Tetanus in the mammalian heart: Studies in the shrew myocardium

j Mol Cell Cardio119, 1247 1252 (1987)

T e t a n u s in the M a m m a l i a n Heart: Studies in the Shrew M y o c a r d i u m O. B i n a h

Rappaport Family Institute for Research in the Medical Sciences and the Department of Physiology and Biophysics, Faculty of Medicine, Technion-Israel Institute of Technology, P.O.B. 9697, Haifa 31096, Israel (Received 5 March 1987, accepted in revisedform 16 October 1987) O. B1NAH.Tetanus in the Mammalian Heart : Studies in the Shrew Myocardium. Journalof MolecularandCellular Cardiology(1987) 19, 1247-1252. The purpose of the present study was to test the possibility that tetanus can occur in the shrew myocardium, in which the ventricular action potential is similar to that of skeletal muscle. Action potentials and tension were recorded from the right ventricular muscle using glass microelectrodes and a force transducer. When the regular twitch (at a cycle length of 2000 ms) was followed by a train of stimui having an internal cycle length of < 50 to 60 ms, unfused and fused tetanus readily developed. Action potentials were generated at the frequency of the stimuli within the train. With trains of sufficient duration, the tension during the tetanus usually reached a steady state and exceeded the tension of the regular twitch preceding the train. Tetanus with similar characteristics was also observed during an arrhythmic train which was occasionally triggered spontaneously by a regular stimulus. Simultaneous recordings of the action potential and the twitch demonstrated that the shrew ventricular muscle can be tetanized, since the action potential is very short (3 to 4 ms) and terminates before any signs of mechanical activity begin. The present study demonstrates that mammalian myocardium can be tetanized when action potential duration is much shorter than the duration of the mechanical event. KEY WORDS:Shrew; Tetanic activity in the heart; Excitation contraction coupling.

Introduction T h e a b i l i t y to be t e t a n i z e d is n o t a c o m m o n f e a t u r e of the m y o c a r d i u m , a n d the m a m m a l i a n h e a r t does n o t m a k e a n y use of this p h e n o m e n o n d u r i n g its n o r m a l function. I n cardiac preparations from commonly studied m a m m a l i a n species such as dog, cat, sheep a n d g u i n e a - p i g , there is a t e m p o r a l o v e r l a p b e t w e e n the a c t i o n p o t e n t i a l a n d the t w i t c h [5, 10, 11, 14], a n d as the effective r e f r a c t o r y p e r i o d outlasts m o s t of the twitch, n e i t h e r s u m m a t i o n n o r t e t a n u s c a n be i n d u c e d . I n skeletal muscle, h o w e v e r , as the a c t i o n p o t e n tial is m u c h shorter (1 to 2 ms) t h a n t h e t w i t c h , a n d the effective r e f r a c t o r y p e r i o d is c o m p l e t e d before a n y signs of m e c h a n i c a l a c t i v i t y begin, s u m m a t i o n a n d t e t a n u s c a n be i n d u c e d a n d are, in fact, p a r t of the n o r m a l m e c h a n i c a l p e r f o r m a n c e [3, 4]. I n a r e c e n t s t u d y p e r f o r m e d in the shrew v e n t r i c u l a r muscle, w e f o u n d t h a t the a c t i o n p o t e n t i a l is v e r y s i m i l a r to t h a t in skeletal muscle, a n d its d u r a t i o n , at a cycle l e n g t h o f 2000 ms, is o n l y 0022-2828/87/121247 + 06 $03.00/0

3 to 4 ms [1, 2]. Since a short r e f r a c t o r y period, w h i c h closely c o r r e l a t e s w i t h a c t i o n p o t e n t i a l d u r a t i o n , is a n essential r e q u i r e m e n t for tetanus, I tested the possibility t h a t t e t a n u s can be i n d u c e d in the s h r e w v e n t r i c u l a r muscle. I p r e s e n t h e r e e x p e r i m e n t a l e v i d e n c e d e m o n s t r a t i n g t h a t w h e n this r e q u i r e m e n t is m e t , the s h r e w m y o c a r d i u m c a n be tetanized.

Materials and Methods

Animals E x p e r i m e n t s w e r e p e r f o r m e d on shrews ( C r o c i d u r a russula) w e i g h i n g 7 to 10 g a n d on g u i n e a pigs w e i g h i n g 300 to 400 g.

