Positive and Negative Inotropic Effects of Elevated ..., Exercises of Physiology

Download Positive and Negative Inotropic Effects of Elevated ... and more Exercises Physiology in PDF only on Docsity! Positive and Negative Inotropic Effects of Elevated Extracellular Potassium Level on Mammalian Ventricular Muscle FREDERIC KAVALER, PAUL M. HYMAN, and ROBERT B. LEFKOWITZ From the Department of Physiology, Downstate Medical Center, State University of New York, Brooklyn, New York 11203 ABSTRACT The effect of moderate elevation in extracellular potassium con- centration (up to 12 mm) on contraction of cat ventricular muscle was exam- ined. Isometric force development was recorded from eight excised trabeculae and from six coronary-perfused in situ papillary muscle preparations. Contrac- tion in the steady state was variably affected, sometimes decreasing mono- tonically, sometimes remaining unchanged, with increasing potassium level. In 11 of these 14 preparations, the steady state was preceded by a transient period in which the contraction was augmented. In addition, eight excised tra- beculae were used in an experimental arrangement designed to distinguish be- tween inotropic effects caused by potassium-induced alterations in the action potential and other, more direct, effects of this ion on contraction. The nega- tive inotropic effect is attributable to a potassium-induced reduction in the amplitude and/or duration of the action potential plateau. The positive ino- tropic effect was found in experimental arrangements where effects of the potas- sium-rich medium on action potential time-course were effectively "buffered." The positive inotropic effect thus depends on the presence of the elevated potassium concentration and can occur independently of effects on the action potential time-course. INTRODUCTION Increases in extracellular potassium concentration to levels still compatible with propagated excitation have been reported by some investigators to de- press contraction of mammalian ventricular muscle (Garb, 1951; Engstfeld et al., 1961; Sarnoff et al., 1966), while others have found it to be unaltered (Green et al., 1952; Goodyer et al., 1964). It is conceivable that potassium exerts its effects through more than one mechanism, which may vary in rela- THE JOURNAL OF GENERAL PHYSIOLOGY · VOLUME 60, 1972 · pages 351-365 351 352 THE JOURNAL OF GENERAL PHYSIOLOGY ·VOLUME 60 · 972 tive magnitude and in time-course, and thus account for these apparently conflicting reports. The present study concerns observations on the effects of moderate elevations in extracellular potassium concentration on contraction of thin excised cat trabeculae and of a coronary-perfused in situ cat papillary muscle preparation. The findings, briefly, are that there are two opposite effects of increased potassium level: (a) a negative inotropic one, which is related to potassium-induced alterations in the time-course of the transmem- brane action potential; (b) a positive inotropic one, which occurs independ- ently of such alterations. METHODS Isometric contraction was recorded from excised cat trabeculae, 0.8 mm or less in diameter, by a technique described previously (Kavaler and Morad, 1966). This in- volved mounting one end between Lucite blocks, with minimal crush of the muscle, and attaching the free end to a force transducer by means of a nylon snare. Eight of the muscles studied were mounted in this "block and snare" and were driven, by 5-msec pulses, at a rate of 15/min. One muscle, similarly mounted (Fig. 3, left), was driven at a faster rate of 30/min. In addition, in order to detect transient changes, a technique was used in which isometric force development was monitored from a papillary muscle, while its coronary circulation was being perfused with Tyrode solu- tion. This preparation is illustrated in Fig. 1. Langendorff perfusion of a cat heart was carried out through an aortic cannula supplying oxygenated Tyrode solution containing 4 % dextran, to minimize tissue edema. Coronary flow by gravity was of the order of 25 ml/min at a pressure head of 100 mm Hg. Right ventriculotomy was carried out and a papillary muscle disconnected from its valve attachments. A close- fitting ring, made of a paper clip, was mounted rigidly on a Prior micromanipulator (Eric Sobotka Co., Inc., Farmingdale, N.Y.) and restrained the septal attachment of the muscle. The plane of the ring was adjusted to make uniform contact with the underlying septal endocardium. Contraction was monitored from the tendinous