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3-2.8 muM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca(2+) channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca(2+) sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 muM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca(2+) influx and activation systems.

The new local anaesthetic ropivacaine (LEA 103) like lidocaine and bupivacaine used as references, blocked cardiac sodium channels in a use-dependent fashion. At equimolar concentrations lidocaine had the lowest efficacy and bupivacaine the highest. The action potential was shortened and the plateau was depressed at high concentrations of each drug. Pacing a papillary muscle at 3.3 Hz in the presence of all three drugs resulted in a marked use-dependent accumulation of block (P less than 0.01). The accumulated block slowly dissipated after rest. At -90 mV holding (= resting) potential, and at a concentration of 10 microM, the time constant for recovery from block was 186 msec. in lidocaine (n = 4), 1.4 sec. in ropivacaine (n = 7), and 2.1 sec. in bupivacaine (n = 7). Lidocaine decreased Vmax progressively at high rates of stimulation, but not significantly at rates below 2 Hz. Ropivacaine progressively decreased Vmax significantly at rates above 1 Hz, but to a lesser degree than bupivacaine. The use-dependent action of the drugs was increased at more depolarized (less negative) holding potentials, whereas at more hyperpolarized potentials the block was diminished. Lidocaine and ropivacaine could be readily dissociated from the receptors at more hyperpolarized membrane potentials (-100 to -120 mV), whereas bupivacaine bound much harder. All three drugs blocked sodium channels more effectively after a long single conditioning pulse. Bupivacaine had the most prominent effect, and lidocaine was least effective. Bupivacaine and ropivacaine seem to interact with the inactivated state of the sodium channels, whereas lidocaine acted on both the open and on the inactivated state of the channels.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY1. We used ouabain-treated guinea-pig and ferret papillary muscles to study transient inward current (Iti), after-contractions, and tonic tension development during voltage-clamp pulses.2. Li, sucrose and choline were used isosmotically as Na substitutes to evaluate the effect of altering the Na equilibrium potential.3. We were unable to detect outward Iti at any potential up to + 30 mV in normal or Na-depleted solutions. However, reduction of Na had a biphasic effect on Iti, initially increasing it and then reducing it at all clamp potentials from -50 to + 20 mV.4. After-contractions were also initially increased and, in sufficiently Na-depleted solutions, decreased by reduction of extracellular Na. However, the peak in the after-contraction always occurred later than the increase in Iti and frequently coincided with the maximum suppression of the current. Complete suppression of after-contractions was not often achieved and always required more complete Na replacement than Iti suppression.5. Tonic tension responses were reduced by Na replacement, usually in synchrony with the reduction of Iti.6. The responses of Iti to Na replacement are consistent with a model of electrogenic Na-Ca exchange over the potential range positive to -50 mV. The responses deviate from the predictions of the model at more negative potentials. The results are consistent with the previous proposal that oscillatory changes in internal free Ca concentration underlie both Iti and after-contractions.

We have studied the responses to a brief interruption of a train of steady state beats, namely: (1) a single prolonged depolarisation within the train; (2) a single short interval within the train; (3) a single long interval within the train. These responses are predicted by a two compartment model of intracellular calcium handling. They are characterised by the following phenomena. (1) Prolongation of one depolarisation/action potential in the steady state train causes potentiation of the following beat. We postulate on the basis of the published evidence that this may be due to "reversed" sodium/calcium exchange during late systole leading to extra calcium entry during the prolonged depolarisation. (2) Postextrasystole potentiation is postulated to share this mechanism when a depolarisation (extrasystole) is introduced immediately after one of the steady state depolarisations (single short interval). The postextrasystolic beat is then potentiated. (3) A single short interval during the steady state train also leads to attenuation of contractile force on the beat immediately after the short interval, that is, the extrasystole. Mechanical restitution is the term applied to the recovery of this force with increasing interval. This consists of two phases. The initial rapid phase is ryanodine and caffeine insensitive, indicating possible independence of sarcoplasmic reticular function. We postulate that a "membrane compartment" of internal calcium may be responsible. The second, slower, phase of mechanical restitution is ryanodine and caffeine sensitive, indicating that it is likely to be a property of the sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)

Inter-relationships between force, membrane voltage and currents were studied in ferret and guinea-pig papillary muscles using the single sucrose gap technique (37 degrees C). The preparations were held at -90 or -40 mV and depolarized (excited) to 0 mV for 180 ms at 1.0 Hz. At regular intervals the shape of a single clamp pulse (called '1') was varied and its effects were investigated during the same test cycle and in two subsequent test cycles ('2' and '3'). Peak force of contraction 1 (F1) increased with the duration of the test clamp up to 90 ms and was constant thereafter. F1 increased with clamp amplitude (V1) between -30 and 10 mV and decreased at greater amplitudes. This relation was similar to the relation between peak second inward current (I1) and V1. The peak force of contractions 2 and 3 rose with the clamp duration and clamp amplitudes of cycle 1. The relation between F3 and F2 was linear (slope 0.40), except at the lowest and highest F2 values where there was a small deviation. There was an inverse relation between I2 and F2. The results support the idea that increased duration or amplitude of the voltage clamp pulse leads to a greater calcium entry which is manifested in the following potentiated contraction. The relation between F3 and F2 implies that about 40% of calcium recirculates between the contractions. The inverse relationship between F2 and I2 indicates that the second inward current is regulated by release from the sarcoplasmic reticulum via negative feedback.