Measuremen'ts of isometric twitch and transmembranepotentials The animals were anaesthetized with pentob a r b i t a l s o d i u m , 40 to 60 m g / k g , i n t r a p e r i toneally, a n d the hearts w e r e r a p i d l y excised a n d p l a c e d in cold T y r o d e ' s solution gassed w i t h 9 5 % 0 2 + 5 % C O 2. S u b s e q u e n t l y , a 9 1987 Academic Press Limited

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O. B i n a h

p r e p a r a t i o n consisting of the right v e n t r i c u l a r free wall strip in the shrew, a n d a thin papillary muscle in the guinea-pig, was dissected a n d m o u n t e d horizontally in a Lucite tissue bath. O n e end of the p r e p a r a t i o n was attached to a force transducer a n d the other end to a stainless steel post-affixed to a m o v a b l e m o u n t , which was controlled by a m i c r o m a n i p u l a t o r a n d used to change the length of the muscle. T h e preparations were studied at the peak of their length tension curve. I n the tissue bath, the preparations were superfused at a rate of 11 m l / m i n with T y r o d e ' s solution gassed with 95% 0 2 + 5% C O 2 and warmed to 36.5 +_0.2~ The T y r o d e ' s solution contained (in mM): NaC1, 131; N a H C O 3 , 18; N a 2 H P O 4 , 1.8; MgC12, 0.5; CaC12, 2.7; dextrose, 5.5; a n d KC1, 4.0. [o)

(b)

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T h e p r e p a r a t i o n s were stimulated by rect a n g u l a r pulses delivered at a basic cycle length of 2000 ms (except where indicated) by means of bipolar silver electrodes insulated to their tips with Teflon. T r a n s m e m b r a n e potentials were recorded using 3 M KCl-filled microelectrodes and s t a n d a r d microelectrode techniques. Action potentials were either photographed with a Poloroid camera from the oscilloscope or recorded on a storage oscilloscope a n d subsequently plotted and analyzed.

Results

Figure 1 depicts recordings of the mechanical activity in the shrew ventricular muscle u n d e r various experimental conditions. Figure 1 (a) illustrates a n isometric twitch recorded at ,,

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FIGURE 1. Mechanical activity recorded from the shrew ventricular muscle under various experimental conditions. (a) Isometric twitch recorded at cyclelengths of 2000 ms. (b) The basic stimulus (cyclelength = 2000 ms), resulting in a twitch, is followed by a train of stimuli (cyclelength = 55 ms), during which unfused tetanus developed. Note the aftercontractions and the potentiation of the twitch that follows the tetanus. (c) and (d) Fused tetanic activity generated in two different preparations. The cycle length within the train was in (c) 15 ms, and in (d) 13 ms. (e) and (f) Tetanic activity developingin two different preparations during an arrhythmic train. The arrhythmia was triggered by a singleregular stimulusdelivered at a cyclelength of 2000 ms.

Tetanic Activity in the Shrew Myocardlum

a cycle length of 2000 ms. At this cycle length, the twitch characteristics were (mean • S.E.M., n = 16): active tension, 0.55 __ 0.07 g; (+dT/dt) . . . . 16.02 • 2.39 g/s; (--dT/dt) . . . . 9.00 __+ 1.25 g/s, time to peak tension, 59.4 _+ 2.0 ms; and twitch duration, 277.9 • 12.1 ms. W h e n a train of stimuli (cycle length = 55 ms) followed the regular stimulus, the tension did not return to the resting level after each twitch, and unfused tetanus developed [Fig. l(b)]. It is seen that the tetanus is followed by several large aftercontractions, and that the twitch that follows the tetanus is stronger than that preceding the tetanus. T h e effects of train duration and extracellular Ca 2 + concentration on the unfused tetanus are seen in Figure 2; as the n u m b e r of stimuli within the train (cycle length = 40 ms) was increased from 80 through 150 up to 300, the tension increased until a steady state level was attained. In addition, as the extracellular Ca 2 + concentration was elevated from 2.7 to 5.4 mM, more tension developed, and the aftercontractions attained higher amplitudes. In general, as the cycle length within the train of stimuli was shortened, the unfused tetanus was converted into fused tetanus. Panels (c) and (d) in BO

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Figure 1 illustrate in two preparations typical patterns of fused tetanus in which the tension during the tetanus exceeded the tension of the preceeding twitch. Altogether, tetanus was observed in all 16 shrews in which the effect of high rates of stimulation on the mechanical performance was investigated. T h a t the tetanus induced by the experimental manipulations might be relevant to physiological, or rather to pathophysiological conditions is suggested by experiments in which tetanus was induced by an " a r r h y t h m i a " that was spontaneously triggered by a regular stimulus. As seen with the induced tetanus, the tetanus resulting from an arrhythmia was also of various forms, two of which are illustrated in Figure 1 [-panels (e) and (f)]. It should be stated that in terms of the basic electrophysiological properties and the mechanical stability and viability, the " a r r h y t h m o g e n i c " preparations were not distinguishable from those that were " n o n - a r r h y t h m o g e n i c " . In order to understand the conditions that allow tetanus to occur in the shrew myocardium, the temporal relationships between the electrical and the mechanical phenomena was studied (Fig. 3). As might be expected, based on the events that occur in skeletal muscle, the 5 0 0 beets

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6s F I G U R E 2. T h e effect o f t r a i n d u r a t i o n a n d e x t r a c e l l u l a r C a z+ c o n c e n t r a t i o n o n unfused tetanus. Basic cycle l e n g t h = 2000 ms. T h e t r a i n d u r a t i o n is reflected b y the i n c r e a s e in the n u m b e r o f beats, delivered a t a cycle l e n g t h = 40 ms.