end of the muscle by means of an RCA 5734 mechanoelectric transducer tube. The muscle was electrically driven by 5-msec pulses applied between the restraining ring and a Teflon-coated wire inserted into the septum 1 or 2 mm away. The drive rate was that which could capture, ranging from 45 to 88/min. Six preparations of this Langendorff type were studied. To distinguish between effects on contraction which arise from the depolarizing consequences of elevated potassium levels and other possible effects of this ion, an experimental technique was devised to minimize potassium-induced depolarization. Results from eight technically satisfactory experiments of this type are reported below. Contraction was recorded from a very short segment of a muscle. The short segment length was 0.2 mm in the instance shown in Fig. 2 and 0.4-0.7 mm in other experi- ments. These lengths reflect some stretch from slack length and are estimated to represent more than 34 of the optimal length for contraction. In this segment, which was 1-4/0 of a length constant, the transmembrane potential was strongly influenced by electrotonic effects from the remainder of the muscle, which was 4 mm or more in length. A thin cat trabecula of 0.2 mm2 cross-section, or less, was drawn through KAVALER, HYMAN, AND LEFKOWITZ Mammalian Ventricular Muscle 355 experiment. The results shown in Figs. 6-10 were obtained during single microelec- trode impalements which remained stable and were free of evident movement arte- facts (one exception: Fig. 10, 15th beat) throughout the entire response to change of perfusing solution. In most cases this included the subsequent return to the control. All other data from excised tissues (Figs. 3-5) were obtained from the "block and snare" preparation referred to above (Kavaler and Morad. 1966). 1.5 -. 5 00 see go .me E 3. E so 010 1.0 h Iorsec c j 0.5 5 3 sec 0.5 0o+.-8M . 6mM Ca + - .8mMCa Ca 1 - m-5.4mMCa+ -. 8MC- Co ++ - FIGURE 3. Estimation of rapidity of alteration of extracellular fluid composition for an excised trabecula driven at 30 beats/min (left) and for a Langendorff-perfused papillary muscle preparation driven at 45 beats/min (right). Ordinate indicates developed force for each beat. RESULTS The Negative Inotropic Effect Fig. 4 shows the negative inotropic effect of moderate elevations in extracellu- lar potassium level for five experiments with excised tissues ("block and snare") (left) and for six coronary-perfused in situ cat papillary muscle prepa- rations (right). In the steady-state values shown in all graphs, contraction is progressively depressed in most muscles, with increasing potassium level, over the range 1.25-12 mEq/liter. In two excised trabeculae, there was no appre- ciable change in contraction over this range of potassium concentrations, and one of the in situ papillary muscles is only slightly affected. This finding applies both to developed force (lower graphs) and to peak rate of force development (upper graphs). The Positive Inotropic Effect In 11 of a total of 14 muscles studied (seven of eight "block and snare" prep- arations, four of six Langendorff-perfused muscles), transient augmentation of contraction occurred on elevation of the potassium level; this was followed, 356 THE JOURNAL OF GENERAL PHYSIOLOGY ·VOLUME 60 · I972 in the steady state, by an ultimate depression of contraction relative to the control. The upper portion of Fig. 5 shows an experiment on a thin trabecula. Within 20 sec after exposure to 10.8 mM potassium Tyrode, peak augmenta- tion of contraction (by 16%) has occurred. The transmembrane resting potential and the duration of the action potential are, as expected, reduced in the potassium-rich medium. In the steady state, as shown on the extreme right, contraction is reduced (by 13%) relative to the control. The tracing on the lower left shows the same effect in a coronary-perfused papillary muscle preparation. Peak augmentation of contraction (a downward-going trace) occurs within 8 sec after exposure to the potassium-rich medium. On W 2 wE o 22 [K']IN MEDIUM (mM KItN MEDIUM (mM) FIGURE 4. Effect of Tyrode potassium level on contraction of five excised trabeculae, mounted in the "block and snare" and of six in situ, coronary-perfused papillary muscles (right). Steady-state isometric values are given for total developed force (lower plots) and for peak rate of force development (upper plots). the lower right are plotted results from an excised trabecula, where the amount of augmentation at the peak is seen to be clearly