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FIGURE 3. Temporal relationships between the mechanical and the electrical events in the shrew ventricular muscle during a single beat and a tetanus. (a) and (c) Simultaneous recordings of the twitch (upper traces) and the action potentials (lower traces) in ventricular preparations from a shrew and a guinea-pig, respectively. (b) ventricular action potential from the shrew, recorded on a fast sweep speed. In (a), (b) and (c) cycle length = 2000 ms. (d) Simultaneous recordings of the tension (upper trace), and of the action potentials (lower trace) during a tetanus induced by a train of stimuli (cyclelength = 30 ms) that followed a basic stimulus (cycle length = 2000 ms).

action potential (and therefore the effective refractory period) t e r m i n a t e d before there was a n y significant d e v e l o p m e n t of tension [Fig. 3(a)], and as such, the requirements for the occurrence of tetanus were met. T h e shrew v e n t r i c u l a r action potential recorded on a fast sweep speed is illustrated in Figure 3(b). Action potential characteristics at a cycle length of 2000 ms (means __ S.mM., n = 16) were: resting potential, - - 7 1 . 0 • 1.2 m V ; action potential a m p l i t u d e , 85.2 • 4.0 m V ; m a x i m a l upstroke velocity of phase 0 d e p o l a r i z a t i o n (Fmax), 262 __ 25 V/s; action p o t e n t i a l d u r a t i o n at 30%, 50% a n d 80% r e p o l a r i z a t i o n (APD3o , APD50 a n d APDso), 1.6 • 0.4, 1.8 • 0.3 a n d 3.4 • 0.4 ms, respectively. T h e t e m p o r a l relationships seen in the shrew m y o c a r d i u m were in m a r k e d contrast

to the events that c h a r a c t e r i z e d the expected b e h a v i o u r of c a r d i a c muscle of m a m m a l i a n species illustrated here in guinea-pig ventricular muscle [Fig. 3(c)]. Finally, to test the possibility that the g r a d u a l increase in tension d u r i n g the tetanus resulted from a m e c h a n i c a l p h e n o m e n o n which was not associated with on going electrical activity, the m e c h a n i c a l a n d the electrical events were recorded simultaneously d u r i n g tetanic activity. T h e representative e x p e r i m e n t shown in F i g u r e 3(d) clearly demonstrates t h a t the d e v e l o p m e n t of tension d u r i n g an i n d u c e d tetanus is closely associated with action potentials g e n e r a t e d at the frequency of the stimuli within the train. O n - g o i n g electrical activity was also recorded simultaneously with tension developing d u r i n g an a r r h y t h m i a - i n d u c e d tetanus.

Tetanlc Activity in the Shrew Myocardium Discussion

T h e present study demonstrates that unlike most other m a m m a l i a n species, the shrew ventricular muscle can be tetanized due to the temporal separation between the electrical and mechanical events. These observations are of interest, since they suggest that the e x c i t a t i o ~ c o n t r a c t i o n coupling in the shrew differs from most other c o m m o n m a m m a l i a n species in a qualitative rather than in a quantitative fashion; that is, the tetanus readily seen in shrew m y o c a r d i u m is generally considered to be a unique feature of skeletal muscle, but not of cardiac muscle. Although not identical, in several aspects the tetanus in shrew is similar to the tetanus in skeletal muscle. As seen in skeletal muscle, unfused and fused tetanus can be induced by increasing the rate of stimulation, the tension during tetanus can attain steady state level, and this tension can exceed the tension of the twitch preceeding the tetanus [3]. T h e requirements that should be met for tetanus to occur in cardiac muscle (and in skeletal muscle), namely, appropriate temporal separation between the electrical and mechanical events, might also be found in other m a m m a l i a n species. As far as the electrical event is concerned, we have recently shown that action potential duration is inversely correlated ( r = - - 0 . 9 2 ) with the metabolic rate (expressed as 0 2 consumption) of different m a m m a l i a n species [1, 2]. We have also shown that alterations in the thyroid state (which modulates 0 2 consumption) in opposite directions in guinea-pigs, resulted in the predicted alterations in action potential duration; hypothyroidism and hyperthy-

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roidism increased and decreased action potential duration, respectively. Based on these relationships, it m a y be hypothesized that in species with high 0 2 consumption, or rather with high thyroid state and therefore with short action potentials, or in species in which the thyroid state has been increased, it will be possible to induce tetanus in the myocardium. At least for the rat and for the mouse, this hypothesis seems to be correct, and in fact, Henderson and his collegues [7] were able to demonstrate tetanus in rat ventricular muscle. As for the mouse in which the ventricular action potential duration is also very short (at a cycle length of 1000 ms, APDs0 = 5.8 ms [2]), our preliminary experiments show that mouse ventricular preparations can also be tetanized. Several studies have shown that cardiac muscle, which under normal experimental conditions has relatively long action potentials, can be tetanized under modified conditions, e.g. when exposed to ryanodine or caffeine in combination with elevated extracellular calcium concentration [6, 12, 15]. Hence, both ryanodine and caffeine inhibit Ca 2+ removal from the myoplasm by the sarcoplasmic reticulum and thereby enable a steady-activation of tension to develop [6, 8,

9, 13]. In summary, the present study demonstrates that the shrew ventricular muscle can be tetanized. These observations and the reports that rat m y o c a r d i u m can also be tetanized suggest that tetanus can occur in m a m malian cardiac muscle when the appropriate temporal relationships exist between the electrical and the mechanical events.

References 1 BINAH,O., ARIELI, R., BECK, R., ROSEN, M. R., PALTI, Y. Modulation of cardiac electrophysiological properties by metabolic rate [Abstract]. Circulation 72 [Suppl 3], 231 (1985). 2 BINAH,O., ARIELI, R., BECK, R., ROSEN, M. R., PALTI, Y. Ventricular electrophysiological properties: Is interspecies variability related to the throid state? A m J Physio1252, H1265 H1274 (1987). 3 BLINKS,J. R., RUDEL, R., TAYLOR, S. R. Calcium transients in isolated amphibian skeletal muscle fibers : detection with aequorin.J Physiol [Lond] 277, 291 323 (1978). 4 CARLSON,F. D., WILKIE, D. R. Muscle Physiology 84. Englewood Cliffs, New Jersey: Prentice~Hall (1974). 5 EHARA, T., INAZAWA, M. Calcium-dependent slow action potentials in potassium-depolarized guinea-pig ventricular myocardium enhanced by barium ions. Naunyn-Schmiedeberg's Arch Pharmaco1314, 47-54 (1980). 6 FORMAN,R., FORD, L. E., SONNENBLICK,E. H. Effect of muscle length on the force-velocity relationships of tetanized cardiac muscle. Circ Res 31, 195-206 (1972). 7 HENDERSON,A. H., FORMAN, R., BRUTSAERT, D. L., SONNENBLICK, E. H. Tetanic contraction in mammalian cardiac muscle. Cardiovasc Res [Suppl] 1, 96 100 (1971). 8 JENDEN,D.J., FAIRHURST,A. S. The pharmacology ofryanodine. Pharmacol Rev21, 1-25 (1969).

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NAYLER,W. G., D~aLE, P., CHIPPERFIELD,D., GANKr K. Effect of ryanodine on calcium in cardiac muscle. A m J Physio1219, 1620-1626 (1970). PELZER,D., TRAUTWEIN,T., McDONALD, T. F. Calcium channel block and recovery from block in mammalian ventricular muscle treated with organic channel inhibitors. Pfl/igers Arch 394, 97 105 (1982). SATUSHI,K., SAKAI,T. Effects of rapid cooling on mechanical and electrical responses in ventricular muscle of guinea pig. J Physiol [Lond] 361, 361-378 (1985). STROBECK,J. E., KRUEGER,J., SONNENBLICK, E. H. Load and time considerations in the force-length relation of cardiac muscle. Fed Proc 39, 175 182 (1980). SUTKO,J. L., KENYON,J. L. Ryanodine modification of cardiac muscle responses to potassium-free solutions: evidence for inhibition ofsarcoplasmic reticulum calcium release. J Gen Physio1112, 385-404 (1983). TRAUTWEIN,W., PELZER, D., McDONALD, T. F., OSTERRIEDER, W. AHA 39, a new bradycardiac agent which blocks myocardial calcium (Ca) channels in a frequency- and voltage-dependent manner. NaunynSchmiedeberg's Arch Pharmaco1317, 928-232 (1981 ). YUE,D. T., MARBAN,E., WIER, W. G. Relationships between force and intracellular [Ca 2+] in tetanized mammalian heart muscle. J Gen Physio187, 223 942 (1986).