related to the con- centration of potassium to which the muscle has been exposed. The range of potassium concentrations which elicit the positive inotropic effect is considerably lower than the potassium levels reported to cause release of tissue catecholamines (Haeusler et al., 1968). In addition, the positive ino- tropic effect was not associated with a decrease in time-to-peak of contraction (e.g., Fig. 5: 510 msec, compared with 505 msec control), a common char- acteristic of the effect of norepinephrine (e.g., Kavaler and Morad, 1966). Finally, the positive inotropic effect was observed in the presence of 3 X 10- 6 M propranolol, a dose which almost totally abolished (i.e., reduced to 7%) the increase in force development brought about by added norepineph- rine. For these reasons, potassium-induced release of chemical mediators KAVALER, HYMAN, AND LEFKOWITZ Mammalian Ventricular Muscle from sympathetic nerve endings is an improbable cause of the positive ino- tropic effect. The Negative Inotropic Effect and Potassium-Induced Alteration of the Action Potential The result of raising the extracellular potassium level to 10.8 m in an entire trabecula (i.e., for both the long and short muscle segments of the preparation described in Fig. 2) can be seen in Fig. 6. There is a progressive reduction in FIGURE 5. The positive inotropic effect, as seen in an excised trabecula ("block and snare," upper Brush recorder tracings, 10.8 mmu potassium Tyrode applied immediately following the 20 sec time calibration) and in a coronary-perfused papillary muscle (lower left, contraction downward-going). A plot of the magnitude of the positive ino- tropic effect as a function of extracellular potassium level is shown on the lower right for one excised trabecula. resting transmembrane potential amounting to a depolarization of 19 my in the steady state. The plateau of the action potential is concomitantly reduced both in amplitude and in duration. Associated with this is a reduction in con- traction, as developed force or as peak rate of force development. The action potential upstroke is markedly reduced in the depolarized tissue, but this introduces no important element of conduction delay. As shown in Fig. 7, essentially the same sequence of events is brought about by raising the potassium to 10.8 mEq/liter only in the Tyrode perfusing the long muscle segment. The short segment was 0.4 mm in length. The depolariz- 357 360 THE JOURNAL OF GENERAL PHYSIOLOGY VOLUME 60 I1972 first two beats following exposure to the 10.8 mEq/liter potassium Tyrode. Such an increase in membrane conductance for potassium might also account for the observed decrease in the rate of rise of the action potential upstroke. The Positive Inotropic Effect and its Independence of Changes in Action Potential In seven preparations contraction was increased on application of the potas- sium-rich Tyrode to only the short segment. In two of these, where the action potential was unaltered in the presence of the 10.8 mM potassium, the positive inotropic effect was maintained at a constant level throughout the period of exposure to the potassium-rich Tyrode. This is shown in Fig. 10. Although some depolarization of the resting level occurs, the plateau and repolarization time-courses are superimposable. A progressive increase in contraction is seen, 15 t h beat 29 t h beat 12 Sec DEPOL. (mv) - 0 3 P 0. 22 0.20 0.17 (g/rm 2 ) P 1.38 1.09 0.90 (g/mm 2 - sec) V 145 110 134 .(v/sec ) FIGURE 9. Negative inotropic effect on application of potassium-rich Tyrode only to the short (0.25 mm) segment, in the presence of a marked alteration in action potential plateau. Preparation is as in Fig. 2, symbols are as in Fig. 6. which was maintained at the level shown in the 15th beat for a subsequent 40 sec period of exposure. Fig. 11 shows the rapid onset and sustained character of the positive ino- tropic effect in another preparation where plateau and repolarization time- course were almost unaltered in the potassium-rich Tyrode. The 90% re- polarization time, for example, was reduced by 11 msec from a control value of 465 msec. The overshoot and the early portion of the plateau were super- imposable on the control record in the manner shown in Fig. 10. An "unbuf- fered" depolarization, equal in magnitude (8.4 my) to that shown in Fig. 10, could be seen during the quiescent period between beats. The slight falloff from the peak value of contraction seen here was not observed in the experi- ment shown in Fig. 10, where action potential time-courses were exactly superimposable. In the other five preparations where potassium-rich medium, applied to the short segment, had a positive inotropic effect, more marked lowering of the action potential plateau and shortening of the repolarization KAVALER, HYMAN, AND LEFrOWITZ Mammalian Ventricular Muscle time were seen. Falloff from a maximal positive inotropic effect was more marked in these trabeculae, resulting in two cases in a net reduction in con- traction in the steady state. Reversal of the positive inotropic effect was regu- larly seen on return of the short segment to the 2.7 mM K+ Tyrode. In two preparations this was seen to occur in the absence of any change in the action potential time-course associated with the return of the extracellular potassium level to 2.7 mM. 8th beat II th beat 15th beat I2 sec DEPOL. (mv) - 0 2 8 P 0.37 0.39 0.39 0.41 (g/mm 2 ) fP 2.0 2.3 2.3 2.3 (g/mm 2 -sec) FIGURE 10. Action potential-independent positive inotropic effect of potassium-rich medium applied to short (0.4 mm) segment. Preparation is as in Fig. 2, symbols are as in Fig. 6. FIGcuRE 11. Contractile record (a downward-going trace) during positive inotropic effect of potassium-rich medium applied to the short (0.7 mm) segment. The highest value for developed force shown here (in the 10.8 mm K Tyrode) is 0.50 g/mrm2. Dura- tion of exposure to the potassium-rich medium is exactly I min. It is reasonable to assume that at the distal site on the short segment from which transmembrane potentials were consistently monitored, voltage control from the long segment would be least adequate. Thus, in the two short segments where plateau and repolarization time-courses in the presence of 10.8 mM potassium Tyrode were superimposable with those of the control, it is likely that the same was true at more proximal sites. In two preparations this was verified by a microelectrode impalement in the middle of the short segment during one application of 10.8 mM potassium Tyrode, and by an impalement at the usual distal site (at 0.95 of the short segment length) during 361 362 THE JOURNAL OF GENERAL PHYSIOLOGY VOLUME 60 - 972 the next application of the potassium-rich medium. Depolarization of resting potential by the potassium-rich medium was less at the short segment's mid- point in both preparations, as was the degree of shortening of action potential duration. One of these experiments is shown in Fig. 12, where it can be seen that depolarization amounted to 10.5 mv at the distal site (0.38 mm of a 0.40 mm short segment) and 7.9 mv at the midpoint (0.18 mm from the rubber partition). The values for 90% repolarization time were 539 msec (at 0.38 mm) and 574 msec (at 0.18 mm). Mid- Segment [018mm End SegmentF[-38 mm Lo.4o m .4 0 mm m 0 .5 g/mm2a /2 se e Control Beat No. 15 Control Beat No. 15 Depol (mv) 7. 9 - 0.5 AP Amp. (mv) 107 94 109 95 90% Repol. Time (msec) 671 574 668 539 FIGURE 12. Microelectrode impalements in the mid-portion of the short segment (left traces) and at a distal point (right traces) during successive exposures of the short seg- ment to 10.8 mM potassium Tyrode. Films during the 15th beat (i.e., the steady state) are in each case superimposed on control (2.7 mm potassium Tyrode) records. The total short segment length (0.40 mm) represents considerable stretch from slack length (0.27 mm). Control action potentials at both sites are comparable, although intra- and extra- cellular electrodes were reversed. The latter accounts for the reversal of the contractile traces on the left (film reversed). DISCUSSION Potassium-rich media can both augment and depress contraction of mam- malian ventricular muscle. This finding provides an explanation for the ap- parently conflicting statements regarding potassium's effect. These effects would be expected to vary in their relative magnitudes in such a way as to cause, occasionally, no change in the steady-state contraction and this was the case for two of the excised trabeculae (Fig. 4, left), where the transient positive inotropic effect was particularly large (33 and 40% of control, re- spectively). That the negative inotropic effect is associated with a reduction in the amplitude and/or duration of the action potential plateau is most convincingly shown by the experiments in which the potassium-rich medium was applied only to the long segment. These changes in plateau, imposed by the long segment, were uniformly associated with diminished contraction of the short segment. Elevation of extracellular calcium in the long segment failed to